SELENIUM Its Molecular Biology and Role in Human Health, Second Edition
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SELENIUM Its Molecular Biology and Role in Human Health, Second Edition
SELENIUM Its Molecular Biology and Role in Human Health, Second Edition
Edited by Dolph L. Hatfield National Cancer Institute, USA Maria J. Berry University of Hawaii, USA and Vadim N. Gladyshev University of Nebraska, USA
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Library of Congress Control Number: 2006924112 lSBN-10; 0-387-33826-8 ISBN-13: 978-0-387-33826-2
e-ISBN-!0: 0-387-33827-6
Printed on acid-free paper.
© 2006 Springer Science+Business Media, LLC. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science-t-Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed in the United States of America. 9 8 7 6 5 4 3 2 1 springer.com
TABLE OF CONTENTS Contributors
xi
Foreword Raymond F. Burk
xvii
Preface
xxi
Dolph L. Hatfield, Maria J. Berry and Vadim N. Gladyshev Acknowledgements
xxiii
Chapter 1 Selenium: A historical perspective James E. Oldfield
1
Part I. Biosynthesis of selenocysteine and its incorporation into protein Chapter 2 Selenium metabolism in prokaryotes August Bock, Michael Rother, Marc Leibundgut and Nenad Ban
9
Chapter 3 Mammalian and other eukaryotic selenocysteine tRNAs 29 Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Robert Irons, Nianxin Zhong, Dolph L. Hatfield, Byeong Jae Lee, Alexey V. Lobanov and Vadim N. Gladyshev Chapter 4 Evolution of selenocysteine decoding and the key role of selenophosphate synthetase in the pathway of selenium utilization 39 Gustavo Salinas, Hector Romero, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield and Vadim N. Gladyshev Chapter 5 SECIS RNAs and K-turn binding proteins. A survey of evolutionary conserved RNA and protein motifs Christine Allmang and Alain Krol
51
vi
Selenium: Its molecular biology and role in human health
Chapter 6 SECIS binding proteins and eukaryotic selenoprotein synthesis Donna M. Driscoll and Paul R. Copeland Chapter 7 The importance of subcellular localization of SBP2 and EFsec for selenoprotein synthesis Peter R. Hoffmann and Maria J. Berry Chapter 8 Selenocysteine biosynthesis and incorporation may require supramolecular complexes Andrea L. Small-Howard and Maria J. Berry
63
73
83
Part II. Selenium-containing proteins Chapter 9 Selenoproteins and selenoproteomes Vadim N. Gladyshev
99
Chapter 10 Deletion of selenoprotein P gene in the mouse Raymond F. Burk, Gary E. Olsen and Kristina E. Hill
Ill
Chapter 11 Selenium and methionine sulfoxide reduction Hwa-Young Kim and Vadim N. Gladyshev
123
Chapter 12 Selenoprotein W in development and oxidative stress Chrissa Kioussi and Philip D. Whanger
135
Chapter 13 The 15-kDa selenoprotein (SeplS): functional analysis and role in cancer 141 Vyacheslav M. Labunskyy, Vadim N. Gladyshev and Dolph L. Hatfield Chapter 14 Regulation of glutathione peroxidase-1 expression Roger A. Sunde
149
Table of Contents
Chapter 15 Selenoproteins of the glutathione system Leopold Flohe and Regina Brigelius-Flohe Chapter 16 New roles of glutathione peroxidase-1 in oxidative stress and diabetes Xin Gen Lei and Wen-Hsing Cheng Chapter 17 Selenoproteins of the thioredoxin system Arne Holmgren Chapter 18 Mitochrondrial and cytosolic tliioredoxin reductase loiocliout mice Marcus Conrad, Georg W. Bornkamm and Marcus Brielmeier
vii
161
173
183
195
Chapter 19 Selenium, deiodinases and endocrine function Antonio C. Bianco and P. Reed Larsen
207
Chapter 20 Biotechnology of selenium Linda Johansson and Elias S.J. Arner
221
Part III. Selenium and human health Chapter 21 Selenium, selenoproteins and brain function Ulrich Schweizer and Lutz Schomberg
233
Chapter 22 Selenium as a cancer preventive agent Gerald F. Combs, Jr. and Junxuan Lii
249
Chapter 23 Peering down the kaleidoscope of thiol proteomics and unfolded protein response in studying the anticancer action of selenium 265 Ke Zu, Yue Wu, Young-Mee Park and Clement Ip
viii
Selenium: Its molecular biology and role in human health
Chapter 24 Genetic variation among selenoprotein genes and cancer Alan M. Diamond and Rhonda L. Brown
277
Chapter 25 Selenium and viral infections MelindaA. Beck
287
Chapter 26 Role of selenium in HIV/AIDS Marianna K. Baum and Adriana Campa
299
Chapter 27 Effects of selenium on immunity and aging Roderick C. McKenzie, Geoffrey J. Becket and John R. Arthur
311
Chapter 28 Selenium and male reproduction 323 Matilde Maiorino, Antonella Roveri, Fulvio Ursini, Regina Brigelius-Flohe and Leopold Flohe Chapter 29 Mouse models for assessing the role of selenium in health and development 333 Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Nianxin Zhong, Dolph L Hatfield, Robert Irons, Cindy D. Davis, Byeong Jae Lee, Sergey V. Novoselov and Vadim N. Gladyshev Chapter 30 Drosophila as a tool for studying selenium metabolism and role of selenoproteins Cristina Pallares, Florenci Serras and Montserrat Corominas Chapter 31 Selenoproteins in parasites Gustavo Salinas, Alexey V. Lobanov and Vadim N. Gladyshev Chapter 32 Incorporating 'omics' approaches to elucidate the role of selenium and selenoproteins in cancer prevention Cindy D. Davis and John A. Milner
343
355
367
Table of Contents
ix
Chapter 33 Selenium-induced apoptosis Ick Young Kim, Tae Soo Kim, Youn Wook Chung and Daewon Jeong
379
Chapter 34 Selenoprotein mimics Junqiu Liu and Guimin Luo
387
Chapter 35 Update of human dietary standards for selenium Orville A. Levander and Raymond F. Burk
399
Index
411
Contributors Christine Allmang
Maria J. Berry
Architecture et Reactivite de I'arN UPR 9002 du CNRS-Universite Louis Pasteur Institut de Biologie Moleculaire et Cellulaire 67084 Strasbourg, France
Department of Cell and Molecular Biology John A. Bums School of Medicine University of Hawaii at Manoa Honolulu, HI 96813, USA
Antonio C. Bianco Elias S. J. Arner Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden
John R. Arthur Division of Vascular Health Rowett Research Institute Bucksbum, Aberdeen, Scotland AB219SB,UK
Nenad Ban Institute of Molecular Biology and Biophysics Swiss Federal Institute of Technology ETH Hfinggerberg, HPK Building CH-8093 Zurich, Switzerland
Marianna K. Baum
Thyroid Section, Division of Endocrinology Diabetes and Hypertension Department of Medicine Brigham and Women's Hospital and Harvard Medical School 77 Avenue Louis Pasteur Boston, MA 02115, USA
August Bdck Lehrstuhl flir Mikrobiologie der Universitat Munchen, D-80638 Munich, Germany
Georg W. Bornkamm Institute of Clinical Molecular Biology and Tumor Genetics GSF-Research Centre for Environment and Health 81377 Munich, Germany
Florida International University Stempel School of Public Health Department of Dietetics and Nutrition 11200 SW 8th Street Miami, FL 33199, USA
Markus Brielmeier
Melinda A. Beck
Regina Brigelius-Flohe
Department of Nutrition University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
Department Biochemistry of Micronutrients German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) Arthur-Scheunert-Allee 114-116 D-14558 Nuthetal, Germany
Geoffrey J. Beckett Department of Clinical Biochemistry University of Edinburgh Combined Laboratories The Royal Infirmary of Edinburgh 51 Little France Cresdent Edinburgh, Scotland, EH 16 4SA, UK
Department of Comparative Medicine GSF-Research Centre for Environment and Health 85764 Neuherberg, Germany
Rhonda L. Brown Department of Human Nutrition University of Illinois at Chicago Chicago, IL 60612, USA
Xll
Selenium: Its molecular biology and role in human health
Raymond F. Burk
Paul R. Copeland
Division of Gastroenterology, Hepatology, and Nutrition Department of Medicine Vanderbilt University School of Medicine Nashville, TN 37232, USA
Department of Molecular Genetics Microbiology and Immunology UMDNJ - Robert Wood Johnson Medical School Piscataway, NJ 08854, USA
Bradley A. Carlson
Montserrat Corominas
Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Departament de Geneica Universitat de Barcelona, Diagonal 645 08028 Barcelona, Spain
Adriana Campa Florida International University Stempel School of Public Health Department of Dietetics and Nutrition 11200 SW 8th Street Miami, FL 33199, USA
Wen-Hsing Cheng Laboratory of Molecular Gerontology National Institute on Aging National Institutes of Health Bahimore, MD 21224, USA
Youn Wook Chung Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea
Gerald F. Combs, Jr. Grand Forks Human Nutrition Research Center, USDA-ARS Grand Forks, ND 58202, USA
Marcus Conrad Institute of Clinical Molecular Biology and Tumor Genetics GSF-Research Centre for Environment and Health 81377 Munich, Germany
Cindy D. Davis Nutritional Science Research Group Division of Cancer Prevention National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Alan M. Diamond Department of Human Nutrition University of Illinois at Chicago Chicago, IL 60612, USA
Donna M. DriscoU Department of Cell Biology Lemer Research Institute Cleveland Clinic Foundation Cleveland, OH 44195, USA
Leopold Flohe MOLISA GmbH Universitatsplatz 2 D-39106 Magdeburg, Germany
Vadim N. Gladyshev Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA
Dolph L. Hatfield Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Contributors
xiu
Kristina E. HiU
Hwa-Young Kim
Division of Gastroenterology, Hepatology, and Nutrition Department of Medicine Vanderbilt University School of Medicine Nashville, TN 37232, USA
Department of Biochemistry University of Nebraska Lincoln, Nebraska 68588, USA
Peter R. Hoffmann John A. Bums School of Medicine Department of Cell and Molecular Biology University of Hawaii at Manoa Honolulu, HI 96813, USA
Ick Young Kim Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea
Arne Holmgren Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden
Clement Ip Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA
Tae Soo Kim Laboratory of Cellular and Molecular Biochemistry School of Life Sciences and Biotechnology Korea University 1,5-Ka, Anam-Dong Sungbuk-Ku Seoul 136-701, Korea
Chrissa Kioussi Robert Irons Nutritional Science Research Group Division of Cancer Prevention and Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Daewon Jeong BK2I HLS, Seoul National University 28 Yeonkun-Dong Chongno-Ku Seoul 110-749, Korea
Linda Johansson Medical Nobel Institute for Biochemistry Department of Medical Biochemistry and Biophysics Karolinska Institute SE-171 77 Stockholm, Sweden
Department of Biochemistry and Biophysics Oregon State University Corvallis, OR 97331, USA
Alain Krol Architecture et Reactivite de I'arN UPR 9002 du CNRS-Universite Louis Pasteur Institut de Biologic Moleculaire et Cellulaire 67084 Strasbourg, France
Vyacheslav M. Labunskyy Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA
XIV
Selenium: Its molecular biology and role in human health
P. Reed Larsen
Junxuan Lii
Thyroid Section, Division of Endocrinology Diabetes and Hypertension Department of Medicine Brigham and Women's Hospital and Harvard Medical School 77 Avenue Louis Pasteur Boston, MA 02115, USA
Hormel Institute University of Minnesota Austin, MN 55912, USA
Guimin Luo Key Laboratory for Molecular Enzymology and Engineering Jilin University Changchun 130023, China
Byeong Jae Lee Laboratory of Molecular Genetics Institute of Molecular Biology and Genetics School of Biological Sciences Seoul National University Seoul 151-742, Korea
Matilde Maiorino
Xin Gen Lei
Roderick C. McKenzie
Department of Animal Science Cornell University Ithaca, NY 14853, USA
Marc Leibundgut Institute of Molecular Biology and Biophysics Swiss Federal Institute of Technology ETH Honggerberg, HPK Building CH-8093 Zflrich, Switzerland
Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy
Laboratory for Clinical and Molecular Virology Royal Dick Veterinary School University of Edinburgh Summerhall, Edinburgh EH9 IQH, UK
John A. Milner
Orville A. Levander
Nutritional Science Research Group Division of Cancer Prevention National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Beltsville Human Nutrition Research Center U. S. Department of Agriculture Agricultural Research Service Beltsville, MD 20705, USA
Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA
Junqiu Liu
James E. Oldfield
Key Laboratory for Supramolecular Structure and Materials Jilin University Changchun 130012, China
Alexey V. Lobanov Department of Biochemistry University of Nebraska Lincoln, NE 68688, USA
Sergey V. Novoselov
Oregon State University Corvallis, OR 97331, USA
Gary £. Olson Division of Gastroenterology, Hepatology, and Nutrition Department of Cell and Developmental Biology Vanderbilt University School of Medicine Nashville, TN 37232, USA
Contributors
XV
Cristina Pallar^s
Ulrich Schweizer
Departament de Geneica Universitat de Barcelona Diagonal 645 08028 Barcelona, Spain
Neurobiology of Selenium Neuroscience Research Center and Institute for Experimental Endocrinology Charit^-Universitatsmedizin Berlin Charite Campus Mitte D-10117 Berlin, Germany
Young-Mee Park Department of Cellular Stress Biology Roswell Park Cancer Institute Buffalo, NY 14263, USA
Hector Romero Laboratorio de Organizacion y Evoluci6n del Genoma Dpto. de Biologia Celular y Molecular Instituto de Biologia Facultad de Ciencias Igua4225 Montevideo, CP 11400, Uruguay
Michael Rother Lehrstuhl fUr Mikrobiologie der Universitat Mflnchen D-80638 Munich, Germany
Antonella Roveri Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy
Aniruddlia Sengupta Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Florenci Serras Departament de Geneica Universitat de Barcelona Diagonal 645 08028 Barcelona, Spain
Rajeev Shrimali Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Andrea L Small-Howard Gustavo Salinas Cdtedra de Inmunologia Facultad de Quimica-Facultad de Ciencias Universidad de la Repiiblica Instituto de Higiene Avda. A. Navarro 3051 Montevideo, CP 11600, Uruguay
Lutz Sclioinburg Institute for Experimental Endocrinology Charit^-Universitatsmedizin Berlin Charity Campus Mitte D-10117 Berlin, Germany
Department of Cell and Molecular Biology John A. Bums School of Medicine University of Hawaii at Manoa Honolulu, HI 96813, USA
Roger A. Sunde 1415 Linden Drive University of Wisconsin Madison, WI53705, USA
Fulvio Ursini Department of Biological Chemistry University of Padova Viale G. Colombo, 3 1-35121 Padova, Italy
xvi
Selenium: Its molecular biology and role in human health
Philip D. Whanger Department of Environmental and Molecular Toxicology Oregon State University Corvallis, OR 97331, USA
YueWu Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA
Xue-Ming Xu Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Min-Hyuk Yoo Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
Nianxin Zhong Molecular Biology of Selenium Section Laboratory of Cancer Prevention Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892, USA
KeZu Department of Cancer Chemoprevention Roswell Park Cancer Institute Buffalo, NY 14263, USA
Foreword The discovery of selenoproteins in 1973 was the starting point for today's flourishing selenium field [1,2]. It provided evidence that selenium had biochemical functions that could account for its nutritional effects [3,4]. Further, it opened the selenium field to investigation by the methods of biochemistry, which led to the identification of several more selenoproteins and showed that selenocysteine was the form of the element in animal selenoproteins and in most bacterial ones. Although noteworthy efforts were made to uncover the mechanism of selenocysteine and selenoprotein synthesis using biochemical methods, the problem yielded only when attacked with the methods of molecular biology [5,6]. The bacterial mechanism was characterized first; characterization of the animal mechanism is a work in progress. It is interesting to note that the only genes that are devoted to selenium metabolism are those that support selenoprotein synthesis and selenocysteine catabolism. Consequently, it seems likely that competition for selenium between selenoprotein synthesis and the production of selenium excretory metabolites [7] controls wholebody selenium homeostasis. The physiological functions of selenium derive fi-om the catalytic and physical properties of selenoproteins. Selenoproteins such as the glutathione peroxidases and the thioredoxin reductases have redox activities that allow them to serve in oxidant defense. The deiodinases use their redox activities to activate and inactivate thyroid hormones. From these two examples, it can be seen that selenoprotein functions are diverse while having in common a redox mechanism. Although a few of the biological functions of selenium have been identified, many have not. Application of bioinformatics techniques to genomic databases has identified 25 genes for selenoproteins in the human genome [8]. Most of the proteins represented by those genes have not been characterized to the point where their functions can be assessed. Thus, one of the major challenges in selenium research is to characterize all the selenoproteins so that their biological activities can be determined. The ultimate goal of selenium research is to improve human health. Veterinary and animal science investigators had already demonstrated that nutritional selenium deficiency occurred in animals fed plants firom areas with low soil selenium availability when, in 1979, Chinese researchers reported the existence of a selenium-responsive disease in such an area. Their study showed convincingly that the occurrence of Keshan disease, a childhood cardiomyopathy, could be prevented by selenium supplementation [9]. Although several other diseases have been postulated to be selenium deficiency conditions, studies to prove those claims have not appeared. Thus,
xviii
Selenium: Its molecular biology and role in human health
Keshan disease, which has almost disappeared from China as economic conditions have improved, remains the extreme example of pathology that can occur in selenium deficient human beings. While selenium deficiency severe enough to allow the occurrence of Keshan disease is rare, people in many areas of the world have selenium intakes that are not sufficient to allow full expression of all selenoproteins. New Zealand and many countries in Europe fall into this category. In response to learning that its selenium status was low, Finland chose in 1985 to add selenium to its fertilizer. It has thereby become a laboratory for studying the effects of supplementing a population with selenium. The selenium status of Firms rapidly became comparable to that of North Americans but without discemable effects on the incidences of major diseases [10]. This type of study without a control population would not be expected to detect subtle health effects or uncommon ones such as altered responses to drugs: so the question of whether full expression of selenoproteins is needed for optimum health must remain open. This issue needs attention from clinical investigators because of the large number of people affected and the implications it has for setting official dietary requirements for selenium. More directly related to basic selenium research, mutations and polymorphisms of selenoprotein genes and of genes involved in selenoprotein synthesis can cause human disease. An example of this is the congenital muscle disease that results from mutation of the gene for selenoprotein N, one of the selenoproteins of unknown function. Perhaps elucidation of the function of selenoprotein N will suggest a treatment for the muscle disease. Phenotypes of mice with deletion of a selenoprotein might be instructive in this respect. For example, deletion of selenoprotein P causes neurological dysfunction that can be prevented by selenium supplements above the nutritional requirement. If an analogous human condition were found, selenium supplements might be efficacious in its treatment. Examples of animal research that support understanding of human diseases stimulate basic selenium research. In addition to research on the physiological functions of selenium, considerable enthusiasm has been generated for studying the effects of pharmacological doses of the element. The results of numerous animal studies and limited human trials have suggested that administration of pharmacological doses of selenium can prevent some kinds of cancer [11]. Additional trials are underway to test this hypothesis. If such a chemopreventive effect of selenium can be proven, it would not likely be linked to the selenoproteins because the subjects in the trials were not selenium deficient before supplementation was started. This means that the selenoproteins would have been at their optimal levels initially and that selenium supplements would not have been expected to affect them. Other
Foreword
xix
metabolic effects of high selenium intake have been noted, however, and might account for its effects on cancer development. It will be important for public health reasons to determine whether selenium is an effective chemopreventive agent in human beings and, if it is, to determine the safety of pharmacological doses of selenium. Many tasks remain in the selenium field. Additional characterization of individual selenoproteins and elucidation of the mechanism of selenoprotein synthesis are needed to facilitate identification of pathological conditions involving selenium. Clinical studies are needed to determine the selenium intake needed to ensure full expression of all selenoproteins and to assess the health implications of selenium intakes that do not allow full expression of all selenoproteins. And, finally, whether selenium is efficacious as a chemopreventive agent needs to be determined. References 1. 2.
DC Turner, TC Stadtman 1973 Arch Biochem Biophys 154:366 JT Rotruck, AL Pope, HE Ganther, AB Swanson, D Hafeman WG Hoekstra 1973 Science 179:588 3. KE McCoy, PH Weswig 1969 JNutr 98:383 4. K Schwarz, CM Foltz 1957 J Amer Chem Soc 79:3292 5. A Bock, K Forchhammer, J Heider, C Baron 1991 Trends Biochem Sci 16:463 6. I Chambers, J Frampton, P Goldfarb, N Affara, W McBain, PR Harrison 1986 EMBO J 5:1221 7. Y Kobayashi, Y Ogra, K Ishiwata, H Takayama, N Aimi, KT Suzuki 2002 Proc Natl Acad Sci U S A 99: 15932 8. GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 Science 300:1439 9. Keshan Disease Research Group 1979 Chinese Medical Journal 92:471 10. M Eurola, G Alfthan, A Aro, P Ekholm, V Hietaniemi, H Rainio, R Rankanen, E-R Venalainen 2003 Agrifood Research Reports 36 Results of the Finnish selenium monitoring program MTT Agrifood Research Finland, FIN-31600 Jokioinen, Finland pp 42 11. LC Clark, GF Combs Jr, BW TumbuU, EH Slate, DK Chalker, J Chow, LS Davis, RA Glover, GF Graham, EG Gross, A Krongrad, JL Lesher, HK Park, BB Sanders, CL Smith, JR Taylor 1996 JAMA 276:1957
Raymond F. Burk
Preface Since the first edition of Selenium: Its Molecular Biology and Role in Human Health was published in 2001, many new insights into the biochemical, molecular, genetic and health aspects of this fascinating element have been elucidated. Several new human clinical trials have also been undertaken examining the role of selenium in protection against different cancers. For example, the National Cancer Institute initiated two new clinical trials involving selenium. One of these is called SELECT, Selenium and vitamin E Cancer Prevention Trial, and it involves examining the role of selenium and vitamin E in protecting against prostate cancer, with a goal of enrolling over 35,000 males in the study. The other trial involves examining the role of selenium in protection against lung cancer, a study incorporating 1960 individuals. The commitment of hundreds of millions of dollars to these trials for examining the role of selenium in protecting humans against different forms of cancer illustrates how highly important this element is regarded by the medical and scientific communities in health issues. What is of such significance to elucidating the role of selenium in health in these human clinical trials is that not only will the effect of selenium on prostate and lung cancers be assessed, but these trials will shed light on the role of many additional aspects of selenium in health such as aging, heart disease, viral inhibition and other forms of cancer including colon, liver and brain malignancies. Many exciting discoveries have occurred in the last five years which are described in the current edition. For example, the entire selenoprotein gene population, designated the selenoproteome, has been identified in humans and rodents. Furthermore, the various selenoproteins described in the last edition have been further characterized and their new features described. Numerous selenoprotein genes have been targeted for removal using standard or loxP-Cre technologies to further elucidate their functions in development and health. Selenoproteins have also been shown to be involved in different human genetic disorders. Many new and novel features have been uncovered on the biosynthesis of selenocysteine, the amino acid that contains selenium, and its incorporation into protein as the 21^' amino acid in the genetic code. Further studies on the various components involved in the biosynthesis of selenocysteine and its insertion into protein have determined that much of this vast selenoprotein machinery exists in supramolecular complexes. Finally, several mouse models that were specifically generated for examining the role of selenium and selenoproteins in health and development have been devised. The rapid expansion and many new discoveries in the selenium field in the last five years are reflected by the addition of many new chapters and a much longer current edition.
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Selenium: Its molecular biology and role in human health
The purpose of the new edition book is to inform the reader of these many new discoveries and to examine our present knowledge of the molecular biology of selenium, its incorporation into proteins as selenocysteine and the role that this element and selenium-containing proteins (selenoproteins) play in health. The book's emphasis is on our understanding of selenium metabolism in mammals and the role of this element in human health. The book begins with a brief history of selenium and how its face has changed through the years from one of a toxin and possible carcinogen to one of an essential micronutrient in the diets of humans and other animals. Indeed, selenium is now touted as an important cancer chemopreventative agent, as well as for its roles in inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, preventing heart disease and other cardiovascular and muscle disorders, slowing the aging process, and having roles in development, male reproduction and immune function. As more of the molecular biology of selenium is unraveled, we are understanding the manner in which this element does indeed have direct roles in each of these health issues. The present book, like the first edition, is divided into three parts with ten more chapters than the earlier edition. The chapters in Part I, which is entitled "Biosynthesis of selenocysteine and its incorporation into protein," define selenocysteine as the 21^' naturally occurring amino acid in the genetic code and describe how this amino acid is incorporated into protein. Interestingly, the inclusion of selenocysteine to the genetic code as its 21'' amino acid marks the first addition to the code since it was deciphered in the mid-1960s. Our current understanding of how selenoprotein expression is regulated and the nucleocytoplasmic shuttling of the selenocysteine biosynthesis and insertion machinery in eukaryotes is also discussed in Part I. Part II is entitled "Selenium-containing proteins" and it discusses our current understanding of selenoproteins, primarily in higher eukaryotes. Part III is entitled "Selenium and human health" and it covers our current understanding of the role of selenium in various diseases, including cancer and heart disease, in HIV infection and AIDS, in male reproduction, and as an antiviral agent. The role of small molecular weight, selenium-containing compounds (selenocompounds) in human health and the dietary selenium requirements for humans are also discussed. In summary, this book provides an up-to-date review of much of the ongoing research in the selenium field. It provides a resource for scientists working in the selenium field, as well as for physicians, other scientists and students who wish to learn more about this fascinating micronutrient.
Dolph L. Hatfield, Maria J. Berry and Vadim N. Gladyshev
Acknowledgements The support and generous help of Bradley A, Carlson throughout the preparation of this book is gratefully acknowledged. The editors also wish to thank Sergey V. Novoselov for his help with the book cover.
Chapter 1. Selenium: A historical perspective James E. Oldfield Oregon State University, Corvallis, Oregon 97331, USA
Summary: The path followed in the biochemistry of selenium has taken some sharp turns during its development. At first, feared as a poisoner of livestock and later impugned as a carcinogen, selenium has about-faced and is now recognized as an essential micronutrient with anti-carcinogenic properties. While early studies on selenium have focused on the role of this trace element in animal physiology and studies with microorganisms, the field has matured to employ molecular biology to explain and employ the protective effects of selenium against a number of human maladies, including cancer and heart disease. The emphasis of this chapter is an examination of selenium's early history as a toxin, its later recognition as an essential micronutrient in the diet of mammals and its impact in the livestock industry that provided the foundations for the vast amount of the current basic and health research on this fascinating element. Even before it had been discovered and named, there were reports of conditions occurring in animals that, in retrospect, must have been caused by an excess of selenium. The Venetian explorer, Marco Polo, wrote of problems encountered by travelers in a mountainous region of what is now Shaan-Xi province in China [1]. He noted that when horses or other beasts of burden grazed on some indigenous plants, their hooves would split and fall off In the light of present knowledge, it would seem that these plants were "selenium accumulators" that concentrate selenium fi'om the soil to levels that are toxic to grazing animals. Then, several hundred years later, Madison [2] an army surgeon stationed at Fort Randall in the Nebraska territory, described a similar condition among dragoon remount horses that had been newly introduced to the area. K.W. Franke, who was a State Chemist at South Dakota State College, headed much of the definitive work on local toxic plants [3]. Actual proof of selenium's involvement in this toxicity problem came when workers in South Dakota identified it as the toxic principle in plants causing what was locally called "alkali disease" in cattle, on range lands of the north-central United States [4].
Selenium: Its molecular biology and role in human health The discovery of selenium, as an element, was made in 1817 by a Swedish chemist, Jons Jakob Berzelius, through what was, at that time, an elegant analytical process [5]. Berzelius was investigating the cause of illnesses among workers at a sulfuric acid manufacturing plant that occurred when copper pyrites from a local mine were used as the source of sulfur. He scraped a red deposit from the walls of the lead chambers in which the pyrites were processed, anticipating that it might contain tellurium, an element he had recently discovered. Tellurium was not present but he isolated another new element which he named selenium, after Selene, the Greek goddess of the moon. Taken together, these three early indications of selenium toxicity were certainly an inauspicious beginning for what was eventually to be recognized as an essential micronutrient. At that time, if anyone thought of selenium at all, and few did, it was as a toxic element. The earliest organized research effort with selenium, then, was directed toward means of avoiding, or coping with its toxicity. It was recognized that certain areas in the United States had seleniferous soils and this, together with the identification of selenium-accumulating plants, spelled trouble for animal agriculture operations. Ranchers learned to identify and remove accumulator plants, to dilute their livestock's forage feed with nonseleniferous materials, and to move their animals around in cycles which included some time on low-selenium forage grazing areas. Then, in 1957, research by a German scientist, Klaus Schwarz, working at the U.S. National Institutes of Health in Bethesda, changed forever the way selenium was assessed by both the scientific community and the general public. Schwarz had been working in Germany on studies of brewers' yeast as a protein source, during World War II, and he continued these studies when he came to America. He found, when he fed torula yeast, rather than brewers' yeast to rats, that they developed necrotic livers and he concluded that the brewers' yeast contained some essential nutrient that the torula yeast did not. He named the unknown substance "factor 3," since two other substances that alleviated liver necrosis had already been identified: vitamin E and (mistakenly) L-cysteine, which were known as factors 1 and 2. In 1957, Schwarz and Foltz armounced that they had fractionated factor 3 and found it to contain selenium [6]. Although its toxicity remained a real and difficult problem, it was now evident that, at lower dietary levels, selenium was harmless and, indeed, was quickly recognized as a dietary essential. The response to this discovery was immediate, and surprisingly extensive, as selenium deficiency was shown to be implicated in a number of animal diseases beyond the original liver necrosis. Studies in Oregon [7] showed that it was the cause of "white muscle disease," a myopathy that affected hundreds of calves and lambs each year in the central part of the state. Then, in quick succession, selenium deficiency was linked to other diseases of domestic animals and birds, including exudative diathesis and pancreatic
Selenium: A historical perspective degeneration in poultry, hepatosis dietetica in pigs and "ill-thrift" in cattle and sheep [8]. Of these, white muscle disease is the most widespread and has the greatest economic impact - involving not only calves and lambs but also deer, goats, horses, poultry and rabbits and occurring in all the major sheepproducing countries in the world [9]. Questions naturally arose about the biochemical function of selenium: how such small amounts of it could produce such profound biological reactions. These were answered, at least in part, by research carried out simultaneously in America and Germany. At the University of Wisconsin, Rotruck and associates [10] discovered selenium's presence in the enzyme, glutathione peroxidase, while Flohe in Germany showed the precise placement of selenium in the enzyme molecule [11]. It seemed that this enzymic involvement might be one way in which selenium could perform its beneficial metabolic functions and would explain how so little selenium could accomplish so much. Farmers and ranchers are often accused of being slow in accepting and applying research results relevant to their operations but this was certainly not the case with selenium supplementation. Its benefits were so dramatic that it soon became an accepted husbandry practice in areas of selenium deficiency, worldwide. Research, too, developed a number of methods by which selenium might be made available to animals, including feed fortification, injection, and with ruminant animals, an ingenious heavy pellet that would remain in the forestomach and gradually make selenium available for periods as long as a year. Selenium was also added to fertilizer mixes used on range and pasture land to improve the selenium status of forage plants grown thereon [12]. So, early biological research with selenium was stimulated by the animal industries, which in countries like New Zealand, were major contributors to the nation's economy. At first thought, the possibility of a selenium deficiency occurring among humans seemed remote on the grounds that the great diversity of the human diet would make an overall selenium deficiency unlikely. Cases of a human selenium deficiency did emerge, however, in some rural areas in China, where the people lived almost entirely on food substances produced on their ovra (selenium-deficient) land. This led to a cardiac myopathy that was first reported in Keshan county, of Heilongjiang province in northeastern China, and was called Keshan disease [13]. It is interesting to compare these symptoms with those of white muscle disease among animals - they have much in common. One of the fascinations of selenium research has been the abrupt changes in direction that have taken place over the years as knowledge of selenium's functions developed. So it was in the latter years of the 20th century when research interest in selenium switched from animals to molecular research and an emphasis on the role of selenium in human health.. This change was
Selenium: Its molecular biology and role in human health fueled by observations that, in addition to its now accepted nutrient function, selenium could also exert beneficial effects on human health at dietary levels somewhat higher than those required for its purely-nutritional activity. In Finland, governmental agencies became concerned about the long-term effects of low-selenium diets in their country on the health of the human population. They authorized the addition of selenium, as selenate, to fertilizers applied in the production of animal and human foods and have shown that this process effectively raises the selenium content of the Finnish diet to levels consistent with good nutrition and human health. They have carefully monitored the situation since Se-fertilization began, in 1984 [14] and we can be grateful to the Finnish scientists for providing much useful information on this type of application of selenium. The Finnish experience, too, has led to studies of the selenium status of other populations where dietary levels have been decreasing, over time [15]. The health-preserving activities of selenium, at about double the dietary levels recommended by the U.S. National Research Council, have been reviewed in detail by Combs [16]. It is interesting that these studies drew on the findings of Clark and associates at the Arizona Cancer Center and this gives rise to another of selenium research's "about faces," since early research had proposed that selenium was a carcinogen [17]. The application of selenium supplementation of livestock feeds to overcome selenium deficiency was prohibited for a time by the U.S. Food and Drug Administration (FDA) because of concerns raised by studies in their own laboratories suggesting that selenium might be a carcinogen [18]. This ruling exasperated American livestock producers who pointed out that they were being denied application of research that their tax dollars had helped pay for, while their strong competitors in New Zealand and Australia were routinely applying selenium in diets of their livestock. This conflict was resolved in this country, and in fact, the use of selenium in livestock feeds has been estimated to have saved this industry hundreds of millions of dollars in preventing muscle disorders and numerous other anomalies including enhancing reproduction as discussed in detail by Combs and Combs [19]. So, to recapitulate, the trail of research with selenium has been a tortuous one, marked by sudden and sometimes dramatic changes in direction. Its discovery, by Berzelius, was serendipitous; he was expecting to find tellurium in the Swedish sulfuric acid vats, but instead, he isolated selenium. There was a corollary to this in the much later studies of its health-protecting properties. When the Oregon workers sought a cure against white muscle disease, they thought it would be vitamin E, which proved ineffective, but selenium worked. Most of the early research, done in the first half of the last century, focused on means of avoiding selenium's toxicity, but Schwarz's carefully controlled studies with yeast opened the door for investigation of its
Selenium: A historical perspective beneficial effects as a micronutrient. Commercial application of supplementary selenium in diets of farm animals was delayed for several years due to fear that it might be a carcinogen but then, in one if its most dramatic about-faces, selenium proved to be anti-carcinogenic. Interestingly, Clark's study aimed against skin cancer where selenium that proved ineffective, but it was found to have significant benefits with other types of cancer, including those of the prostate, lung and intestine/colon (see [17] and references therein). It is understandable, certainly, because of its dreaded consequences in human health that cancer should have received the major attention by investigators of this new area of selenium's activity. It is exciting, however, that it has been shown to be a useful strategy against a number of other human diseases, and the Antioxidant Vitamins newsletter published by Hoffinan La Roche company listed 50 diseases against which selenium may play a protective role [20]. These include diseases of the heart, long recognized as major killers of the world's human populations [21,22] and ADDS, which has been called the "greatest catastrophe in human history" [23]. But most importantly, these earlier studies showing the importance of selenium in the diets of laboratory animals and livestock and the finding of selenium in protein as the amino acid selenocysteine in the 1970s have provided the foundations for the remarkable transformation that this field witnessed in the last 20 years. Indeed, the basic research described in this edition specifies selenium as a preventative agent in cancer, heart disease and other cardiovascular and muscle disorders, as an inhibitor of viral expression and as a factor delaying the aging process and the progression of AIDS in HIV positive patients. Furthermore, selenium is identified as,an essential element in mammalian development, male reproduction and immune function. These many health benefits now attributed to selenium highlight the serrated road fi-om a toxin to what may now be designated as a magic bullet. References 1.
2. 3. 4. 5. 6. 7. 8.
Polo, Marco. 1967. The Travels of Marco Polo Translated by EW Marsden and revised by T Wright pp 100-101 Everymans Library, London (Cited in C Reilly 1996 Selenium in Food and Health Chapman & Hall London p 3) TC Madison 1860 Statistical Report on the Sickness and Mortality in the Army of the United States RH Cooledge ed Ex Doc 52:37 KW Franke 1934 J Nutrition 8:597-608 AL Moxon 1937 Bull. 311, S. Dakota AgExp Sta 81 pp JJ Berzelius 1818 Serie 2 7:194 (Cited in C Reilly 1996 Selenium in Food and Health Chapman & Hall London p 2) K Schwarz, CM Foltz \951JAm Chem Soc 78:3292 OH Muth, JE Oldfield, LP Remmert, JR Schubert 1958 Science 128:1090 C Reilly 1996 Selenium in Food and Health Chapman & Hall ed London 338
Selenium: Its molecular biology and role in human health
9. E Wolf, V Kollonitsch, CH Kline 1963 Agr & Food Chem 11:355 10. JT Rotruck, AL Pope, HE Ganther, AB Swanson, DG Hafeman, WG Hoekstra 1973 Science 179:588 11. L Flohe, WA Gunzler, HH Schock 1973 FEES Letters 32:132 12. JE Oldfield 1997 Biomed & Environ 10:280 13. B Gu 1993 Chinese Med J 96:25\ 14. P Koivistoinen, K Huttunen 1986 Ann Clin Res 18:13 15. MP Rayman 2000 Lancet 356:233 16. GF Combs Jr 2001 Nutrition and Cancer 40:6 17. LC Clark, GF Combs Jr, BW Tumbull, EH Slate, D Alberts, D Abele, R Allison, J Bradshaw, D Chalker, J Chow, D Curtis, J Dalen, L Davis, R Deal, M Dellasega 1996 J Am Med Assoc 216:1957 18. AA Nelson, OG Fitzhugh, HO Calvery 1943 Cancer Res 3:230 19. GF Combs Jr, SB Combs 1986 The Role of Selenium in Nutrition Academic Press Inc New York 20. 1993 Antioxidant Vitamins Newsletter Hoffinan LaRoche Co New York 7:12 21. AFM Kardinaal, FJ Kok, L Kohlmeier, M Martin-Moreno, J Ringstad, J Gomez-Aracena, VP Mazaer, M Thamm, BC Martin, P Van'tVeer, JK Huttunea 1997 Am J Epidemiology 145:373 22. JT Salonen 1985 Trace Elements in Health and Disease H Bostrom, N Ljungstedt ed Almquist and Wiksell International Stockholm 172 23. HD Foster 2002 What Really Causes AIDS? Trafford Publishing Victoria, Canada 197
Parti
Biosynthesis of selenocysteine and its incorporation into protein
Chapter 2. Selenium metabolism in prokaryotes August Bock and Michael Rother Lehrstuhlfiir Mikrobiologie der Universitat Munchen, D-80638 Munich, Germany
Marc Leibundgut and Nenad Ban Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, ETH Honggerberg, HPK building, CH-8093 Zurich, Switzerland
Summary: The biosynthesis and specific incorporation of selenocysteine into protein requires the function of a UGA codon determining the position of selenocysteine insertion and a secondary/tertiary structure within the mRNA, designated the SECIS element, following the UGA at its 3'side in bacteria and located in the 3 'non-translated region in archaea. Biosynthesis of selenocysteine takes place on a unique tRNA species, tRNA^**", which is charged by seryl-tRNA synthetase and serves as an adaptor for the conversion of the seryl moiety into the selenocysteyl product by selenocysteine synthase. Monoselenophosphate, provided by selenophosphate synthetase, is the selenium donor. Selenocysteyl-tRNA^**^ is bound by the special translation factor SelB, which in bacteria via its Cterminal extension interacts with the apical part of the SECIS stem-loop structure. Crystallographic and NMR structural analyses of this extension from Moorella thermoacetica SelB, either free or complexed with the SECIS element, showed that it is made up of four winged helix domains from which only the C-terminal one interacts with the RNA ligand. Structure of the entire SelB molecule from Methanococcus maripaludis in the apo- and GDP/GTP bound forms revealed that it is a chimera between elongation factor Tu and initiation factors. Comparison of the structures in the GDP and GTP forms and modelling of the interactions between selenocysteyl-tRNA and SelB provided information on how SelB may discriminate tRNA^**^ from canonical tRNAs and may differentiate between the selenocysteyl moiety and the serylresidue of the precursor. A scenario for the major steps in the decoding process is postulated and arguments are given why the interaction of SelB with the mRNA is crucial. Reasons are also presented for the necessity of a balanced ratio of the components of the selenocysteine insertion apparatus and how it is regulated in E. coli via translational repression implicating a SECIS-like element located at the ultimate 5 'end ofselAB mRNA.
Selenium: Its molecular biology and role in human health
10
Introduction When bacteria are challenged with low molecular weight selenium compounds in the medium, they can process selenium in a nonspecific or a specific manner. The nonspecific metabolism rests on the chemical similarity between selenium and its neighbor element in the periodic table, sulfijr. When present above a critical concentration in Escherichia coli, i.e., at selenite concentrations higher than 1 ^M, selenium intrudes the sulfur pathways and is metabolized along the routes of sulfur metabolism [1,2] (Figure 1). Thus, selenium in the form of selenate is taken up by the sulfate transport system and reduced to selenide via the assimilatory sulfate reduction system. When offered as selenite, reduction appears to proceed chemically by interaction with thiol compounds like glutathione (see [3] for review).
Sulfate/Selenate
4
Sulfate/Selenate '
Selenite
V
Selenite . R-SH
•f
Sulfide/Selenide O-Ac-Ser
"V
Seryl-tRNAS^":
Cysteine/Se-Cysteine^ Pool mnm^s^U S/Se-Cystationine S/Se-Cys-tRNACv^
i I
ucu uco
S/Se-Methionine
S/Se-Met-tRNA'^^t
Se-Cysteyl-tRNASe
Scicnoprotcins — » I Selenylated Proteins AUG
Figure 1. Scheme for the specific and nonspecific metabolism and incorporation of selenium into macromolecules. The specific pathway is highlighted in bold. mnm's^U is the abbreviation for 5-methylamino-methyl-2-thiouridine and mnm'se^U for 5-methylaminomethyl-2-selenouridine. 0-Ac-Ser: 0-acetylserine, [Se] designates the reactive selenium species used by the selenophosphate synthetase as a substrate for the synthesis of selenophosphate; its possible metabolic origin is indicated by dashed arrows (see Chapter 4).
Selenium metabolism in prokaryotes
11
The first organic selenium compound formed is free selenocysteine, which can be converted to selenocystathionine and eventually to selenomethionine. On the other hand, selenocysteine has been shown to be a substrate for cysteyl-tRNA synthetase, which forms selenocysteyl-tRNA'^^^ and in this way incorporates selenocysteine at cysteine positions in proteins [4-6]. The decision whether selenium is incorporated nonspecifically as either selenocysteine or selenomethionine, therefore, should be dependent on the relative catalytic efficiencies of cysteyl-tRNA synthetase and cystathionine synthetase for the substrate cysteine and its analog selenocysteine. Nonspecific incorporation into macromolecules is drastically reduced when the cysteine biosynthetic pathway is interrupted by mutations or when it is fully repressed [6]. When selenomethionine is provided in the medium, it is almost indiscriminately incorporated into protein in place of methionine. This replacement is frequently used in x-ray analysis of protein crystals by multiwavelength anomalous dispersion [7] or in NMR spectroscopy [8]. Selenomethionine as the major selenium compound has also been detected when bacteria were grown on excessive amounts of selenite [9,10]. Free selenocysteine, on the other hand, is highly toxic and therefore growth inhibitory. Its incorporation in place of cysteine requires an overexpression system like the promoter-polymerase system of phage T7 to circumvent toxicity [11,12]. The specific incorporation of selenocysteine, on the other hand, is effective at much lower concentrations of selenite in the medium. With the aid of a fdhF-lacZ fusion reporter gene, in which readthrough into lacZ is dependent on the availability of selenium (see below), saturation has already occurred by 0.1 |iM selenite [13]. Specific incorporation does not involve free, low molecular weight selenocysteine since the biosynthesis of the molecule takes place from a precursor amino acid esterified with tRNA. It should be emphasized that the capacity to synthesize selenoproteins by the specific pathway is not ubiquitous. Actually, it is absent in the majority of microorganisms [14]. In this chapter we will discuss the specific incorporation of selenocysteine by bacteria, mainly E. coli and by members of archaea. Identification of the components involved in selenocysteine biosynthesis and specific insertion rests to a considerable degree on the early work of several groups studying the anaerobic formate metabolism oiE. coli [15-20]. Genes had been analyzed which, when mutated, abolished the ability of E. coli to synthesize active isoenzymes of formate dehydrogenase known as formate dehydrogenase N and formate dehydrogenase H which couple formate oxidation to the reduction of nifrate or protons, respectively. Thus, some mechanism must have been affected in the mutants that is required for generating activity of both enzymes. The genes had been mapped on the
12
Selenium: Its molecular biology and role in human health
chromosome oiE. coli and some of them (fdhAfdhB andfdhC) turned out to be involved in selenium metabolism [21]. Merits also go to two technical developments, namely the establishment of a plate overlay technique for screening large numbers of colonies for formate dehydrogenase activity [17] and the set-up of a procedure for specific incorporation of radioactive selenium into selenopolypeptides [22]. With the aid of these techniques, it was easy to differentiate between specific and nonspecific incorporation (see Figure 1). Specific incorporation of selenocysteine by bacteria The first genes discovered to contain an in-fi-ame UGA codon directing selenocysteine insertion were gpx, coding for glutathione peroxidase fi"om mouse [23], and fdhF fi-om E. coli, coding for the selenopolypeptide of formate dehydrogenase H [24]. Whereas an amino acid sequence was available for glutathione peroxidase showing colinearity between the UGA in the mRNA and selenocysteine in the protein, this was not the case for the bacterial enzyme. Evidence was obtained, however, by leading truncations from the 3'end into the gene and showing that removal of the segment containing the UGA also abolished selenium incorporation into the truncated gene product. Definite proof for the cotranslational insertion was then provided by fusion of the /acZ reporter gene upstream and downstream of the UGA in fdhF and the demonstration that readthrough of the UGA required the presence of selenium in the medium [13]. Analysis of mutations that affected readthrough led to the identification of the genetic elements involved in selenium metabolism in E. coli [21]. After the discovery that UGA also directs selenocysteine insertion into proteins in archaea [25], and with the results of the bioinformatic analysis of whole genome sequences from several hundred organisms, it has become an accepted notion that UGA is the universally conserved codon for selenocysteine [26]. tRNA^" The key element for specific selenocysteine insertion in E. coli was identified as the product of the fdhC gene, now designated as the selC gene [27]. It codes for a tRNA with unusual sequence and structural properties (Figure 2A). With 95 nucleotides, tRNA^'' is the largest tRNA in E. coli mainly because of an aminoacyl acceptor stem of eight possible base pairs and a 22 nucleotide long extra arm. There are also a number of deviations fi-om the consensus structure characteristic of canonical elongator tRNAs, namely a G at position 8, an A at position 14, a Y-R pair at the 10-25 sites and an R-Y base pair at positions 11-24. Moreover, the R-Y Levitt pair between the positions 15-48 is missing. As expected, extensive enzymatic and chemical
Selenium metabolism in prokaryotes
13
probing of the solution structure of tRNA^^*^ from E. colt, compared with that of canonical tRNA^^', showed that these deviations, plus the fact that the D stem is closed to a six base pair helix minimizing the D loop to four nucleotides, also restrict the types of tertiary interactions within the molecule [28]. Whereas the canonical G19-C56 interaction is still present, there are new interactions between CI6 of the D loop and C59 of the T loop and the canonical A21-(U8-A14) triple pair is substituted by a G8-(A21-U14) triple interaction. The extra arm is closed by a G45-A48 pair and connected to the anticodon coaxial helix only by interaction of A44 with U26. All these unusual sequence and structural properties are conserved in the sequences of other bacterial tRNA^^*^ species [29]. In view of the still open discussion on the structure of the eukaryal (see Chapter 3) and archaeal (Figure 2B) counterparts and of the lack of an x-ray structure, the conclusions can be concentrated on three characteristic features: (i) the acceptor-T stem stacked helix is extended to 13 base pairs made up of 8 plus 5 base pairs in bacteria and 9 plus 4 in archaea and eukarya, (ii) the closure of the D stem and the deviations from the sequence in canonical positions restrict the possibilities for tertiary interactions within the molecule, and (iii) the extra arm appears to be less well fixed to the body of the molecule than in classical elongator tRNAs. k
A C C i' G G—C 6—C A—U A—U
c
G
C^G
C G G^C G—C
c—e
m u C U GM C C
c u c uG c m • M i l 1 11 • G fiS «*"uV c !0a
ll|f G A GI-GPx >pGPx » P H G P x : 1) The k2 values, which reflect affinity to GSH are at least one order of magnitude higher for cGPx [12] than for pGPx [13,14] and PHGPx [15]; 2) pGPx is equally active with GSH, thioredoxin and glutaredoxin [16]; 3) PHGPx oxidises various dithiols [17], but nevertheless prefers GSH and acts on various protein thiols only when GSH becomes limiting [18-20] (see Chapter 28). 4) the P. falciparum GPx homolog clearly prefers thioredoxins over GSH [21]; and 5) the GPx homolog of Trypanosoma brucei uses tryparedoxin as reductant [22]. Differential response to selenium Glutathione peroxidases occupy extreme positions in the "hierarchy of selenoproteins'' , with cGPx ranking lowest [5]. The extracellular GPx behaves similar to cGPx [5]. PHGPx, is fairly resistant against selenium deficiency and is rapidly synthesized when selenium is replenished [5,23]. GI-GPx is most resistant to selenium deficiency [24]. The relative position of the selenoproteins within this hierarchy was believed to reflect their relative biological importance. The selenium-responsiveness of the four GPx types, however, conflicts with this assumption. GI-GPx and cGPx, reflecting the two extremes on the scale, have been knocked out without creating any overt pathological effects in unstressed mice [25-27].
Selenoproteins of the glutathione system
163
The molecular mechanisms leading to differential synthesis of selenoproteins under selenium restriction are far from being clear. As a rule, the ranking parallels the stability of the pertinent mRNA [24,28-30]. The instability of cGPx mRNA has been attributed to "cytoplasmic nonsensemediated decay'' , a phenomenon describing the elimination of mRNA species having a stop codon located at a certain distance upstream from a premRNA splice site [31]. However, the mRNAs of PHGPx and GI-GPx have their UGA in homologous positions and remain stable or are even increased in selenium deficiency. Differences in translational efficiencies do not account for the different stabilities either and maximum SECTS efficiencies do not correlate with the selenium-responsiveness of SECTS efficiencies [58]. Likely, the mRNAs are specifically stabilized by a factor binding to its SECTS in a selenium-dependent manner. The SECTS-binding protein SBP2 itself can hardly be responsible for the differential mRNA stabilities, since it is not known to directly interact with any cellular selenium compound. Also, the dissociation constants for the reaction of SBP2 with different GPx 3'UTRs, as measured by mobility shift assays [32] or by surface plasmon resonance (own unpublished data), are similar. Likely, the stabilizing factor is the eukaryotic SelB homolog EFsec [33,34] that may modulate the SECTS affinity of SBP2 in response to binding selenocysteyl-loaded tliNA^^"'^*'^ in the ribosomal super complex [33-35]. According to this hypothesis, selenocysteine-loaded tRNA[^''^^*'^ would reflect the cellular selenium status and EFsec is the selenium sensor (see Chapters 6 and 7). Se-independent regulation of glutathione peroxidases cGPx: The cGPx gene has been reported to contain an oxygen-responsive element in the 5'-flanking region, and the targeting binding protein 'OREBP' responding to oxygen tension has been characterized. Further, human cardiomyocytes [37] and endothelial cells [38] respond to hyperoxia with an up-regulation of cGPx. However, convincing data on a transcriptional response of gpx-1 to oxidative stress in general does not exist [39]. Typically, the capacity of the cGPx/GSH system is enhanced by induction of yglutamyl-cysteine synthetase upon exposure to peroxides or redox cyclers via the NrG/Keapl system [40,41]. Most consistently, cGPx is up-regulated by estrogens [42-45]. A typical estrogen-responsive element is, however, not detectable in gpx-1. The estrogen response of gpx-J is thought to result from estradiol-mediated activation of N F K B that targets a putative binding site of gpx-1 [43,45]. Tn neutrophils cGPx was shown to depend on the transcription factor PU.l. Binding of PU.l to putative PU.l sites in the 5' promoter and the 3' flanking region of gpx-1 was demonstrated by gel shift assays and transactivation [46]. The gpx-1 promoter was further reported to be activated by p53 in human osteogenic cell lines [47]. Tn RXR-negative mice cGPx expression was
164
Selenium: Its molecular biology and role in human health
reduced to 30%, as was that of y-glutamyl-cysteine synthetase, suggesting a regulation of cGPx via the retinoid X receptor [48]. At the translational level, cGPx biosynthesis is down-regulated by homocysteine, which may interfere with the selenium-dependent readthrough at the UGA codon [49]. Homocysteine-induced endothelial dysfunction can be ameliorated by overexpression of cGPx [50] and basal endothelial dysfunction is more severe in cGPx knockout mice [51]. pGPx: pGPx was found to be increased in plasma of patients with bowel disease and of mice with experimental colitis [52] as well as in the epithelial lining fluid in the lungs of asthmatic patients [53]. The increased levels of pGPx were considered to result from transcriptional up-regulation due to oxidative stress. A functional consensus sequence for the redox-regulated transcription factor AP-1 is located in the 5' promoter region of gpx-3 [53]. More recently, an alternative transcription start in gpx-3 was identified [54]. The pertinent promoter contains putative binding sites for SP-1 and the hypoxia-inducible factor-1 (HIF-1), a redox-sensitive metal response element and an antioxidant response element. Experimentally, hypoxia was demonstrated to strongly up-regulate gpx-3 expression in Caki-2 cells [54]. GI-GPx: Several caudal homeobox protein binding sequences and two retinoic acid responsive elements were identified in gpx-2 [55], and GI-GPx could be induced by retinoic acid in some (MCF-7), but not all (HT29) cells. Further, GI-GPx was down-regulated in hepatoma cells infected with hepatitis C virus subgenomic RNA. Inversely, induction of GI-GPx by retinoic acid suppressed the HCV replicon [56] suggesting a therapeutically interesting inverse relationship of GI-GPx levels and viral replication. In several microarray studies, gpx-2 transcripts were found to be elevated together with phase II enzyme transcripts upon exposure to the Nrf2 activator sulforaphane or to hyperbaric oxygen [57,58], In gpx-2 a ATG-proximal conserved ARE proved to be indispensable to endogenous and sulforaphaneinduced gpx-2 expression at the transcript and protein level. The relevance of the pertinent NrfZ/Keapl system was corroborated by enhancement of a gpA;2 promoter-driven reporter gene expression, by transfection with Nr£2 and suppression thereof by transfection with Keapl [59]. PHGPx: Related studies of regulatory phenomena do not yet yield a comprehensive picture that could explain the extraordinary tissue distribution, the differentiation-specific expression of PHGPx or the relative abundance of its isoforms. Functionality of putative hormone-responsive elements in gpx-4 could not be verified in somatic cell lines. Instead, the known dependence of testicular PHGPx on gonadotropic hormones [60] could be explained by abundant expression of PHGPx in spermatogenic cells that proliferate under stimulation by testosterone [61]. A selective upregulation of PHGPx in the bovine oviduct by 17p estradiol was, however, verified, although without a mechanistic explanation [62]. In rat casein-
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elicited neutrophils, PHGPx was found up-regulated. The results were interpreted as induced self-protection against the oxidant products of these cells. The effect could be mimicked by recombinant growth-regulated oncogene (GRO) and abrogated by anti-GRO antibodies [63]. Most recent studies focus on the expression of the nuclear isoform of PHGPx. Maiorino et al [64] and Moreno et al [65] demonstrated a basal promoter activity of the first intron oigpx-4 resulting in the nucleus-specific transcript, while Borchert et al [66] and Ufer et al [67] found no or marginal activity of this region and postulated that also the expression of nuclear PHGPx is essentially triggered by the promoter region upstream of the ATG codons that represent the translation starts of the cytosolic and the mitochondrial forms. Here, binding sites for SPl, nuclear factor Y (NF-Y) and members of the Smad family were identified [67], while expressiondepressing sites for EGRl and SREBPl were detected in the first intron [66]. Tramer et al [68] confirmed the promoter activity of the first intron and there identified the cAMP-response element modulator x (CREM-x) as an essential activator. According to these authors, the expression of nuclear PHGPx in spermatogenic cells is explained by high levels of CREM-t in pachytene spermatocytes and spermatids [68]. None of the quoted studies, however, explains the burst of normal PHGPx expression in spermatids that is evidently more important to sperm function than the faint expression of nuclear PHGPx (see Chapter 28). Functional diversification of glutatliione peroxidases Lessons from knock out and overexpression of gpx-1 The cGPx knock-out mouse reveals that cGPx is little else than an emergency device to cope with hydroperoxide challenge. Unchallenged cGFx^''^ mice developed normally, even grew faster and tolerated elevated oxygen tension [25]. The lack of any overt phenotype is not surprising in light of the clinical phenotype of patients with deficiencies in GSH regeneration. Such patients are normal as long as they are unchallenged with hydroperoxides and the related genetic defects were accordingly rated as "non-diseases'' [70]. The complete lack of cGPx in mice is tolerated like the more or less pronounced impairment of GSH regeneration in patients. Like these, the cGPx^"'"' mice are however highly susceptible to oxidative damage, as has been shown by exposure to redox-cycling herbicides [26,71,72] and to lipopolysaccharides that trigger oxidative burst in phagocytes [73]. The cGPx^"'"^ mouse also demonstrates that the seemingly normal life without cGPx is threatened by certain environmental hazards. Most importantly, the cGPx*''^ mouse proved to be a model for the classical human selenium-deficiency syndrome Keshan disease. When these mice were exposed to a non-virulent Coxsackie strain, the virus rapidly mutated into a
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virulent form [74], as had been observed before with selenium-deficient mice [75]. Based on these studies, the debate whether Keshan disease is caused by selenium-deficiency itself or a viral infection [76] can likely be settled. A decrease in cGPx, the enzyme responding fastest to selenium deprivation, results in elevated steady-state-levels of hydroperoxides which in turn accelerate the mutation rate of usually benign virus strains and allow virulent mutants to become dominant. The experiments with oxidatively challenged cGPx^"'"' mice were performed in selenium-deprived and selenium-adequate animals and, as a rule, the selenium-status did not significantly alter the results. This surprising outcome allows two alternative interpretations: Either the selenium deficiency was not sufficient to decrease other selenoproteins to any relevant degree or, more likely, none of the remaining selenoproteins can efficiently substitute for cGPx in balancing a systemic oxidative challenge. The latter conclusion may be viewed as provocative in light of the occurrence of four more selenoperoxidases and of the metabolic link of thioredoxin reductases to peroxiredoxin-type peroxidases. Optimization of cGPx activity, e.g., by selenium supplementation or GPx mimics, is widely considered to be beneficial. Some observations, however, cast doubt on this view: 1) in HIV-infected tissue culture, cGPx overexpression enhanced viral spread [77]; 2) knockout of cGPx enhanced resistance against kainic acid-induced epileptic seizures [78]; 3) overexpression of cGPx reduces TNFa-induced NPKB activation in tissue cultures [79], as does selenium-supplementation [80,81]; 4) overexpression of cGPx promoted acetaminophen toxicity [82]; 5) hepatocytes of cGPx^'"^ mice, while being highly sensitive to oxidative stress, were largely protected against peroxynitrite challenge [83]; 6) strikingly, an increase of skin cancer tumor incidence in GPx overexpressing transgenic mice was induced by DMBA/TPA treatment, an experimental design commonly believed to involve ROS-induced/promoted carcinogenesis [84]; and 7) cGPx*'"^ mice consistently developed hyperglycemia and insulin resistance, as is typical of type II diabetes that is commonly believed to be facilitated by oxidative stress [85]. Evidently evolution has created a ratio of peroxide generating and detoxifying systems that meets the demands of most, though not all, endogenous and environmental conditions, and it would be unwise to disturb this delicate balance without proven need. pGPx and the extracellular peroxide tone pGPx has only a limited opportunity to counter a serious challenge by hydroperoxides, since the tiny concentrations of extracellular GSH or thioredoxin would quickly be consumed in absence of any regenerating system. The biological role of pGPx therefore still remains speculative. pGPx has been implicated in the reduction of lipid hydroperoxides LDL [3] and
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thus might be relevant to Steinberg's ideas on atherogenesis [86]. In view of the low steady-state level of peroxidized lipoproteins, the limited reduction capacity of pGPx may just suffice. The substrate specificity of pGPx, however, is not ideal for the reduction of peroxidized LDL, since it does not reduce peroxidized cholesterol esters [11]. Alternatively, the enzyme could reduce soluble lipid hydroperoxides, which have been implicated in the activation of cyclooxygenase, the key enzyme of prostaglandin synthesis [87]. Similarly, the activation of other lipoxygenases, which typically remain dormant in the absence of any hydroperoxides, could be prevented by pGPx, as occurs in vitro by cGPx [88] and PHGPx [89-92]. Similarly, the extracellular pGPx/GSH system could be regarded as a redox buffer that is required to discriminate between irrelevant and serious inflammatory stimuli [5]. Specifically, a fast acting peroxidase in a compartment with low reducing capacity could make the best use of the oxidative host defense machinery, which is indispensable for survival in a hostile environment, but selfdestructive when over-reacting [5]. GI-GPx as modulator of inflammatory responses of the intestine GI-GPx, because of its preferred localization in the intestine, has been proposed to prevent systemic access of food-bom peroxides, which could pre-exist in food or are generated by the intestinal flora or by the mucosa when metabolising xenobiotics [93]. Experimental evidence in support of this concept is scarce. GI-GPx^"'"' mice appear to have a normal phenotype [27], but mice deficient in both, cGPx and GI-GPx, showed retarded growth after weaning, suffered from ileocolitis starting at day 11 of age [94], and develop intestinal cancer [95]. Interestingly, the development of both, ileocolitis and tumors depended on, or was essentially aggravated by, colonization of the intestine by bacteria. A single allele of gpx-2 in the double transgenic mice was sufficient to ameliorate the pathologies, while a single allele of gpx-1 was ineffective [95-97]. The observations point to a pivotal role of GI-GPx in counteracting inflammatory responses elicited by the intestinal microflora. The intimate relationship of GI-GPx and the gastrointestinal flora is further evidenced by an induction of GI-GPx upon colonization of the intestine [96]. Pathological changes that occur in the double knockout suggest a mutual complementation of GI-GPx and cGPx. The enzymes could, e.g., cooperate in redox-regulated processes such as proliferation or apoptosis. Inhibition of apoptosis has been documented for the overexpression of cGPx [77] and PHGPx [98-100] and may be common to all selenoperoxidases. The intestinal epithelium displays a steep gradient of GI-GPx that declines from the proliferating stem cells in the lower crypts to the luminally exposed cells gradually undergoing apoptosis [101,102]. If this delicate balance of events is indeed regulated by GI-GPx, the absence of cGPx could unmask a " dysregulation'' that is still compensated in the isolated GI-GPx knock-
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Selenium: Its molecular biology and role in human health
out. The intriguingly high concentration of GI-GPx in Paneth cells, which are involved in mucosal immunity and not in absorption, and its association in colon cells with vesicular structures [102] also suggest highly specialized functions that still remain to be elucidated. PHGPx in lipid peroxidation, inflammation, and differentiation PHGPx was discovered and characterized as an enzyme preventing progression of lipid peroxidation in biomembranes due to its unique ability to reduce hydroperoxo-groups in complex lipids [103]. In this context, it is synergistically supported by -tocopherol, which reduces lipid peroxy radicals to lipid hydroperoxides. The latter, if not reduced by PHGPx, would reinitiate free radical-mediated lipid peroxidation by Haber-Weiss- or Fentontype chemistry. Over the years, however, growing evidence indicated that protection of biomembranes against unspecific lipid peroxidation is possibly not the most important role of PHGPx. A knockout mouse model, which could shed light on the multiple roles of PHGPx, has not yet been obtained. Homozygous PHGPx^''^ mice died between day 7.5 and 8.5p.c.[104,105]. Specific functions of the enzyme are however evident from overexpression and selenium supplementation studies. With other glutathione peroxidases, PHGPx shares the ability to silence lipoxygenases [106,107], to inhibit apoptosis [98-100], and to suppress cytokine-induced N F K B activation [108]. In several cases, however, PHGPx was demonstrated to be the biologically most relevant regulator: 1) Seleniumdeficient rat basophilic leukemia cells as well as whole animals overproduce 5-lipoxygenase products comprising the potent pro-inflammatory leukotrienes [106]. Similarly, leukotriene biosynthesis is suppressed in transformed rat basophilic leukemia cells selectively overexpressing PHGPx [109]. PHGPx thus appears to be the principal selenoperoxidase in charge of silencing 5-lipoxygenase. 2) A moderate overexpression of PHGPx in the human ECV cell line completely abrogated interleukin-1-induced N F K B activation, while a huge variation of cGPx activity achieved by deprivation and re-supplementation of selenium did not [108]. N F K B activation induced by hydroperoxides is similarly suppressed by overexpression of PHGPx in rabbit aortic smooth muscle cells [99] and UV-stressed human skin fibroblasts [110]. 3) The pivotal role of PHGPx in dampening inflammation is further underscored by its ability to suppress COX-2 expression/activity, to prevent COX2 product-dependent malignant growth [111] and to inhibit VCAM-1 expression by inducing heme oxygenase-1 [112]. The dampening of inflammatory responses thus is common to all the GPxs, but is in part achieved by different, although complementary mechanisms. A potential cross-talk between PHGPx and GI-GPx in the management of exogenous stressors is outlined in Scheme 1. How the peculiar specificity of PHGPx in regulating inflammatory processes is achieved is unknown. It may
Selenoproteins of the glutathione system
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be due to its preference for hydrophobic lipids or to subcellular micro compartmentation. The observation that
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Scheme 1. Role of GI-GPx and PHGPx in inflammatory (right) and adaptive (left) responses. The N F K B system, which is activated by pro-inflammatory cytokines and bacterial toxins via Toll-like receptors TLR), is activated by reactive oxygen species, in particular by hydroperoxides, and dampened by selenium-containing peroxidases, in particular PHGPx. The Nr£2/Keapl system is activated by dietary electrophiles and moderate oxidative challenge, promotes ARE-mediated expression of phase 2 enzymes and GI-GPx, and is supposed to enforce resistance against inflammation and cancer.
PHGPx is sometimes found oxidatively cross-linked to itself or other proteins [18,19] suggests another possibility: PHGPx, by a reaction of its oxidized selenium with accessible protein thiols, could also act as a peroxidedependent thiol modifying agent. By an analogous reaction, PHGPx polymerizes in absence of GSH and can thereby be transformed into a
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Selenium: Its molecular biology and role in human health
structural protein. This process has been shown to be pivotal to the differentiation of mammalian spermatids into mature spermatozoa as is described in detail in Chapter 28. Acknowledgements The preparation of this article was supported by the Deutsche Forschungsgemeinschaft DFG (grants Fl 61/12-3 and Bri 778/5-3). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
L Floh6, JR Andreesen, R Brigelius-Flohe et al 2000 lUBMB Life 49:411 RF Burk, RA Lawrence 1978 Functions of glutathione in liver and kidney H Sies, A Wendel (Eds) Springer Verlag, Berlin, p 114 Y Saito, T Hayashi, A Tanaka et al 1999 J Biol Chem 274:2866 JW Chen, C Dodia, SI Feinstein et al 2000 J Biol Chem 275:28421 L Flohe, R Brigelius-Floh^ 2001 Selenium Its molecular biology and role in human health 1st "dition DL Hatfield (Ed) Kluwer Academic Publishers, Boston, Dordrecht, London, p 157 O Epp, R Ladenstein, A Wendel 1983 EurJBiochem 133:51 B Ren, W Huang, B Akesson et al 1997 JMol Biol 268:869 L Floh6, KD Aumann, R Brigelius-Floh6 et al 1993 Active Oxygen, Lipid Peroxides, and Antioxidants K Yagi (Ed) CRC press, Boca Raton, p 299 A Grossmann, A Wendel 1983 EurJBiochem 135:549 A Sevanian, SF Muakkassah-Kelly, S Montestruque 1983 Arch Biochem Biophys 223:441 Y Yamamoto, K Takahashi 1993 Arch Biochem Biophys 305:541 L Floh6, G Loschen, WA Gunzler et al 1972 Hoppe Seylers ZPhysiol Chem 353:987 RS Esworthy, FF Chu, P Geiger et al 1993 Arch Biochem Biophys 307:29 G Takebe, J Yarimizu, Y Saito et al 2002 J Biol Chem 277:41254 F Ursini, M Maiorino, C Gregolin 1985 Biochim Biophys Acta 839:62 M Bjoemstedt, J Xue, W Huang et al 1994 J Biol Chem 269:29382 A Roveri, M Maiorino, C Nisii et al 1994 Biochim Biophys ^cto 1208:211 C Godeas, F Tramer, F Micali et al 1996 Biochem Mol Med 59:118 F Ursini, S Heim, M Kiess et al 1999 Science 285:1393 M Maiorino, L Flohe, A Roveri et al 1999 BioFactors 10:251 H Sztajer, B Gamain, K-D Aumann et al 2001 J Biol Chem 276:7397 T Schlecker, A Schmidt, N Dirdjaja et al 2005 J Biol Chem 280:14385 F Weitzel, F Ursini, A Wendel 1990 Biochim Biophys Acta 1036:88 K Wingler, M Bocher, L Flohe et al 1999 EurJBiochem 259:149 YS Ho, JL Magnenat, RT Bronson et al 1997 J Biol Chem 272:16644 JB de Haan, C Bladier, P Griffiths et al \99% J Biol Chem 273:22528 RS Esworthy, JR Mann, M Sam et al 2000 Am J Physiol Gastrointest Liver Physiol 279:G426 RA Sunde, JA Dyer, TV Moran et al 1993 Biochem Biophys Res Commun 193:905 XG Lei, JK Evenson, KM Thompson et al 1995 JNutr 125:1438 G Bermano, JR Arthur, JE Hesketh 1996 FEBSLett 387:157 X Sun, PM Moriarty, LE Maquat 2000 EMBO J19:4734 JE Fletcher, PR Copeland, DM Driscoll et al 2001 RNA 7:1442 RM Tujebajeva, PR Copeland, XM Xu et al 2000 EMBO Rep I ASS D Fagegaltier, N Hubert, K Yamada et al 2000 EMBO J19:4796 AM Zavacki, JB Mansell, M Chung et al 2003 Mol Cell 11:773
Selenoproteins of the glutathione system 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87.
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Selenium: Its molecular biology and role in human health M Haurand, L Flohe 1988 Biol Chem Hoppe Seyler 369:133 WC Chang 2003 JBiomed Sci 10:599 CJ Chen, HS Huang, WC Chang 2003 FASEBJMA 694 CJ Chen, HS Huang, SB Lin et al 2000 Prostaglandins Leukot Essent Fatty Acids 62:261 H KOhn, A Borchert 2002 Free Radic Biol Med 33:154 DA Parks, GB Bulkley, DN Granger 1983 Surgery 94:428 RS Esworthy, R Aranda, MG Martin et al 2001 Am J Physiol Gastrointest Liver Physiol 281:0848 FF Chu, RS Esworthy, PG Chu et al 2004 Cancer Res 64:962 RS Esworthy, SW Binder, JH Doroshow et al 2003 Biol Chem 384:597 FF Chu, RS Esworthy, JH Doroshow 2004 Free Radic Biol Med 36:1481 K Nomura, H Imai, T Koumura et al 1999 J Biol Chem 274:29294 R Brigelius-Floh6, S Maurer, K L6tzer et al 2000 Atherosclerosis 152:307 H Imai, T Koumura, R Nakajima et al 2003 Biochem J 371:799 FF Chu, RS Esworthy 1995 Arch Biochem Biophys 323:288 S Florian, K Wingler, K Schmehl et al 2001 Free RadRes 35:655 F Ursini, M Maiorino, M Valente et al 1982 Biochim Biophys Acta 710:197 H Imai, F Hirao, T Sakamoto et al 2003 Biochem Biophys Res Commun 305:278 LJ Yant, Q Ran, L Rao et al 2003 Free Radic Biol Med 34:496 F Weitzel, A Wendel 1993 J Biol Chem 268:6288 K Schnurr, J Belkner, F Ursini et al 1996 J Biol Chem 271:4653 R Brigelius-Flohe, B Friedrichs, S Maurer et al 1997 Biochem J 328:199 H Imai, K Narashima, M Aral et al 1998 J Biol Chem 273:1990 J Wenk, J SchUller, C Hinrichs et al 2004 J Biol Chem 279:45634 I Heirman, D Ginneberge, R Brigelius-Flohe et al 2005 Free Radic Biol Med, in press: A Banning, R Brigelius-Floh6 2005 Antioxid Redox Signal 7:889
Chapter 16. New roles of glutathione peroxidase-1 in oxidative stress and diabetes Xin Gen Lei Department of Animal Science, Cornell University, Ithaca, NY 14853, USA
Wen-Hsing Cheng Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
Summary: Glutathione peroxidase-1 (GPXl) was identified as an antioxidant enzyme in 1957 and as a selenoprotein in 1972. In the last ten years, ample data have been generated from several lines of GPXl knockout mice, showing an essential role of GPXl in defending against severe oxidative stress mediated by pro-oxidants. This protection is associated with attenuated oxidation of NADPH, NADH, lipid, and protein. When Sedeficient mice are under mild oxidative stress, minute amounts of GPXl activity in tissues are able to protect them against the pro-oxidant-induced lethality and hepatic aponecrosis. Strikingly, GPXl prevents apoptosis of hepatocytes caused by reactive oxygen species, but potentiates cell death caused by reactive nitrogen species. Mice overexpressing GPXl developed insulin resistance and obesity. These new data illustrate mixed roles of GPXl in coping with different types of oxidative stress, and suggest a possible deleterious impact of GPXl up-regulation on glucose metabolism. Introduction Selenium (Se) is of fundamental importance to human health, and performs its metabolic functions presumably in the form of selenoproteins. It was not until 1957 that Se was considered essential in the diet for rats [1]. Coincidentally, this was the same year that cellular glutathione peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9, GPXl) was found to protect erythrocytes against oxidative hemolysis, as erythrocytes from Se-deficient rats were more prone to hemolysis upon hydrogen peroxide exposure than those from Se-adequate rats [2]. In the 1970s, Se was shown to be cofractioned with GPXl activity in Se-deficient rats after Se^^ administration [3,4]. Consequently, GPXl became the first identified selenoprotein and was
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Selenium: Its molecular biology and role in human health
considered the biochemical mediator of Se in protecting erythrocytes against oxidative hemolysis. GPXl is expressed in virtually all tissues and represents the majority of body Se [5]. As a homotetramer of 88-kDa, the enzyme shares 30-60% sequence homology with other Se-dependent glutathione peroxidases. The human gpxl gene was mapped on chromosome 3ql l-3ql3.1[6]. The protein sequence of human GPXl is 86 and 83% homologous to its counterparts in mice and rats, respectively. Cloning of the mouse Gpxl gene in 1986 led to the identification of employing the stop codon UGA for translation of selenocysteine, the 21*' amino acid [7]. The process requires a stem-loop structure called a selenocysteine insertion sequence element in the 3' untranslated region of mRNA [8,9]. Nutritionally, Se deficiency in rats results in a 90% loss of liver GPXl mRNA and even greater loss of GPXl activity [10]. The sensitivity of GPXl activity to body Se fluctuation renders it a convenient and responsive biochemical marker to assess body Se status and dietary Se requirements. However, manipulating tissue GPXl activity by altering dietary Se intake in conventional animal models does not allow studies for specific or exclusive roles of GPXl, due to possible confounding effects of multiple selenoproteins. Several GPX mimics and inhibitors are available, but are not highly specific or completely satisfactory [11,12]. Thus, it was necessary to develop GPXl knockout [GPXl(-/-)] and overexpression [GPX1(+)] mice for an accurate assessment of the physiological functions of GPXl. Up to now, three lines of GPX1(-/-) mice [13-15], and at least two lines of GPX1(+) mice [16,17] have been generated. Effect of GPXl null or overexpression on selenoprotein expression Two features of GPXl argued against an essential role for the enzyme in vivo. First, GPXl activity was more susceptible to dietary Se deprivation than other selenoproteins [18,19]. Second, there was no apparent adverse effect on animal health when GPXl activity in tissues dropped to < 1% of controls. In addition, GPXl is the predominant biochemical form of Se in many tissues [5]. Thus, GPXl was proposed as a storage or buffer of body Se that could mobilize its own Se for the synthesis of other selenoproteins in Se deficiency, but takes up cellularfireeSe in Se adequacy [20,21]. To test this hypothesis, we determined expression of several selenoproteins and concentrations of total Se in tissues of GPX1(-/-) and GPX1(+) mice fed Se-deficient, -adequate, or -excessive diets for 5-13 weeks [17,22,23]. We observed that knockout of GPXl reduced liver Se concentration by 60% in Se-adequate mice [22,23]. However, knockout or overexpression of GPXl in mice did not alter the mRNA and (or) activity expression of GPX3, GPX4, thioredoxin reductase, and selenoprotein P in various tissues, irrespective of their dietary Se concentrations. If GPXl were truly a buffer of body Se, there should have been more Se available for a higher activity expression of GPX3,
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GPX4, or thioredoxin reductase in the GPX1(-/-) than in the wild-type mice fed Se-deficient diets. Likewise, decreases in activity expression of these selenoenzymes in dietary Se deficiency could have been, at least, partially corrected by GPXl overexpression compared with wild-type controls. Clearly, our data do not support the buffer role of GPXl in Se partitioning for selenoprotein synthesis, but suggest that expression of GPXl is independent of other selenoproteins. Recent searching for selenoprotein genes among the human genome by Gladyshev and colleagues indicates that there are a total of 25 selenoproteins in humans and 24 in rodents [24]. It remains to be seen whether knockout or overexpression of GPXl affects the expression of the newly identified selenoproteins. Overall metabolic impact of GPXl null or overexpression When GPX1(-/-) mice were fed a Se-adequate diet in our animal facility, they were healthy and fertile, showing normal body weight gain, food intake, and no abnormal histology in various organs [13,23]. On the contrary, we observed insulin resistance and obesity in male, GPX1(+) mice 24 weeks of age, compared to their age-matched wild-type controls [25]. Although the GPX1(-/-) mice developed in another study showed a 20% reduction of body weight by 8 months of age compared to controls [15], the GPX1(-/-) mice from a third study did not display a body weight change [14]. Another reported phenotype of GPX1(-/-) mice is the age-related onset of cataracts. Reddy et al. [26] observed a higher incidence of cataracts in GPX1(-/-) mice (90%) than in wild-type mice (20%) of 15 months at age. However, Spector et al. [27] failed to detect cataracts in old GPX1(-/-) mice. Early research on GPXl function Overexpression of GPXl in cultured human cells enhanced their resistance to pro-oxidant-induced oxidative stress [28-30]. Approximately an 80% reduction in GPXl by antisense RNA in cells increased their sensitivity to selective genotoxic oxidants [31]. Apparently, these in vitro results show the biological potential of GPXl as an antioxidant enzyme, but could not be extrapolated as to its physiological function. The in vivo antioxidant roles of GPXl need to be verified using whole animal models. Paraquat and diquat have been used to study antioxidant roles of Se in several species [11,32,33]. Following acute exposures of animals to these compounds, the major target organ for diquat and paraquat is liver and lung, respectively [34]. Both pro-oxidants initiate reactive oxygen species (ROS) generation via the conversion of molecular oxygen into superoxide radicals [34,35]. The toxic superoxide radicals are converted into hydrogen peroxide by superoxide dismutase. Glutathione peroxidase and catalase then reduce hydrogen peroxide into water. Excessive hydrogen peroxide may react with iron to produce highly reactive hydroxyl fi-ee radicals that destroy large
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biomolecules, leading to cellular dysfunction, multiple organ failures, and death in animals. Thus, the paraquat or diquat-mediated hydrogen peroxide production offers an excellent test of the in vivo antioxidant role of GPXl. It was clear that prior intraperitoneal (ip) injections of Se into Se-deficient rats prevented diquat or paraquat-induced lipid peroxidation and liver necrosis [32]. However, the metabolic mediator conferring the Se protection remained unclear. Because of differences in their repletion profiles after the Se administration, selenoprotein P, but not GPXl, was considered responsible for the protection by the injected Se [36]. In addition, selenoprotein P was found in endothelial cells of liver in rats [37] and was shown to exhibit peroxidase activity [38]. Although these data suggested a possible involvement of selenoprotein P in the Se protection against diquat or paraquat toxicity, the inherent limitations of the animal model could not allow a conclusion on the role of selenoprotein P or an exclusion of GPXl from the protection. In contrast, the recently-developed GPXl [13-15] or selenoprotein P knockout mice [39,40] provide us with unprecedented models for direct answers to these questions. Role of GPXl in severe oxidative stress We used GPX1(-/-) and GPX1(+) mice to determine the importance and contribution of GPXl, in relation to other selenoproteins, to the body defense against severe or lethal oxidative stress [14,41-44]. After an ip injection of paraquat at a dose of 50 mg/kg body weight, all GPX1(-/-) mice, regardless of their body Se status, died within 4-6 hours [41]. While the Se-deficient wild-type mice also died within 4-6 hours after paraquat injection, the Seadequate wild-type mice survived 3 days [41]. Apparently, GPXl was the mediator of body Se for the protection against the paraquat-induced lethality, and the survival time of mice was a function of tissue GPXl activity. Consistently, de Hann et al. [14] showed hypersensitivity of GPX1(-/-) mice to paraquat injection (ip) at a dose of 30 mg/kg body weight. In another experiment conducted by us, Se-adequate wild-tjqje mice survived an ip injection of diquat at a dose of 24 mg/kg body weight, whereas Se-adequate GPX1(-/-) and Se-deficient wild-type mice died within 3-4 hours [42]. When Se-adequate GPX1(+) mice were given an ip injection of paraquat at 125 mg/kg body weight, their mean survival time was 10-fold longer (59 vs. 5.8 h) than that of wild-type mice. Altogether, the resistance of mice to prooxidant-induced lethality was decreased by GPXl knockout, but enhanced by GPXl overexpression. Undoubtedly, GPXl is antioxidative in vivo and is important for the body to defend against severe, acute oxidative stress. Because GPX1(-/-) mice died of high doses of paraquat or diquat acutely, without showing tissue lesions seen in Se-adequate wild-type mice that survived much longer [41-43], we conducted time course experiments to determine redox status (ratios of NADPH/NADP and NADH/NAD [45]),
New roles of glutathione peroxidase-1
111
lipid peroxidation (F2-isoprostanes [46]), and protein oxidation (protein carbonyl [47]). Decreases in hepatic NADPH/NADP and NADH/NAD ratios occurred in all groups of mice at 1 hour post paraquat injection, and the drop was much sharper in GPXl-deficient mice than the GPXl-adequate mice [48]. Tissue F2-isoprostanes and protein carbonyl contents were also sharply increased after the paraquat injection in these GPXl-deficient mice. An increased liver F2-isoprostanes in GPXl-deficient mice injected with lethal doses of diquat preceded the plasma ALT activity rise, an indicator of liver injury [42]. Obviously, the GPXl protection against mouse lethality induced by high levels of paraquat or diquat was associated with the attenuated protein oxidation, lipid peroxidation, and redox shift. Likely, GPXl is important to impede the redox shift toward oxidation, driven by overproduction of ROS under severe oxidative stress, so that the NADPHdependent metabolic systems are not largely disturbed. Role of GPXl in mild oxidative stress Deprivation of Se potentiates paraquat toxicity in several species [11,12,32]. In rats, Se deficiency shifts the LD50 of paraquat from 30 to 10 mg/kg body weight and the target organ from lung to liver [33]. To determine the role of GPXl in coping with mild oxidative stress in Se deficiency vs. Se adequacy, we injected Se-deficient and Se-adequate mice with 12.5 mg of paraquat/kg body weight (Figure 1, [49]). Lrespective of the genotypes, all Se-adequate mice survived fi^om this insult and showed no rise in plasma ALT activity, whereas 90% of the Se-deficient mice died within 10 hours after the injection with approximately 1,000-fold increase in plasma ALT activity over the baseline. It was striking that an ip injection of Se (50 (Ag/kg body weight, as Na2Se03), at 6 hours before the paraquat injection, had little effect on the responses of Se-deficient GPX1(-/-) mice to the pro-oxidant insult, but reduced the mortality rate from 90 to 50% (P < 0.05) and plasma ALT activity from 24,000 to 8,300 U/L. Hepatic aponecrosis, the combined apoptosis and necrosis, was attenuated by the Se injection in the Se-deficient WT mice, but not in the Se-deficient GPX1(-/-) mice. All these key differences between the two genotypes were associated with only a 4% increase in tissue GPXl activity in the Se-deficient wild-type mice by the Se injection [49]. A time-course study indicated that this minute amount of GPXl activity repletion in the Se-deficient wild-type mice delayed the appearance and decreased the severity of the paraquat-mediated hepatic aponecrosis, compared with that in the Se-deficient GPX1(-/-) mice. Consistently, the former mice had lower levels of hepatic phosphor-c-Jun Nterminal kinase (phospho-JNK), p53, and phosphor-p53 than the latter. In contrast, the paraquat-mediated gene and/or protein expression of proapoptotic Bax, Bcl-w, and Bcl-Xs, cell survival/death factors GADD45, MDM2, c-Myc, and caspase-3 were up- regulated, but that of antiapoptotic
Selenium: Its molecular biology and role in human health
178
Se (50 i-ig/ka)
T
.ft.
*
\
ime (li)
Se-deficieiit
-6 61i Paraquat (12.5 mg/kg) 0 G;-;;
WT
4l
6 10
Assays Biochemical analyses Iimuiiiioliistopatliclogy Western analyses of p53- JKK. and p38 Iiuiiiuiiiocomplex kinase assay Microarray of apoprosis gene expression Figure 1. Model for assessing GPXl function in moderate oxidative stress. The small boxes represent injections of Se or paraquat. GPXl"'", GPXl knockout; WT, wild-type.
Bcl-2 was down-regulated in the GPX1(-/-) mice compared with wild-type mice. Interestingly, we conducted kinase assays using anti-JNK immunoprecipitates, and found that phosphor-JNK catalyzed phosphorylation of endogenous or purified p53 on Ser-15, and the event was promoted by the minute amount of GPXl activity repletion in the Sedeficient wild-type mice [50]. A previous study has shovwi that JNKl
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depletion by antisense technology abolished Ap-induced p53 phosphorylation on Ser-15 [51]. Our findings suggest a modulating role of GPXl on the JNK-dependent p53 phosphorylation on Ser-15 under mild oxidative stress. Functional interactions between GPXl and vitamin E The nutritional essentiality of Se was initially recognized by its vitamin Esparing role in the prevention of liver injuries, and symptoms of Se deficiency are generally confounded with those of vitamin E deficiency [1]. Vitamin E is known as an antioxidant that quenches free radicals in biological membranes. While vitamin E was shown to inhibit paraquatinduced cell death and lipid peroxidation in cultured rat hepatocytes [52,53], it played only limited roles in diquat cytotoxicity [54,55]. Moreover, supplementing vitamin E did not alleviate acute oral paraquat lethality in chicks [56]. To determine whether high levels of dietary vitamin E replaced the protection of GPXl against paraquat-induced oxidative stress in mice, we challenged Se-adequate GPX1(-/-) and wild-type mice with an ip injection of paraquat (50 mg/kg body weight) after feeding these mice with various levels of dietary vitamin E (up to 100-fold of daily needs) [57]. Although high levels of dietary vitamin E attenuated the paraquat-mediated hepatic lipid peroxidation, mouse survival time or rate was affected by only the GPXl knockout, but not dietary vitamin E levels. Clearly, the protection conferred by GPXl against this lethal oxidative stress can not be replaced by high levels of dietary vitamin E. Contrasting roles of GPXl in coping with ROS vs. RNS Past research on the antioxidant roles of GPXl or any other enzymes has heavily skewed to ROS. However, reactive nitrogen species (RNS) are constantly generatedfirommetabolism such as the formation of peroxynitrite from superoxide anion and NO. Using purified bovine GPXl protein and a cell-free system [58], Sies et al. suggested that GPXl was a peroxynitrite reductase. To test the biological relevance of their view, we isolated hepatocytes from the GPX1(-/-) and wild-tj^je mice and treated them with diquat (a superoxide generator), S-nitroso-iV-acetyl-penicillamine (SNAP, a nitric oxide donor), 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator), and peroxynitrite (a potent RNS) [59,60]. We measured DNA strand breaks, cytochrome c release, and caspase-3 activation as indicators of apoptosis. It was very striking that the diquat-induced apoptosis was significantly greater in the GPX1(-/-) than in wild-t5rpe cells, whereas the complete opposite was true for the peroxynitrite-induced apoptosis. The GPX1(-/-) cells were not more susceptible to the treatments of SNAP or SIN1 with diquat than wild-type cells [59,60]. Instead, there was less protein nitrotyrosine formation in the GPX1(-/-) cells treated with SNAP and diquat
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Selenium: Its molecular biology and role in human health
than that in wild-type cells [60]. Although stimulated macrophages isolated from GPX1(-/-) mice produced more NO than those from the wild-type mice, and GPXl protected NO-associated protein carbonyl formation in these cells [61], our results do not support the notion that GPXl was a peroxynitrite reductase [58]. In fact, our data suggest that GPXl may potentiate RNSrelated oxidative sfress. This is completely opposite to its protection against ROS-related oxidative stress. Our view of the contrasting roles of GPXl in ROS vs. RNS-related oxidative stress is supported by a number of animal experiments [62-66]. It has been shown that GPXl protects against ischaemia/reperfusion injury [6264], virus-induced myocarditis [65], and endotoxemia [66]. Increased endotoxemia in GPX1(-/-) mice was in association with increases in the expression of genes whose products regulate levels of ROS [67]. Furthermore, GPX1(-/-) mice are more susceptible than wild-type mice to pro-oxidant-induced neurotoxicity [68,69], despite variations from studies with neurons isolated from GPX1(-/-) mice [70,71]. In contrast, toxicities induced by RNS-generated drugs were prevented or attenuated by GPXl null, but aggravated by GPXl overexpression. Mice overexpressing GPXl became more susceptible to acetaminophen-induced lethality and hepatic GSH depletion than wild-type mice [72]. Meanwhile, GPX1(-/-) mice were more resistant to acetaminophen-induced plasma ALT activity increases [73] and to kainic acid-induced mortality and seizures than wild-type mice [74]. It seems to be overly simplifying to call GPXl an antioxidant enzyme as its role in coping with any given stress may depend upon the nature of oxidants. Impacts of GPXl overexpression on insulin function It has been reported that Se exhibits insulin-mimetic property, and that there is a linkage between oxidative stress and diabetes [75]. Surprisingly, we found that GPX1(+) mice developed insulin resistance and obesity, along with hyperglycemia, hyperinsulinemia, and elevated plasma leptin [25]. After insulin stimulation, GPX1(+) mice exhibited attenuated phosphorylations of insulin receptor and Akt on Ser^°* and Ser'*^^, compared with wild-type controls. It is likely that overexpression of GPXl overquenched intracellular ROS, resulting in an accelerated dephosphorylation of proteins in the insulin cascade. A recent human study has shown a positive association between increases in erythrocyte GPXl activity and incidences of insulin resistance during pregnancy [76]. Although knockout of GPXl did not affect renal damage associated with type 1 diabetic nephropathy [77], our results imply a possible involvement of GPXl in type 2 diabetes. Perspectives The successful generation of the GPX1(-/-) and GPX1(+) mice have helped us in elucidating our understanding of GPXl regulation and fimction. From
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the initial verification of m vivo antioxidant roles of GPXl to the more recent illustration of GPXl overexpression on insulin function, we have begun to get glimpses of the complex networks of GPXl actions. Unequivocally, GPXl is a bona fide antioxidant in vivo, and a regulator of ROS and RNS metabolism. It is important to recognize that metabolic functions of GPXl in oxidative stress are not unilateral. It will be fascinating to find out: 1) how GPXl exerts its differential roles in coping with ROS and RNS; 2) how GPXl modulates the nature of necrotic and apoptotic cell deaths, and 3) how GPXl overexpression intervenes in insulin signaling and function. Answers to these critical questions will definitely enrich our knowledge of GPXl biology and help in developing diagnoses and therapies related to oxidative injuries. Acknowledgements Research was supported in part by NIH grant DK53018 to X.G.L. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
K Schwarz, CM Foltz 1957 J Am Biol Soc 79:3292 GC Mills 1957 J Biol Chem 229:189 L Flohe, WA Gunzler, HH Schock 1973 FEES Lett 32:132 JT Rotruck, et al. 1973 Science 179:588 D Behne, W Wolters 1983 JNutr 113:456 S Chada, MM Le Beau, L Casey, PE Newburger 1990 Genomics 6:268 I Chambers, et al. 1986 EMBO J5:\22\ Q Shen, FF Chu, PE Newburger 1993 J Biol Chem 268:11463 MJ Berry, et al. 1991 Nature 353:273 MS Saedi, et al. 1988 Biochem Biophys Res Commm 153:855 SD Mercurio, GF Combs Jr 1986 J Nutr 116:1726 SD Mercurio, GF Combs Jr 1986 Biochem Pharmacol 35:4505 YS Ho, et al. 1997 J Biol Chem 272:16644 JB de Haan, et al. 1998 J Biol Chem 273:22528 LA Esposito, et al. 2000 Free Radic Biol Med 28:754 O Mirochnitchenko, U Palnitkar, M Philbert, M Inouye 1995 Proc Natl Acad Sci USA 92:8120 17. WH Cheng, et al. 1997 JNutr 127:675 18. XG Lei, JK Evenson, KM Thompson, RA Sunde 1995 JNutr 125:1438 19. G Bermano, et a/. 1996 Biol Trace Elem Res 51:211 20. RF Burk 1991 FASEB J 5:2274 21. RA Sunde 1994 Selenium in Biology and Human Health Springer-Verlag, New York pp 45 22. WH Cheng, GF Combs Jr, XG Lei 1998 Biofactors 7:311 23. WH Cheng, et al. 1997 JNutr 127:1445 24. GV Kryukov, et al 2003 Science 300:1439 25. JP McClung, et al. 2004 Proc Natl Acad Sci USA 101:8852 26. VN Reddy, et al. 2001 Invest Ophthalmol Vis Sci 42:3247 27. A Spector, W Ma, RR Wang, Y Yang, YS Ho 1997 Exp Eye Res 64:477 28. ME Mirault, A Tremblay, N Beaudoin, M Tremblay 1991 J Biol Chem 266:20752 29. DM Hockenbery, ZN Oltvai, XM Yin, CL Milliman, SJ Korsmeyer 1993 Cell 75:241
182 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77.
Selenium: Its molecular biology and role in human health MJ Kelner, RD Bagnell, SF Uglik, MA Montoya, GT MuUenbach 1995 Arch Biochem Biophys m-AO SD Taylor, LD Davenport, MJ Speranza, GT MuUenbach, RE Lynch 1993 Arch Biochem Biophys 305:600 RF Burk, RA Lawrence, JM Lane 1980/ Clin Invest 65:1024 SZ Cagen, JE Gibson 1977 Toxicol Appl Pharmacol 40:193 LL Smith 1987 Hum Toxicol 6:31 JA Farrington, M Ebert, EJ Land, K Fletcher 1973 Biochim Biophys Acta 314:372 RF Burk, et al. 1995 //epato/ogv 21:561 JB Atkinson, KE Hill, RF Burk 2001 Lab Invest 81:193 Y Saito, et al 1999 J Biol Chem 274:2866 L Schomburg, et al 2003 Biochem J 2,10:2,91 KE Hill, et al 2003 J Biol Chem 278:13640 WH Cheng, et al.i 199S JNutr 128:1070 Y Fu, WH Cheng, JM Porres, DA Ross, XG Lei 1999 Free Radic Biol Med 27:605 Y Fu, WH Cheng, DA Ross, X Lei 1999 Proc Soc Exp Biol Med 222:164 RH Van, et al. 2004 Free Radic Biol Med 36:1625 H Witschi, S Kacew, KI Hirai, MG Cote 1977 Chem Biol Interact 19:143 JD Morrow, et al 1990 Proc Natl Acad Sci USA 87:9383 RL Levine, JA Williams, ER Stadtman, E Shacter 1994 Methods Enzymol 233:346 WH Cheng, YX Fu, JM Porres, DA Ross, XG Lei 1999 FASEB y 13:1467 WH Cheng, FW Quimby, XG Lei 2003 Free Radic Biol Med 34:918 WH Cheng, X Zheng, FR Quimby, CA Roneker, XG Lei 2003 Biochem J 370:927 MP Fogarty, EJ Downer, V Campbell 2003 Biochem J31\:1S9 MG Traber, H Sies 1996 Annu Rev Nutr 16:321 N Watanabe, Y Shiki, N Morisaki, Y Saito, S Yoshida 1986 Biochim Biophys Acta 883:420 MS Sandy, MD Di, MT Smith 1988 Toxicol Appl Pharmacol 93:288 L Eklow-Lastbom, L Rossi, H Thor, S Orrenius 1986 Free Radic Res Commun 2:57 GF Combs Jr, FJ Peterson 1983 JNutr 113:538 WH Cheng, BA Valentine, XG Lei 1999 JNutr 129:1951 H Sies, VS Sharov, LO Klotz, K Briviba 1997 J Biol Chem 272:27812 Y Fu, H Sies, XG Lei 2001 J Biol Chem 276:43004 Y Fu, JM Porres, XG Lei 2001 Biochem J 359:687 Y Fu, CC McCormick,C Roneker, XG Lei 2001 Free Radic Biol Med 3\:A50 N Maulik, T Yoshida, DK Das 1999 Mol Cell Biochem 196:13 T Yoshida, et al 1997 Circulation 96:11 PJ Crack, et al 2001 JNeurochem 78:1389 MA Beck, RS Esworthy, YS Ho, FF Chu 1998 FASEB J \2:\\43 H Jaeschke, YS Ho, MA Fisher, JA Lawson, A Farhood 1999 Hepatology 29:443 C Li, J Liu, MP Waalkes, H Jaeschke 2003 Toxicol Lett 144:397 P Klivenyi, et al 2000 J Neurosci 20:1 J Zhang, DG Graham, TJ Montine, YS Ho 2000 JNeuropathol Exp Neurol 59:53 K Nakamura, et al.200O JNeurochem 74:2305 JM Taylor, U Ali, RC lannello, P Hertzog, PJ Crack 2005 JNeurochem 92:283 O Mirochnitchenko, et al 1999 J Biol Chem 274:10349 TR Knight, A Kurtz, ML Bajt, JA Hinson, H Jaeschke 2001 Toxicol Sci 62:212 D Jiang, G Akopian, YS Ho, JP Walsh, JK Andersen 2000 Exp Neurol 164:257 OEzakil990y5jo/aeOT265:1124 X Chen, TO SchoU, MJ Leskiw, MR Donaldson, TP Stein 2003 J Clin Endocrinol Metab 88:5963 JB de Haan, et al 2005 Am J Physiol Renal Physiol 289:F544
Chapter 17. Selenoproteins of the thioredoxin system Ame Holmgren Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden
Summary: The three isoenzymes of mammalian thioredoxin reductase are dimeric selenoproteins containing an essential catalytically active selenocysteine (Sec) residue. In contrast to the enzymes from bacteria, yeast and plants, the mammalian enzymes are larger and entirely different in structure and mechanism. They are homologous to glutathione reductase, but with a C-terminal elongation of 16 residues containing the conserved Cterminal active site sequence -Gly-Cys-Sec-Gly. The active site is a selenneylsulfide formed from the conserved Cys-Sec sequence, which is reduced to a selenolthiol by electrons from the redox active disulfide of the other subunit, as revealed by a three-dimensional structure of the rat enzyme. The essential role of Sec in thioredoxin reductase explains the very broad substrate specificity including reduction of thioredoxin, selenite, dehydroascorbic acid and ascorbyl free radical, hydrogen peroxide and lipid hydroperoxides. The essential role of selenium in human thioredoxin reductases further explains roles of this trace element in cell growth via pleiofropic effects in reduction of thioredoxin with its multiple roles in elecfron transport to essential biosynthetic enzymes, thiol redox confrol of transcription factors, or in defense against oxidative sfress. Clinically used inhibitors of cell growth or inflammation like gold thioglucose are targeted to the reduced Sec residue of the enzyme. Introduction The thioredoxin system comprised of NADPH, thioredoxin (Trx) and the flavoprotein thioredoxin reductase (TrxR) is ubiquitously present from Archaea to man [1, 2]. Thioredoxin with a redox-active dithiol/disulfide is an electron donor for essential enzymes such as ribonucleotide reductase and a general protein disulfide reductase with numerous functions in confrol of infracellular redox potential, defense against oxidative sfress and signal fransduction by thiol redox control [2]. Thioredoxin reductases from mammalian cells and higher eukaryotes are selenoenzymes [3,4] and very different from the smaller selenium-independent enzymes of Archaea,
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bacteria, yeast and plants [5]. This chapter will discuss reactions between selenium compounds and the thioredoxin system and some of the structurefunction relationships of mammalian thioredoxin reductases. General properties of thioredoxin systems All thioredoxin reductases reduce oxidized thioredoxin (Trx-S2) at the expense of NADPH [1,2] (Reaction 1). Reduced thioredoxin [Trx-(SH)2] is reoxidized by disulfides in proteins generating thiols (Reaction 2): TrxR Trx-(SH)2 + NADP^
Trx-S2 + NADPH + H^
Trx-(SH)2 + Protein-S2
spontaneous Trx-S2 + Protein-(SH)2
(1)
(2)
Generally, the Km-value for NADPH is low or in the range below 10 nM and that of Trx-S2 is typically from 1 to 3 (xM. Isolation and characterization of mammalian thioredoxin and thioredoxin reductase started about 30 years ago [6-8]. As shown in Table 1, there are some major differences between the thioredoxin systems of prokaryotes like E. coli and that of mammalian organisms.
Table 1. Properties of Thioredoxin Systems E. coli
Human
Thioredoxin
U,= 12,000 108 aa -CGPC-active site Trx-S2 stable upon storage
Mr= 12,000 104 aa -CGPC-active site +3 structural SH-groups, Trx-activity reversibly lost by additional disulfide formation upon aerobic storage
Tliioredoxin reductase
Mr= 70,000 2 subunits High specificity Stable
Mr= 114,000 or larger; 3 genes 2 subunits Broad specificity, selenoenzymes Labile to oxidation - reduction cycles
E. coli and mammalian cytosolic thioredoxins are homologous proteins with a conserved -Cys-Gly-Pro-Cys- active site. However, mammalian thioredoxin must be purified in the fully reduced form since they contain structural SH-groups which form additional disulfides upon oxidation. This may have autoregulatory function of thioredoxin activity in resting cells or
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upon oxidative stress, yet incompletely known in vivo. Thioredoxin reductases from mammalian cells have very different properties when compared with the enzymes from E. coli, yeast or plants (reviewed in [5]). The cytosolic en2yme has subunits with 55 kDa or larger instead of the 35 kDa in the E. coli enzyme with a known three-dimensional structure [5]. As will be described below, the mammalian enzyme also has a very broad substrate specificity entirely different from the generally subsfrate-specific enzymes only reducing Trx-S2 that are present in prokaryotes, yeast and plant cytosol. Selenium reduction by the thioredoxin system The fact that administration of selenium compounds like selenite (SeOs^) cause the inhibition of tumor cell proliferation in vivo and the knowledge that thioredoxin reductase appeared to be more highly expressed in malignant cells prompted us to start investigations on the reactions of selenium compounds with the mammalian thioredoxin system. Contrary to expectations, we discovered that selenite is a direct substrate for thioredoxin reductase as well as an efficient oxidant of thioredoxin [9,10]. With 200 ^M NADPH and 50 nM calf thymus thioredoxin reductase, addition of 10 ^M selenite caused oxidation of 40 jxM NADPH in 12 min and 100 jxM NADPH after 30 min demonstrating a direct reduction of selenite with redox cycling by oxygen [9,10]. This was demonstrated by incubation under anaerobic conditions where only 3 mol of NADPH was oxidized per mol of selenite according to Reaction 3:
SeOa'+ 3 NADPH + 3H^
TrxR -^ Se^'+ 3 NADP^ + 3 HjO
(3)
Addition of thioredoxin stimulated the reaction further since selenite rapidly reacts with Trx-(SH)2 to oxidize it to Trx-S2 [11-13]. Since glutathione reductase will not react with selenite [13], Reaction 3 should provide cells with selenide, a required precursor for selenophosphate and selenocysteine (Sec) synthesis [14]. Selenite and glutathione react to form selenodiglutathione (GS-Se-SG) which has been suggested to be a major metabolite of inorganic selenium salts in mammalian tissues [15]. Reduction of selenodiglutathione by NADPH and glutathione reductase was demonstrated by Ganther [16] and it has been proposed to be a source of selenide in cells as well as an inhibitor of neoplastic growth [17]. We synthesized GS-Se-SG [11,18] and discovered that this compoimd is a direct efficient substrate for mammalian thioredoxin reductase and a highly
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Selenium: Its molecular biology and role in human health
efficient oxidant of reduced thioredoxin. Since GSSG is not a substrate for mammalian thioredoxin reductase [7,8] the insertion of the selenium atom in the GSSG molecule to form GS-Se-SG makes this molecule highly reactive with the enzyme. Reduction of GS-Se-SG to yield selenide by glutathione reductase requires two mol of NADPH. We found only the first stoichiometric reduction to be fast with GS-Se" as a product [11]. The second reaction was slow and relatively inefficient. These results strongly suggest that the major selenide generation in cells is via thioredoxin reductase and thioredoxin. Thus, in mammalian cells the selenoenzyme thioredoxin reductase is also responsible for the generation of selenide required for its own synthesis. An oxygen dependent and non-stoichiometric consumption of NADPH is given by the thioredoxin system in the presence of selenite, selenodiglutathione and selenocystine [9-11,18]. The latter oxidized form of Sec is an efficient substrate for mammalian thioredoxin reductase with a Km of 6 \iM [18]. The mechanism may be that the XSe" reacts with a dithiol (or selenolthiol) to catalyze oxidation according to Reaction 4:
XSe'+ R-(SH)2 + (0)
-^
XSe+R-Sz + HaO
(4)
The effect will be 02-dependent consumption of NADPH and the results demonstrating autoxidation of the selenium compounds provide an explanation for the lack of a free pool of Sec as well as the acute toxic effects of selenium compounds on cells, e.g., leading to oxidative stress and apoptosis. Substrate specificity of thioredoxin reductase Mammalian thioredoxin reductases display a surprisingly wide substrate specificity as first observed during purification [7,8]. This is in contrast to the smaller prokaryotic thioredoxin reductases, which do not use mammalian thioredoxins as substrates despite their conserved active sites and closely related three-dimensional structures [19]. As summarized in Table 2, a truly wide range of direct reductions are catalyzed by mammalian cytosolic thioredoxin reductases. Thioredoxin from E. coli is a substrate with a similar Kcat, but with a 15-fold higher Km-value (35 \xM) compared with the rat liver protein [8]. Mammalian cytosolic thioredoxins generally show full crossreactivity with thioredoxin reductases fi-om different mammalian sources and vice versa, hi many instances, fi-ee selenocyst(e)ine will stimulate reduction of substrates [24,13,23,29].
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187
Table 2. Reactions catalyzed by cytosolic mammalian thioredoxin reductases (rat, bovine and human) 5,5-dithiobis-(2-mtrobenzoic acid) reduction Thioredoxin-Sa reduction Protein disulfide isomerase (PDI) Selenite (SeOj^") and Sec reduction Selenodiglutathione reduction Nitrosoglutathione (GSNO) reduction Electron donor to plasma glutathione peroxidase H2O2 and lipid hydroperoxide reductase Reduction of alloxan and vitamin K NK-lysin disulfide reduction and inactivation of cytotoxic activity Lipoic acid and lipoamide reduction Reduction of dehydroascorbic acid Reduction of ascorbylfl-eeradical
[7] [8] [20] [ 10,13] [11] [21 ] [22] [23,24] [8,25] [26] [27] [28] [29]
Structure and mechanism of mammalian thioredoxin reductase Recent biochemical studies, sequencing and cloning of mammalian thioredoxin reductases have revealed that the enzymes are selenoproteins and entirely different from the corresponding enzymes in bacteria, yeast and plants (review in [5]). Stadtman and coworkers serendipitously discovered that human tumor cell thioredoxin reductase is a selenoprotein using labeling of selenoproteins with radioactive selenite [3]. This also explained [30] why a previously putative clone of the human enzyme [31] where the TGA codon for Sec was interpreted as the stop codon (Figure 1) gave no enzyme activity. The TGA will be acting as a stop codon in E. coli due to the species-specific machinery for synthesis of selenoproteins which is different in bacteria and mammalian cells [14]. By sequencing large parts of the cytosolic bovine enzyme, we also directly identified the C-terminal peptide as containing Sec. The bovine peptides were used to identify a rat cDNA clone which was sequenced [4]. The results showed a polypeptide chain with a high homology to glutathione reductase [4,23] including an identical active site disulfide (CVNVGC) (Figure 1) but with a 16-residue elongation containing the conserved Cterminal sequence, Gly-Cys-Sec-Gly. A Sec insertion sequence (SECIS) was identified in the 3'-untranslated region [4]. Furthermore, digestion of thioredoxin reductase by carboxypeptidase after reduction by NADPH released Sec with loss of activity; the oxidized form of the enzyme was resistant to carboxypeptidase digestion [4]. Redox titrations with dithionite and NADPH demonstrated that the mechanism of the human placenta enzyme is similar to that of lipoamide dehydrogenase and glutathione
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Selenium: Its molecular biology and role in human health
reductase and distinct from the mechanism of thioredoxin reductase from E. coli [32]. The results also demonstrated that the Sec residue of human thioredoxin reductase is redox active and communicates with the redox active disulfide since more than 4 elecfrons per subunit are required to completely reduce the FAD of the oxidized enzyme. Furthermore, the Sec residue is alkylated with loss of activity only after reduction by NADPH [4,33,34]. The Sec residue is also the target of the irreversible inhibitor 1chloro-2,4-dinifrobenzene only after reduction by NADPH [35] as shown by peptide analysis [34].
CVNVGC
GCUG
r^v^: H jN -I
V FAD
NADPH
Intertaoe
|- COOH
a n-Al a - a y-Cys-Sec-Q y-Ter (human TrxR) CAG GOT GGC TGC TQA GGT TAA GCC CCA . . . CAG TOT GGC TGC TGA GGT TAA GCC CCA . . . a n - S e r - Q y-Cys-Sec-Q y-Ter ( r a t TrxR) Figure 1. Schematic structure (upper small, rectangular box) and C-terminal sequences (lower large, rectangular box) of human and rat thioredoxin reductases [4,30,31]. The N-terminal glutathione reductase-like active site disulfide (CVNVGC) is shown as well as the FAD, NADPH and interface domains. The active site is shown in the C-terminal region with GCUG denoting Gly-Cys-Sec-Gly. The lower part of the figure also shows the TGA codon encoding Sec.
The essential role of selenium in the catalytic activities of mammalian thioredoxin reductase was revealed by characterization of recombinant enzymes with Sec mutations [23]. This was done by removing the Sec insertion sequence in the rat gene and changing the Sec498 encoded by TGA to Cys or Ser codons by mutagenesis. The truncated protein having the Cterminal dipeptide deleted, expected in selenium deficiency, was also engineered. All three mutants were successfully overexpressed in E. coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. All three mutant proteins rapidly generated the A540 absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR. Only the Sec498 Cys enzyme showed catalytic activity in reduction of thioredoxin, with a 100-fold lower Kcat and a 10-fold lower K^ compared to
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the wild type rat enzyme. The pH-optimum of the Sec-containing wild type enzyme was 7 whereas the Sec498 to Cys mutant showed a pH optimum of 9. This strongly suggested the involvement of the low pKa Sec selenol in the enzyme mechanism. Also selenium was required for hydrogen peroxide reductase activity [23]. Thus, selenium is required for the catalytic activities of thioredoxin reductase explaining the essential role of this trace element in cell growth. Based on the homology to glutathione reductase we proposed a model of mammalian thioredoxin reductase (Figure 2).
NADPH
domain
Interface
16 aa with
elongation Cys-SeCys
I'AD
i'AD
domain
domain 16 aa elongation with Cys-SeCys
Thioredoxin
dotnilin
NADPH
domain
Reductase
Figure 2. Structural model of mammalian thioredoxin reductase based on the homology to glutathione reductase. The 16-residue C-terminal extension with the active site is shown as well as the head to tail arrangement of the subunits in the dimer. Taken from [36]. The FAD, NADPH and interface domains are shown (see also Figure 1).
The enzyme is a head to tail dimer with the 16-residue elongation in principle taking the place of GSSG in glutathione reductase. The active site of the enzyme is a selenolthiol in its reduced form and a selenenylsulfide formed from the conserved cysteine-Sec sequence in the oxidized form [36]. The selenenylsulfide was isolated by peptide sequencing and also confirmed by mass spectrometry [36]. Mechanisms of the enzyme have also been postulated involving a reductive half-reaction similar to that of glutathione reductase leading to reduction of the active site disulfide (Figures 1 and 2). Electrons are thereafter transferred from the redox-active dithiols to the selenenylsulfide of the other subunit generating the selenolthiol. Characterization of the Cys mutant enzyme revealed that the selenium atom
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with its larger radius is critical for the formation of the unique selenenylsulfide [36] since the C-terminal dithiol stays reduced in the Cys mutant [36]. Similar results confirming these data have also been obtained by others [37]. The structure of the enzyme has been solved by X-ray crystallography after the Cys mutant enzyme has been crystallized [38]. Crystal structure of the Sec498Cys mutant of rat TrxRl in complex with NADPH was determined to 3.0-A resolution. The overall structure is similar to that of glutathione reductase, including the conserved amino acid residues that bind the cofactors FAD and NADPH. The redox active disulfide in the N-terminal portion of the enzyme is identical to that of glutathione reductase. Residues directly binding the substrate glutathione disulfide in glutathione reductase are conserved despite the fact that glutathione disulfide is not a substrate for thioredoxin reductase [38]. The 16-residue Cterminal tail, a unique feature on mammalian thioredoxin reductases, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer (see schematic drawing in Figure 2). A unique feature of the Sec498Cys mutant of rat TrxRl is that the thiols do not form a disulfide [38]. A model of the complex of TrxR read Trx allows docking of oxidized thioredoxin to the structure without large conformational changes [38]. This is in great contrast to the large conformational change required for the prokaryotic Cys-residue enzymes [5]. The model suggests specific interactions between residues in thioredoxin (D60, D61 and K72 and corresponding charges in TrxR forming electrostatic interactions). The Xray structure particularly explains how the 16-residue C-terminal extension conserved in all three mammalian isoenzymes of thioredoxin reductase. It extends the electron transport chain from the catalytic disulfide to the enzjone surface, enabling reaction with Trx and a range of other substrates (Table 2). It acts to prevent the enzyme from acting as a glutathione reductase by blocking access to the redox active disulfide. The results of the X-ray study [38] strongly suggest that mammalian thioredoxin reductase evolved from a glutathione reductase scaffold rather than from its prokaryotic coimterpart. Such an evolutionary switch will render cell growth dependent on selenium in the form of Sec and it may have advantages for cells using reactive oxygen species like hydrogen peroxide in cell signaling. Isoenzymes of thioredoxin reductase Apart from the cytosolic thioredoxin reductase, TrxRl, two additional genes encoding novel forms of human and mouse selenoprotein thioredoxin reductases have been identified [39]. One is a mitochondrial enzyme [40,41] and the other thioredoxin-glutathione reductase carrying an N-terminal glutaredoxin domain; the latter is preferentially expressed in testis. All these enzymes have extensions in the N-terminal region but share the C-terminal
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active site sequence. Additional complexity is given by the identification of enzymes with mRNA variants differing in the 5'-untranslated region [42] and by 5'-exon splicing [43]. The nematode C. elegans contains two homologues related to mammalian thioredoxin reductase, one with Cys and the other with Sec. The Sec containing enzyme with 74 kDa subunits is the major selenoprotein in C. elegans. Medical aspects of selenium in thioredoxin reductase Human thioredoxin reductase is a general reducing enzyme with a wide substrate specificity contributing to cellular redox homeostasis and is a major pathophysiological factor and drug target. Together with thioredoxin, it is involved in prevention, intervention and repair of damage caused by hydrogen peroxide-based oxidative stress. As a selenite reducing enzyme with a selenol containing active site human thioredoxin reductase plays a central role in selenium physiology. A range of human diseases and conditions are now known or suspected to be related to the activity and function of thioredoxin reductase (in-depth review in [45]). This involves diseases like reumatoid arthritis, Sjogren's syndrome, AIDS and malignancies. The close homology between human thioredoxin reductase and glutathione reductase has lead to the realization that several clinically used drugs like nitrosurea derivatives are targeted to thioredoxin reductase [45]. Furthermore, studies on the regulation of thioredoxin reductase mRNA [46] and the development of specific inhibitors for use in antitumour therapy [47-49] make the enzyme a major target for drug development. In this context it will be important in future studies to establish also the role of the glutathione-dependent glutaredoxin system [50,51] which is an alternative non-selenium pathway of transferring electrons to essential biosynthetic reactions like ribonucleotide reductase. Thus, determining if a malignant cell is dependent on the thioredoxin system or the glutaredoxin system should be essential in drug selection in tumor therapy. The fact that the thioredoxin system is ubiquitous and present in quite highly variant forms in pathogenic bacteria makes the enzyme a particularly attractive drug target. There is a surprising diversity in the structure and mechanism of the enzyme in several important pathogenic bacteria (reviewed in [45]). This may lead to the development of specific inhibitors of bacterial infections as in Lepra, parasitic diseases and malaria. Treatment of an inflammatory disease like reumatoid arthritis with drugs like gold thioglucose and aiu-anofin which are strong inhibitors of thioredoxin reductase likely occur by binding to the reduced Sec residue in the enzyme. Since thioredoxin reductase is involved in central biosynthetic reactions and defense against oxidative stress via thioredoxin, it has a high priority in the hierarchy of synthesis of selenoproteins. Of particular interest is whether
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the truncated en2yme expected in selenium deficiency is present in cells. This may be of great importance for understanding the effects of selenium supplementation as an anticancer agent [52]. The modification of the Sec residue in TrxR or the truncated enzyme gives rapid induction of cell death [53]. A number of clinically used anticancer compounds, including alkylating and platinum-containing drugs, inhibit thioredoxin reductase, but not glutathione reductase [54]. Obviously, thioredoxin reductase is a novel and important molecular target for cancer therapy [55]. Thioredoxin reductase gene targeting Reactive oxygen species (ROS) are generated as by products of the respiratory chain or by NADPH oxidases. ROS are implicated in the pathogenesis and pathophysiology of a variety of human diseases, such as cardiovascular and degenerative disorders and cancer. ROS is also implicated in cellular signaling. Peroxiredoxins working together with thioredoxins and thioredoxin reductases are controling the levels of reactive oxygen species and free radicals. A complete thioredoxin system, including thioredoxin reductase, Trx and peroxiredoxin (Prx III) is present in mitochondria. To address the fimction of mitochondrial thioredoxin reductase (TrxR2), a ubiquitous Cre-mediated inactivation of TrxR2 was shown to be associated with death at embryonic day 13 [56]. TrxR2" embryos are smaller and severely anemic and showed increased apoptosis in liver [56]. Also, the size of hematopoietic colony cultures ex vivo was dramatically reduced. TrxR2-deficient embryonic fibroblasts showed high sensitivity to endogeneously produced oxygen radicals when glutathione synthesis was inhibited [56]. Also, the ventricular heart wall of the mitochondrial thioredoxin reductase knockout embryos was thinner and the proliferation of cardiomyocytes was decreased. Cardiac specific ablation of TrxR2 resulted in fetal cardiomyopathy with symptoms similar to those of Keshan disease and Friedreich's ataxia [56]. Thus, mitochondrial thioredoxin reductase plays an essential role in hematopoiesis, heart development and heart function [56]. A similar study on the cytoplasmic thioredoxin reductase (TrxRl), using a conditionally target deletion of the Txnrdl gene showed that the gene was essential for embryogenesis [57]. Ubiquitous Cre-mediated inactivation of Txnrdl leads to early embryonic lethality [57]. Embryos of the homozygous mutant displayed severe growth retardation and failed to turn [57]. Also, Txnrdl-deficient embryonic fibroblasts do not proliferate in vitro in line with growth impairment. Surprisingly, in contrast, ex v/vo-cultured embryonic Txnrdl-deficient cardiomyocytes were not effected and mice with a heart specific inactivation of Txnrdl developed normally and appeared healthy [57]. The conclusion from these studies is that the TrxRl
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enzyme is essential during embryogenesis in most developing tissues except for the heart. Obviously, the role of thioredoxin, thioredoxin reductase to provide electrons for the synthesis of deoxyribonucleotide by ribonucleotide reductase is the suspected reason for the essential role of TrxRl. The association of TrxRl with proliferation make this enzyme a specifically interesting drug target for cancer therapy. The combined effects of the studies on gene targeting [56,57] stress the importance of inhibiting TrxRl specifically without affecting mitochondrial TrxR2. Drugs affecting TrxR2 will also have serious cardiac side effects [56]. Selective RNA interference may in the future be used to selectively target TrxRl, since drugs are likely to effect both the cytoplasmic and mitochondrial thioredoxin reductases with their identical active sites. Acknowledgement The research support from the Swedish Medical Research Council (3529), the Swedish Cancer Society and the K A Wallenberg foundation is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
A Holmgren 1985 Annu Rev Biochem 54:237 ES J Am6r, A Holmgren 2000 Eur J Biochem 161:6102 T Tamura, T C Stadtman 1996 Proc Natl Acad Sci USA 93:1006 L Zhong, ESJ Amer, J Ljung, F Aslund, A Holmgren 1998 J Biol Chem 273:8581 CH Williams Jr, LD Arscott, S Muller, BW Lennon, ML Ludwig, P-F Wang, DM Veine, K Becker, RH Schirmer 2000 Eur J Biochem 267:6110 NE EngstrSm, A Holmgren, A Larsson, S Soderhall 1974 J Biol Chem 249:205 A Holmgren 1977 J Biol Chem 252:4600 MLuthman, A Holmgren 1982 SiocAe/««//y 21:6628 A Holmgren, S Kumar 1989 Selenium in Biology and Medicine A Wendel (Ed) Springer-Verlag, Berlin, 47 S Kumar, M BjOmstedt, A Holmgren 1992 Eur J Biochem 207:435 M BjSmstedt, S Kumar, A Holmgren 1992 J Biol Chem 267:8030 X Ren, M Bjdmstedt, B Shen, M Ericson, A Holmgren 1993 Biochemistry 32:9701 M BjOmstedt, S Kumar, L Bjorkhem, G Spyrou, A Holmgren 1997 Biomed Environ Sci 10:271 TC Stadtman 1996 Annu Rev Biochem 65:83 HS Hsieh, HE Ganther 1975 Biochemistry 14:1632 HE Ganther 1971 Biochemistry 10:4089 RJ Shamberger 1985 Mutat Res 154:29 M BjOmstedt, S Kumar, A Holmgren 1995 Methods Enzymol 252:219 A Holmgren 1995 Structure 3:239 J Lundstrdm, A Holmgren 1990 J Biol Chem 265:9114 DNikitovic,AHolmgrenl996J5/o/C/iem271:19180 M Bjfimstedt, J Xue, W Huang, B Akesson, A Holmgren 1994 J Biol Chem 269:29382 L Zhong, A Holmgren 2000 J Biol Chem 275:18121 M Bjfimstedt, M Hamberg, S Kumar, J Xue, A Holmgren 1995 J Biol Chem 270:11761 AHolmgren,C Lyckeborg 19S0 Proc Natl Acad Sci USA 77:5149 M Andersson, A Holmgren, G Spyrou 1996 J Biol Chem 271:10116 ESJ Amer, J Nordberg, A Holmgren 1996 Biochem Biophys Res Commun 225:268
194 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.
57.
Selenium: Its molecular biology and role in human health JM May, S Mendiratta, KE Hill, RF Burk 1997 J Biol Chem 272:22607 JM May, CE Cobb, S Mendiratta, KE Hill, RF Burk 1998 J Biol Chem 273:23039 VN Gladyshev, K-T Jeang, TC Stadtman 1996 Proc Natl Acad Sci USA 93:6146 PY Gasdaska, JR Gasdaska, S Cochran, G Powis 1995 FEBSLetters 373:5 LD Arscott, S Gromer, RH Schirmer, K Becker, CH Williams Jr 1997 Proc Natl Acad Sci USA 94:9621 SN Gorlatov, TC Stadtman 1998 Proc Natl Acad Sci USA 95:8520 J Nordberg, L Zhong, A Holmgren, ES J Am6r 1998 J Biol Chem 273:10835 ESJ Amer, M Bjfimstedt, A Holmgren 1995 J Biol Chem 270:3479 L Zhong, ESJ Am6r, A Holmgren 2000 Proc Natl Acad Sci USA 97:5854 SR Lee, S Bar-Noy, J Kwon, RL Levine, TC Stadtman, SG Rhee 2000 Proc Natl Acad Sci USA 97:2521 T Sandalova, L Zhong,Y Lindqvist, A Holmgren, G Schneider 2001 Proc Natl Acad Sci USA 98:9533 Q-A Sun, Y Woo, F Zappacosta, K-T Jeang, BJ Lee, DL Hatfield, VN Gladyshev 1999 J Biol Chem 274:24522 SR Lee, JR Kim, KS Kwon, HW Yoon, RL Levine, A Ginsburg, SG Rhee 1999 J Biol Chem 274:4722 A Miranda-Vizuete, AE Damdimopoulos, JR Pedrajas, J-A Gustafsson, G Spyrou 1999 Eur J Biochem 261:405 A-K Rundief, M Carlsten, MMJ Giacobini, ESJ Am^r 2000 Biochem J 347:661 QA Sun, F Zappacosta, VM Factor, PJ Wirth, DL Hatfield, VN Gladyshev 2001 J Biol Chem 276:3106 VN Gladyshev, M Krause, X-M Xu, KV Korotkov, GV Kryukov, Q-A Sun, BJ Lee, JC Wootton, DL Hatfield 1999 Biochem Biophys Res Commun 259:244 K Becker, S Gromer, RH Schirmer, S Muller 2000 Eur J Biochem 267:6118 DL Kirkpatrick, S Watson, M Kunkel, S Fletcher, S Ulhag, G Powis 1999 Anticancer Drug Res 5A21 JR Gasdaska, JW Harney, PY Gasdaska, G Powis, MJ Berry 1999 J Biol Chem 274:25379 MM Berggren, JF Mangin, JR Gasdaska, G Powis 1999 Biochem Pharmacol 57:187 G Powis, DL Kirkpatrick, M Angulo, A Baker 1998 Chem Biol Interact 111-112:23 A Holmgren 1999 Redox Regulation of Cell Signaling and its Clinical Application L Packer, J Yodoi (Eds) Marcel Dekker, New York, 279 A Holmgren 1989 J Biol Chem 264:13963 S Gromer, JH Gross, 2002 J Biol Chem 277:9701 K Anestal, ESJ Am6r 2003 J Biol Chem 278:15966 A-B Witte, K Anestal, E Jerremalm, H Ehrsson, ESJ Amer 2005 Free Radio Biol Med 39:696 P Nguen, RT Awwad, DDK Smart, DR Spitz, D Gius 2005 Cancer Letters in press M Conrad, C Jakupoglu, SG Moreno, S Lippl, A Banjac, M Schneider, H Beck, AK Hatzopoulos, U Just, F Sinowatz, W Schmal, KR Chien, W Wurst, GW Bomkamm, M Brielmeier 2004 Mol Cell Biol 24: 9414 C Jakupoglu, GKH Przemeck, M Schneider, SG Moreno, N Mayr, AK Hatzopoulos, M Hrab6 de Angelis, W Wurst, GW Bomkamm, M Brielmeier, M Conrad 2005 Mol Cell Biol 25:1980
Chapter 18. Mitochondrial and cytosolic thioredoxin reductase knockout mice Marcus Conrad and Georg W. Bomkamm Institute of Clinical Molecular Biology and Tumor Genetics, GSF-Research Centre for Environment and Health, 81377 Munich, Germany
Markus Brielmeier Department of Comparative Medicine, GSF-Research Centre for Environment and Health, 85764 Neuherberg, Germany
Summary: To address the role of the thioredoxin system in redox regulation of apoptosis and proliferation, mice with targeted deletions of both the cytosolic (Txnrdl) and the mitochondrial (Txnrd2) thioredoxin reductases were generated. These two selenoproteins are key enzymes governing the activities of cytosolic and mitochondrial thioredoxins, respectively, which are, in turn, implicated in a variety of cellular functions, such as cell-cell communication, proliferation and apoptosis. Ubiquitous and heart-specific inactivation revealed widely non-redundant functions of Txnrdl and Txnrd2. A significant drop in cell proliferation rates throughout the embryo (except in the heart), but not increased apoptosis, was the underlying cause of embryonic death of Txnrdl knockout embryos at E10.5. Perturbed cardiac development and increased apoptosis of fetal blood cells in the liver caused severe anemia, growth retardation and embryonic death (E13.5) in Txnrd2 knockout embryos. Cardiac-tissue restricted inactivation of Txnrd2 led to biventricular dilated cardiomyopathy and postnatal death; in contrast heartspecific inactivation of Txnrdl had no apparent effect on the viability of the knockout mice. In conclusion, Txnrdl contributes to cell proliferation, whereas Txnrd2 is rather involved in apoptosis regulation. Introduction Three distinct thioredoxin reductases are known in mammals, each encoded by individual genes. Thioredoxin reductase 1 (Txnrdl) is primarily localized in the cytosol [1,2], thioredoxin reductase 2 (Txnrd2) in mitochondria (Txnrd2) [3],and thioredoxin reductase 3 (Txnrd3), also called thioredoxinglutathione-reductase (TGR), is mainly expressed in testis [4]. Thioredoxin reductases are homodimeric flavoproteins with each subunit of approximately 54-58 kDa in size, members of the pyridine nucleotide-disulfide
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oxidoreductase family and possess two N- and C-terminally located interacting redox-active centres [5,6]. Txnrd3 contains an N-terminal glutaredoxin-like domain giving the enzyme an additional protein-disulfide isomerase function [7]. NADPH/H^ is a cofactor of thioredoxin reductases and reducing equivalents are first transferred to the prosthetic group FAD, from where they are passed to the N-terminal -CVNVGC- catalytic centre of one subunit and subsequently to the C-terminally located redox-active selenenylsulfide of the other subunit [8-11]. Selenocysteine (Sec), which is part of a conserved (-GCUG) motif, is crucial for Txnrd function [12-14]. Due to the easily accessible C-terminal catalytic centre, thioredoxin reductases have a broad range of substrates including hydrogen peroxide, selenite, lipoic acid, NK-lysin, ascorbate, and ubiquinone [15-17]. Cytochrome C was recently shown to be a Txnrd2 substrate [18]. But the main substrates of thioredoxin reductases are thioredoxins (Txn). Thioredoxins are small redox-reactive proteins and involved in numerous physiological processes including cell-cell communication, redox metabolism, proliferation, and apoptosis [19]. For instance, Txn exert a cytokine-like influence on blood cells [20], modulate the activity of redoxregulated transcription factors, such as N F - K B [21] and AP-1 [22], are putatively involved in DNA synthesis, and efficiently protect cells from oxidative damage by acting through peroxiredoxins [16,23]. Several gene targeting approaches in mice have been performed to investigate the participation of the thioredoxins in development and adult physiology. Deletion of either cytosolic thioredoxin (Txnl) or mitochondrial thioredoxin (Txn2) revealed both genes are indispensable for murine embryonic development [24,25]. In Txnl knockout mutants, early embryonic death (E6.5) is associated with dramatically reduced proliferation of the inner mass cells. Txn2-deficient embryos develop exencephaly, show markedly increased apoptosis, and die during midgestation around El0.5. Moreover, in the chicken B cell line DT40, Txn2 is critically involved in the regulation of mitochondria-dependent apoptosis [26]. Heart-specific overexpression of dominant negative Txnl was shown to be associated with increased oxidative sfress and cardiac hypertrophy in mice [27]. While the catalytic mechanisms and structural properties of thioredoxin reductases have been extensively studied in the past, little is known about the individual contribution of the different thioredoxin reductases in living organisms, or about possible redundancies among the different redox systems. A number of reports have linked the thioredoxin/thioredoxin reductase-system to cell proliferation, cancer development, angiogenesis, invasiveness, and drug resistance of tumor cells [28,29], and diseases, such as rheumatoid arthritis. All of these argue for the development and potential therapeutic use of Txnrd inhibitors. Various chemicals, such as 1,3-bis-(2chloro-ethyl)-l-nitrosourea, antirheumatic gold compounds, cisplatin, and a
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number of other platinum and organotellurium compounds have been shown to inactivate Txnrd [6,30-32]. However, the presence of several thioredoxin reductases with virtually identical redox-centres may impose a great challenge for the development of drugs selectively inhibiting the activity of one isoform without affecting the other ones. Therefore, mice with targeted deletions of either Txnrd 1 or Txnrd2 might thus prove a most valuable tool to decipher the contribution of the thioredoxin/thioredoxin reductase network in physiology, pathophysiology and disease development. Mouse models with conditional alleles for Txnrdl and Txnrd2 Anticipating that loss of Txnrdl or Txnrd2 might be associated with embryonic death, as already observed in Txnl and Txn2 null mice, and to be able to investigate their functions in specific organs and at defined time points, mice with conditional alleles were established. Txnrdl and Txnrd2 are encoded by genes spanning regions of approximately 39 [33] and 53 kb [34], and are composed of 15 and 18 exons, respectively. The last exon of the Txnrdl gene encodes the final 22 amino acids including the C-terminally located Sec-containing redox-centre. The 1.7 kb long 3' untranslated region additionally contains the Sec insertion sequence (SECTS) element essential for co-translational Sec incorporation at the UGA codon [35], AU-rich mRNA instability elements, and the endogenous transcription termination signal. AU-rich elements have been found in several cytokines and protooncogenes and are responsible for rapid mRNA turnover. Txnrd2 mRNA lacks AU-rich elements. The Sec codon of Txnrd2 is encoded by exon 17 whereas the SECIS element and the poly-adenylation signal are localized on the last exon of the Txnrd2 gene. The gene targeting strategies for both genes aimed at flanking the last exon(s) with loxP sites, which upon Cre-mediated removal, leads to nonfunctional alleles. Only the last exon of Txnrdl was flanked by loxP sites, whereas in case of Txnrd2, the last 4 exons were flanked by loxP sites. The fit-flanked neomycin phosphotransferase gene (neo) required for selection of homologous recombination in embryonic stem (ES) cells was placed downstream of the genes. Several considerations were taken into account to specifically target the 3' regions of both genes: (i) extensive alternative first exon usage, as reported for both Txnrdl [36,37] and Txnrd2 [36], may restrict gene targeting of the 5' regions; (ii) the 5' region of Txnrd2 overlaps with the first exon of the catechol-o-methyltransferase gene [34]; (iii) mutational and biochemical modification of the Sec codon UGA and the Sec moiety [12-14], respectively, as well as deletion of the SECIS element [38] result in inactivation of Txnrd activity. Homologous recombination of both targeting constructs in mouse ES cells and Flp-mediated removal of the neo gene in the loxP flanked (floxed) Txnrd2 alleles was performed [39,40].
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Backcross of chimeric animals [39] to C57BL/6J mice gave rise to germline transmission of the floxed Txnrdl [41] and Txnrd2 alleles [42]. Embryonic lethality of both Txnrdl and Txnrd2 knockout mice Floxed mice were crossed to congenic C57BL/6J Cre deleter mice resulting in deletion of the floxed alleles in any tissue including the germline [43]. Txnrdl^'" and Txnrd2^'' mice are viable, fertile, show no overt phenotype, and have a normal life span compared to wild-type littermates. Mice with either the floxed or the deleted alleles were backcrossed on a C57BL/6J background to obtain congenic mice. Intercross of hemizygous Txnrdl and Txnrd2 knockout mice never resulted in viable homozygous mutant mice. Genotyping of embryos dissected from hemizygous intercrosses at different days of gestation revealed that the expected Mendelian ratio was maintained up to E10.5 for Txnrdl and E13.5 for Txnrd2; resorption of Txnrdl knockout embryos was frequently observed between gestational days 9.5 and 10.5. RTPCR analysis using embryonic mRNA isolated at E9.5 or El3.5 did not yield products with mRNA's from knockout embryos with primer pairs specific for the deleted exons. However, faint products were obtained with primer pairs covering the central regions indicating very low levels of truncated Txnrdl and Txnrd2 messages. Embryonic expression profile of Txnrdl and Txnrd2 To better understand the basis of the null phenotypes of Txnrdl and Txnrd2 knockout embryos, the expression profile of both genes was studied. At E8.5, Txnrdl is expressed throughout the entire embryo with the exception of the primitive heart with the highest levels detected in neuronal tissues such as the developing forebrain and the rhombomeres. At E9.5, Txnrdl expression is confined to the neural tube, the forebrain, branchial arches, somites, and to the limb buds. At E10.5, Txnrdl is present in developing somites, in the apical ectodermal ridge of the limb buds, in the first and second branchial arches, and in the lateral edges of the nasal pit. Thus, Txnrdl shows a complex and dynamic expression pattern in early developmental stages. To study embryonic Txnrd2 expression at E12.5, sections of mice with a conditional lacZ knock-in into the Txnrd2 locus were used. Anti-lacZ immunohistochemistry of hemizygous Txnrd2 lacZ knock-in embryos revealed strong expression in the embryonic heart, especially in the myocardium and atrial walls, and to a lower extent in the embryonic liver. These findings reflect Txnrd2 expression with mRNA data obtained from adult tissues [44], and associate Txnrd2 function with organs characterised by high metabolic activity. This may further corroborate a crucial role for Txnrd2 in the control of harmful intracellular reactive oxygen species. In short, the distinct expression patterns of Txnrdl and Txnrd2, especially the
Chapter 19. function
Selenium, deiodinases and endocrine
Antonio C. Bianco and P. Reed Larsen Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
Summary: The three iodothyronine deiodinases catalyze the initiation (Dl, D2) and termination (D3) of thyroid hormone effects in vertebrates. A 3dimensional model predicts that these enzymes share a similar structural organization and belong to the thioredoxin (TRX) fold superfamily. Their active center is a selenocysteine-containing pocket defined by the pi-al-p2 motifs of the TRX fold and a domain that shares strong similarities with the active site of iduronidase, a member of the clan GH-A fold of glycoside hydrolases. While Dl and D3 are long-lived plasma membrane proteins (tl/2 10-12 h), D2 is an endoplasmic reticulum resident protein that is inactivated by selective conjugation to ubiquitin, a process that is mediated by WSB-1, a Hedgehog-inducible gene. Remarkably, D2 ubiquitination is reversible and activity restored after deubiquitination by the pVHL-interacting deubiquitinating enzymes (VDUl and VDU2). Deiodinases play a major role in development as well as in adults as critical players in thyroid hormone homeostasis, particularly during hypo- and hyperthyroidism. In addition to playing an important part in energy homeostasis, changes in deiodinase activity explain the alterations in thjroid economy observed during illness and in the recently described syndrome of consumptive hypothyroidism. Introduction The three deiodinases, enzymes that activate thyroxine (T4) and inactivate both T4 and T3, are present in all vertebrates. Their relevance resides in the fact that T4 is a long-lived (tl/2 is ~7 days in humans) pro-hormone molecule that must be activated by deiodination to the short-lived biologically active T3 (tl/2 is ~1 day) in order to initiate thyroid hormone action. T3 modulates gene expression in virtually every vertebrate tissue through ligand-dependent transcription factors, the thyroid hormone receptors. The deiodination of T4 to T3 occurs in the phenolic (outer or 5')ring of the T4 molecule and is catalyzed by two iodothyronine deiodinases, i.e. Dl and D2. As a counter point to the activation pathway, T4 activation
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can be prevented and T3 can be irreversibly inactivated by deiodination of the tyrosyl (inner or 5)-ring, a reaction catalyzed by D3 (and Dl). In both experimental animals and humans, the coordinated changes in the expression and activity of these enzymes ensures thyroid hormone homeostasis and the constancy of T3 production, constituting a major mechanism for adaptation to changes in the ingestion of iodine, starvation and changes in environmental temperature (reviewed in [1]). The study of animals with deficiency of Dl (C3H mouse) or targeted disruption of D2 (Dio2''") or D3 (D3'") genes has not only confirmed but revealed new intricacies about the critical role played by these enzymes in thyroid hormone homeostasis [2-5]. The 3D structure of the deiodinases is conserved The three deiodinase proteins (Dl, D2 and D3) show considerable similarity (~ 50 % sequence identity). All are integral membrane proteins of 29-33 kDa, and have regions of high homology in the area surrounding the active center [6-8]. Insights into the structures of these proteins were obtained through protein modeling using hydrophobic cluster analysis (HCA) [9]. Based on the HCA analysis it is clear that the three deiodinases share a common general structure composed of a single trans-membrane segment, which is present in the N-termini of Dl, D2 and D3, and several clusters, typical of a-helices or P-strands, corresponding to core secondary structures of the deiodinase globular domains. A striking common feature is the presence of the thioredoxin (TRX) fold, defined by the paP and ppgt motifs. It is interesting that, within the canonical TRX fold, the relationship between the pap and ppa motifs is locally interrupted by interfering elements. These sequences correspond to distinct secondary structure elements added to the canonical TRX fold core, a feature also observed in other proteins of the TRX fold family [10]. A unique aspect of the deiodinases, however, is that one of these highly conserved intervening elements shares similarities with a-L-iduronidase (IDUA; 47 % identity with Dl and D3, 60% with D2), a lysosomal enzyme that cleaves a-linked iduronic acid residues fi-om the nonreducing end of glycosaminoglycans [11]. The 3D general model of the deiodinases predicts that the active center is a pocket defined by the pi-al-P2 motifs of the TRX-fold and the IDUA-like insertion. A striking feature of this pocket is the rare amino acid selenocysteine (Sec), critical for the deiodination reaction catalyzed by all three deiodinases. This was first identified when the rat Dl cDNA became available, the analysis of which revealed the presence of the Sec encoded by UGA, which is recognized in the vast majority of mRNAs as a STOP codon [12]. However, a specific RNA stem-loop immediately downstream of the UGA codon allows for the Sec incorporation in the STOP codon. This structure is termed the Sec Insertion Sequence, or SECIS element, which is present in the deiodinases and all other selenoproteins [13].
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The ubiquitination pathway inactivates D2 D2 is considered the critical homeostatic T3-generating deiodinase due to its substantial physiological plasticity (see [1] for review). A number of transcriptional and post-translational mechanisms have evolved to ensure limited expression and tight control of D2 levels, which is inherent to its homeostatic function. The D2 mRNA in higher vertebrates is more than 6 kb in length, containing long 5' and 3' untranslated regions (UTRs). The D2 5'UTRs are greater than 600 nucleotides and contain 3-5 short open reading frames (sORFs), which reduce D2 expression by as much as 5-fold [14]. Alternative splicing is another mechanism that regulates D2 level as mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs, but not encoding an active enzyme, are present in both human and chicken tissues. D2 levels can also be regulated by AUUUA instability motifs located in the 3'UTR of D2 mRNA as deletion of 3.7-kb from this region increases D2 activity ~3.8-fold due to an increase in D2 mRNA half-life [14]. D2 activity/mRNA ratios are variable, indicating that there is significant post-translational regulation of D2 expression [15]. In fact, the decisive D2 property that characterizes its homeostatic behavior is a short half-life (-40 min) [16] that can be further reduced by exposure to physiological concentrations of its substrate, T4, and in experimental situations, reverse T3 or even high concentrations of T3 [16-22]. This constitutes a rapid, potent generalized regulatory feedback loop that efficiently controls T3 production and intracellular T3 concentration based on how much T4 is available. Important metabolic pathways often contain key rate-limiting enzymes whose half-lives can be modified by selective proteolysis. This process is mediated by the ubiquitin (Ub)-proteasome system by which target proteins are marked for destruction by conjugation to Ub, a ~8kDa protein. The ubiquitinated proteins are subsequently recognized and degraded by the proteasomes [23,24]. Indeed, ubiquitination and proteasomal degradation are deeply implicated in the post-translational regulation of D2 activity. The first evidence was obtained in GH4C1 cells in which the half-life of the endogenous D2 was noted to be stabilized by MG132, a proteasome inhibitor [25]. Substrate-induced loss of D2 activity was also inhibited by MG132 in such cells, indicating that both pathways affecting loss of D2 activity were mediated by the proteasomes. This implies that the loss of D2 activity is, at least partially, due to proteolysis, a premise that was confirmed after the levels of immunoprecipitable labeled D2 were shown to parallel D2 activity, both under basal conditions and after exposure to substrate [26]. Selection of specific proteins for proteolysis is usually achieved at the level of Ub conjugation, a process that involves recognition of amino acidsequences in the target protein by the ubiquitinating enzymatic machinery. The first step is activation of Ub by ATP, a process catalyzed by the El enzyme. The next step, target recognition, is coordinated by the combined
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actions of a series of Ub-conjugating enzymes (E2s) and Ub-ligases (E3s). Individual E2s are involved in different cellular processes and, therefore, in the ubiquitination of different classes of substrate proteins. In the case of D2, we have identified UBC6 and UBC7, two E2s that specifically assist in the transfer of activated Ub to D2 [27,28]. E3s, on the other hand, are more abundant and with no overt sequence homology, are thought to be largely responsible for the high degree of specificity of protein ubiquitination [24]. Using a yeast-two hybrid system to screen a brain library we identified a novel D2-interacting protein, WSB-1, which is a SOCS-box-containing WD-40 protein of unknown function that is induced by hedgehog signaling in embryonic structures during chicken development [29]. We subsequently showed that WSB-1 acts as an E3 ubiquitin ligase for D2. The WD-40 propeller of WSB-1 recognizes an 18amino acid loop in D2 that confers metabolic instability, while the SOCSbox domain mediates its interaction with an ubiquitinating catalytic core complex, modeled as Elongin BC-Cul5-Rbxl (ECS^^°"'). In the developing tibial growth plate, hedgehog-stimulated D2 ubiquitination via ECS^^^"' induces parathyroid hormone related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS*^^"' mediates a novel mechanism by which "systemic" thyroid hormone can effect local control of the hedgehogPTHrP negative feedback loop and thus skeletogenesis [29]. Using the same yeast two hybrid system that identified WSB-1, we identified D2 as the only known specific substrate of VDUl and VDU2 [30], which in turn are the first ubiquitin-specific processing proteases (UBP) known to specifically deubiquitinate an ERAD substrate. These results show that protein recognition is not only involved in the E3-mediated ubiquitination process but also in the deubiquitination pathway catalyzed by UBPs. Both VDUs are downstream targets for ubiquitination by pVHL E3 ligase, and VHL mutations that disrupt the interaction between the VDUs and pVHL abrogate their ubiquitination [31,32]. Although hundreds of UBP enzymes have been cloned, only a few examples of substrate recognition by UBP enzymes have been reported and, to our knowledge, none are ERresident proteins [33-37]. Confocal studies indicate that both VDUs colocalize with D2, itself an integral membrane ER-resident protein. Although present in the particulate fi-action and not in cytosol, it is not clear, based on their hydrophobic profile, whether VDUl/2 are integral membrane proteins [30]. Their physical colocalization with D2, however, provides the opportunity for catalysis and D2 deubiquitination. Thus, due to the intrinsic inefficiency of the selenoprotein synthesis, the availability of a reversible ubiquitination-dependent mechanism to control the activity of D2 constitutes an advantage that allows for rapid control of thyroid hormone activation. The finding that VDUl and VDU2 are coexpressed with D2 in many human tissues, including brain, heart and skeletal
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muscle [1,31], indicates that the importance of this mechanism may extend well beyond thermal homeostasis to include brain development, cardiac performance, glucose utilization and energy expenditure. Role of deiodinases in thyroid hormone homeostasis T3 can be produced by two different and relatively independent sources, namely the direct product of the thyroid secretion or as a result of extrathyroidal deiodination of T4. The relative contributions of the two sources of T3, thyroid secretion and T4 5' deiodination, can be quantified by determining the T4 to T3 conversion rate, which is on average about 36 % [38]. Hence, with a normal T4 production rate of 110 nmol Id, 40 nmol of T3 are produced by peripheral deiodination of T4 and the remaining 10 nmol are secreted directlyfi-omthe thyroid gland. Extrathyroidal T3 can derive from T4 via two different deiodination pathways, namely Dl or D2. To quantitate the role of Dl in catalyzing the production of plasma T3, it is informative to review the results of two studies performed in patients with primary hypothyroidism receiving fixed doses of exogenous T4 [39,40]. In these patients, administration of PTU (1000 mg/day) caused about a 25 % decrease in serum T3. In a third study, the production of labeled plasma T3 from T4 was not reduced in patients given 1200 mg/day of PTU [41]. Results of these three studies favor the concept that, except for during hyperthyroidism, Dl-catalyzed T3 production is not a major component of exfrathyroidal T3 production in euthyroid humans. In a more recent study, we modeled in vitro the in vivo situation and calculated the rate of T4 to T3 conversion by intact cells fransiently expressing Dl or D2 at low (2 pM), normal (20 pM), and high (200 pM) free T4 concentrations. Deiodinase activities were then assayed in cell sonicates. The ratio of T3 production in cell sonicates (catalytic efficiency) was multiplied by the tissue activities reported in human liver (Dl) and skeletal muscle (D2). From these calculations, we predicted that in euthyroid humans, D2-generated T3 is 29 nmol/d, while that of Dl-generated T3 is 15 nmol/d, from these major deiodinase-expressing tissues. The total estimated extrathyroidal T3 production, 44 nmol/d, is in close agreement with the 40 nmol T3/d based on previous kinetic studies. D2-generated T3 production accounts for approximately 71% of the peripheral T3 production in hypothyroidism, but Dl for approximately 67% in thyrotoxic patients [42]. Deiodinases mediate tissue-specific control of thyroid hormone action Given the generalized metabolic sensitivity to thyroid hormone documented during hypo- and hyperthyroidism, one would anticipate a major physiological role of this hormone in energy homeostasis. However, serum T3 concentration is remarkably constant, thus precluding a major role of T3 in the basal metabolic rate (BMR) variations observed after a meal or during
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sleep. In the last 20 years, light was shed on this problem by studies demonstrating that in some tissues the cellular actions of thyroid hormone are determined not only by serum T3. Thyroid hormone action is initiated through its binding to nuclear receptors, which are high-affinity nuclear T3 binding proteins that regulate transcription of T3-dependent genes. Receptor occupancy is determined by the affinity of the receptor for T3 and the T3 concentration in the nucleus. These values are such that, at normal serum T3 concentration, the contributionfi^omserum T3 alone results in an approximately 50% saturation of thyroid hormone receptors in most tissues. However, tissues expressing D2 have an additional source of T3 contributed by the conversion of intracellular thyroxine (T4) to T3 [43,44]. As a result, receptor saturation can reach as high as 100 %, with more than half of this T3 produced locally [4547]. While we still do not understand all the intricacies of this system, we do know that generation of T3 by D2 occurs in the perinuclear region, a cellular compartment with preferential access to the nucleus. This is in contrast to Dl, which is localized to the plasma membrane, from which the T3 produced more readily enters the plasma [48]. Thus, for cells lacking D2, intracellular thyroid status is determined predominantly by the serum T3 concentration. In contrast, cells expressing D2 have the ability to generate intracellular T3 fi-om T4. Thus, cells expressing D2 have two potential sources of nuclear T3: plasma T3, or T3(T3), and T3(T4). On the other hand, D3 is localized to the plasma membrane and to undergo recycling in the endosomes. Remarkably, D3 expression causes cell hypothyroidism as a result of its inactivating effect on thyroid hormone [49], thus creating a virtual barrier that prevents thyroid hormone from entering the cell nucleus. Thus, despite steady serum T3 levels, intracellular thyroid status varies along a wide range according to the type and level of deiodinase expression. Thyroid hormone receptor saturation is expected to be minimum in cells expressing D3 and maximum in cells expressing D2. In addition, because of the plasticity of deiodinase expression, particularly that of D2, receptor saturation of a single cell type might change rapidly and dramatically without affecting serum T3. Thus, deiodinase expression in metabolically active tissues is a potent mechanism by which energy dissipation can be controlled. Significance of D2 to adaptive tliermogenesis in Iiumans The potential role of D2 in human energy homeostasis has been ignored because human newborns grow less dependent on BAT thermogenesis, and adult humans, unlike small mammals, do not have substantial amounts of BAT [50]. However, since the cloning of the human D2 cDNA and the finding of cAMP-inducible D2 mRNA and activity in human skeletal muscle [51,52], the role of D2 in controlling human adaptive thermogenesis has been
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revisited. Thyroid hormone per se is known to increase energy expenditure in skeletal muscle (see [53] for review) and could also regulate local energy homeostasis through its interaction with the SNS. Accordingly, human skeletal muscle is under the influence of the thyroid-adrenergic synergism and an increase in local cAMP production is known to activate glycolytic enzymes, sarcolemmal Na^/K^ pumps, phospholamban, and voltage-sensitive and sarcolemmal Ca^^ channels [54,55], resulting in increased glucose uptake and utilization [56,57]. The expression of GLUT4, the insulinresponsive-glucose transporter that mediates glucose metabolism in skeletal muscle, is also up-regulated by thyroid hormone [58]. Various studies support a previously unrecognized role of D2 in determining the thyroid status and metabolic rate of the skeletal muscle, analogous to its role in BAT. Earlier experimental studies of humans [59] have consistently found diet-induced changes in serum thyroid hormones that could be explained by changes in D2 activity. As an example, the increase in BMR observed in subjects fed a high carbohydrate diet is typically associated with an increase in the serum T3/T4 ratio [59], a condition that is also observed in adult subjects chronically treated with terbutaline, a Padrenergic receptor (P-AR) stimulator [60]. This indicates the existence of a relevant cAMP-dependent T4-to-T3 conversion pathway in humans that plays a role in energy homeostasis. That this pathway is predominantly through D2 is supported by the finding that the D2 gene is up-regulated several fold by adrenergic stimulators and cAMP [61]. Studies of patients receiving T4 replacement at various dosages have shown a direct correlation of the BMR with free T4 and inversely with serum TSH but not with serum T3 [62]. Together, these data indicate that D2-produced T3 in skeletal muscle might be a significant determinant of energy expenditure in humans. Recent studies describing a Dio2 polymorphism in which a threonine (Thr) change to alanine (Ala) at codon 92 (D2 Thr92Ala) provides is additional support to a role of D2 in glucose uptake and utilization. Of note, in humans, skeletal muscle is the primary site of insulin-dependent glucose disposal [63]. Remarkably, this Dio2 polymorphism was associated with an -20 % lower rate of glucose disposal in obese women than in non-obese women [64]. In addition, the fi-equency of the variant allele was found to be increased in some ethnic groups, such as Pima Indians and MexicanAmericans, with a higher prevalence of insulin resistance [64]. The possible role of the D2 Thr92Ala polymorphism on insulin resistance was also investigated in patients with type 2 diabetes Mellitus (DM2). Studies of these patient offers a practical approach to the investigation of energy expenditure because they require intense metabolic monitoring and are subjected to a detailed scrutiny of fuel utilization. In accordance with the previous study in obese individuals, individuals homozygous for the variant allele have an increased insulin-resistance index as assessed by the HOMA
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(homeostasis model assessment) index. The increased insulin resistance observed in the DM2 patients homozygous for the Ala allele could be explained by a decrease in D2 activity, as has been found in thyroid and skeletal muscle samples from individuals with this genotype [65]. A lower D2 activity would decrease D2-generated T3 in skeletal muscle and create a state of relative intracellular hypothyroidism, decreasing the expression of genes involved in energy utilization, such as GLUT4, leading to insulin resistance. Supporting this hypothesis is the remarkable finding that the UCPl knock-out mouse develops, as a compensatory mechanism, increased D2 activity in white adipose tissue [66], stressing the importance of understanding the D2-generated T3-dependent thermogenic mechanisms. Changes in iodothyronine deiodination during fasting or illness It has been recognized for decades that there are significant changes in the concentrations of circulating thyroid hormones during illness or starvation in human plasma. Despite numerous studies, there remains much controversy regarding both the precise etiology of these changes and what, if anything, should be done therapeutically regarding them [67-69]. The hallmark of these changes is a decrease in circulating free T3 and an increase in total reverse T3, although there is not complete agreement even on these most basic changes [70-72]. The similarity of the changes in illness to those of fasting or caloric deprivation suggest that the decrease in thyroid hormone activation is a beneficial physiological response designed to reduce metabolic rate and conserve energy during stress [73]. In patients postcoronary artery bypass grafting, however, there is disagreement about the effectiveness of T3 supplementation with one study showing a positive effect [74,75]. The changes in circulating thyroid hormones and TSH during illness are a continuum with progressively more abnormalities with more severe illness. Patients with mild illnesses, such as after uncomplicated surgery, or who are fasting, generally have a reduction of up to 50% in circulating T3, a reciprocal increase in serum reverse T3 and no changes in serum T4 or TSH [72,76]. With moderately severe illness, the clearance of T4 is slowed while T4 secretion persists, leading to increases in free T4 accompanied by further decreases in serum T3 and increases in reverse T3. When the abnormalities in T3 and reverse T3 were first described, the initial assumption was that these reciprocal changes reflected diversion from T4 activation to inactivation. This raised the possibility that the changes could be attributed to an alteration specificity of Dl from 5' to 5 T4 deiodination since this is the only deiodinase with the capacity to catalyze both outer and inner ring deiodination of T4. Subsequent studies indicated that the elevation in reverse T3 was due to a reduction in the clearance of this T4 byproduct and its production rate is unchanged as long as T4 remains
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normal [76]. This indicates that the tissues in which reverse T3 is produced from T4, largely by the action of D3, are processing T4 normally during illness or fasting. On the other hand, since the principal pathway for reverse T3 clearance is via Dl, these results indicate either that the Dl enzyme or its co-factor is reduced, or that the uptake of reverse T3 into Dl-expressing tissues is impaired [77]. Decreased transport of reverse T3 into the Dlcontaining liver or kidney during fasting or illness has been attributed to either ATP depletion or interference with reverse T3 transport by competing substances circulating in the plasma [67]. In moderate to severe illness, the serum T3 can fall to 20-30 % of baseline. About 20% of T3 in human plasma derives from the thyroid with the T3 derived from extrathyroidal Dl and D2 catalyzed T4 5' monodeiodination accounting for about 25 and 55% of the plasma T3, respectively. Since TSH, and therefore T3 secretion, is not suppressed unless illness is prolonged and/or severe, the severely reduced T3 in ill patients is due to decreased peripheral T4 deiodination by Dl, D2 or both. The fact that the fall in T3 substantially exceeds what we can currently assign to Dl (~30 %) suggests that T3 generation by D2 must also be inhibited. With respect to Dlcontaining tissues, T4 uptake into the rapidly equilibrating pool, primarily liver and kidney, is significantly reduced in obese patients on a 240 kcal diet and similar observations have been made in uremia [78]. This can explain the decrease in T3 production via Dl and, again, either inhibition of T4 transport by unknown circulating compounds or by ATP depletion could be to blame [67]. In addition, entry of T4 into the slowly equilibrating pool, likely to be the one in which D2-catalyzed T3 production occurs, is also reduced in obese patients on a hypocaloric diet [78]. In addition to reduced T4 transfer, a second important consideration with respect to D2-catalyzed T4 to T3 conversion is the rapid proteolysis of D2 through the ubiquitin-proteasome pathway. Thus, persistent D2 synthesis is required to maintain D2 levels normal. Protein synthesis is impaired during fasting or in severe illness. It is tempting to speculate that a rapid fall in D2 protein can explain the abrupt decrease in plasma T3 associated with these conditions. The possibility that D3 action is also increased during illness has also been considered. In a study that determined serum thyroid hormone levels and the expression of Dl, D2, and D3 in liver and skeletal muscle from deceased intensive care patients, liver Dl was down-regulated and D3 was induced in liver and skeletal muscle [79]. Dl and D3 mRNA levels corresponded with enzyme activities, suggesting regulation of the expression of both deiodinases at the pre-translational level. Liver Dl was down-regulated and D3 (which is not present in liver and skeletal muscle of healthy individuals) was induced, particularly in disease states associated with poor tissue perfusion. These observations may represent tissue-specific ways to reduce thyroid hormone bioactivity during illness [79].
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D3 overexpression in hemangiomas D3 activity in the normal uteroplacental unit regulates the transfer of maternal thyroid hormone to the fetus [80]. D3 is expressed in multiple fetal structures, but the endometrium and the placenta are the only normal tissues known to express high levels of D3 activity in the mature human. D3 has also been found in vascular anomalies, in human brain tumors, and in some malignant cell lines. These studies have led to the categorization of D3 as an oncofetal protein, but recent data indicate that postnatal expression can be reactivated in normal tissues during critical illness [79]. D3 expression at high levels occurs in infantile hemangiomas [81]. If these tumors are sufficiently large, the rate of thyroid hormone inactivation can exceed the maximal rate of thyroid hormone synthesis. The first patient documented with this condition was 3 months old, presenting with severe hypothyroidism with an elevation in serum TSH, undetectable serum T4 and T3 concentrations and high reverse T3 and thyroglobulin. The relationship between infantile hemangiomas and D3 expression is especially significant since it identifies a previously unrecognized cause of hypothyroidism, which usually occurs at a critical age for neurological development. While extensive hepatic hemangiomas can be fatal, a significant fraction of these infants survive due to therapy and the natural tendency of these tumors to regress. Accordingly, these patients may require replacement with large quantities of thyroid hormone in addition to therapy directed at their hemangiomas. Thyroid hormone treatment is also imperative to prevent the complication of irreversible mental retardation later in life. Hemangiomas produce high quantities of basic fibroblast growth factor, which has been shown to activate the expression of D3 in rat glial cells via ERK activation [82]. It seems likely that this is one mechanism for the high D3 expression in these tumors. Using nontransformed human cells, it has been shown that TGF-beta stimulates transcription of the hDio3 gene via a Smad-dependent pathway. Combinations of Smad2 or Smad3 with Smad4 stimulate hDio3 gene transcription only in cells that express endogenous D3 activity, indicating that Smads are necessary but not sufficient for D3 induction. TGF-beta induces endogenous D3 in human cell types such as fetal and adult fibroblasts from several tissues, hemangioma cells, fetal epithelia, and skeletal myoblasts. Maximum stimulation of D3 by TGF-beta also requires MAPK and is synergistic with phorbol ester and several mitogens known to signal through transmembrane receptor tyrosine kinases but not with estradiol. These data reveal a previously unrecognized interaction between two pluripotent systems, TGF-beta and thyroid hormone, both of which have major roles in the regulation of cell growth and differentiation [83].
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Dl overexpression contributes to the relative excess of T3 production in hyperthyroidism It has been well established that the production rate of T3 and its circulating concentration is about 2-fold higher relative to that of T4 in hyperthyroid patients [84]. This is reflected in the markedly greater elevation in free T3 than in free T4 in such patients. Since the human Diol promoter is T3responsive, one would anticipate that Dl activity or mRNA would be significantly increased in hyperthyroid patients. This has been demonstrated in Graves' thyroid tissue and in mononuclear leukocytes of patients with Graves' disease [85-87]. It would be expected that PTU, a drug that blocks Dl, but not D2 activity, would have a greater effect on plasma T3 production in thyrotoxic than euthyroid individuals in whom Dl activity should be increased and D2 activity reduced. That PTU inhibits T4 to T3 conversion was demonsfrated in a series of patients comparing the acute changes in serum T3 between Graves' patients freated with a combination of iodide and PTU with a group of similar patients treated with methimazole and iodide [84]. These results indicate that a PTU-inhibitable process, Dl-catalyzed T4 to T3 conversion, is more active in the hyperthyroid than the euthyroid subject in which PTU causes 0-25 % decrease in T3. This has led to the recommendation that large doses of PTU or other agents which block T4 to T3 conversion, such as iopanoic or ipodipic acid, be used in the acute treatment of the severely hyperthyroid individual [88-91]. A paradoxical observation in the Graves' thyroid is that thyroidal D2 mRNA and activity is increased despite systemic thyrotoxicosis [92]. This is due to the effect of the thyroid immunostimulator to activate the cAMPdependent hDio2 promoter, which must overwhelm the negative transcriptional effect of T3 on hDio2. Furthermore, the presence of D2 activity in the Graves' or TSH-stimulated human thyroid raises the possibihty that a portion of the excess T3 secretion in Graves' disease results from intrathyroidal T4 to T3 conversion catalyzed by D2 [92]. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
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MJ Berry, L Banu, PR Larsen 1991 Nature 349:438 MJ Berry et al 1991 Nature 353:273 B Gereben, A KoUar, JW Harney, PR Larsen 2002 Mol Endocrinol 16:1667 Burmeister, LA, Pachucki, J, DL St Germain 1997 Endocrinology 138:5231 DL St Germain 1988 Endocrinology 122:1860 JL Leonard, MM Kaplan, TJ Visser, JE Silva, PR Larsen 1981 Science 214:571 RJ Koenig et al 1984 Endocrinology 115:324 JE Silva, JL Leonard 1985 Endocrinology 116:1627 Y Halperin, LE Shapiro, MI Surks 1994 Endocrinology 135:1464 JL Leonard, et al 1984 Endocrinology 114:998 MJ Obregon, PR Larsen, JE Silva 1986 Endocrinology 119:2186 O Coux, K Tanaka, AL Goldberg 1996 Annu Rev Biochem 65:801 A Hershko, A Ciechanover 1998 Annu Rev Biochem 67:425 J Steinsapir, JW Harney, PR Larsen \99S J Clin Invest 102:1895 J Steinsapir et al 2000 £nrfocnno/ogy 141:1127 BW Kim et al 2003 Mol Endocrinol 17:2603 D Botero et al 2002 Mol Endocrinol 16:1999 M Dentice et al 2003 J Clin Invest 112:189 Z Li, X Na, D Wang, SR Schoen, EM Messing, G Wu 2002 J Biol Client 277:4656 Z Li et al 2002 Biochem Biophys Res Commun 294:700 S Taya, T Yamamoto, M Kanai-Azuma, SA Wood, K Kaibuchi 1999 Genes Cells 4:757 N Gnesutta et al 2001 J Biol Chem 276:39448 S Taya et al 199% J Cell Biol 142:1053 X Chen, B Zhang, JA Fischer 2002 Genes Dev 16:289 H Ideguchi et al 2002 Biochem J i6T.87 PR Larsen, TF Davies, ID Hay 1998 In Williams Textbook of Endocrinology JD Wilson, DW Foster, HM Kronenberg, PR Larsen, editors Philadelphia: WB Saunders Co 389-515 DL Geffher, M Azukizawa, JM Hershman 1915 J Clin Invest 55:224 M Saberi, FH Sterling, RD Utiger 1975 J Clin Invest 55:218 JS LoPresti et al 1989 J Clin Invest 84:1650 AL Maia, BW Kim, SA Huang, JW Harney, PR Larsen 2005 J Clin Invest 115:2524 JE Silva, PR Larsen 1977 Science 198:617 JE Silva, JL Leonard, FR Crantz, PR Larsen 1982 y Clin Invest 69:1176 JE Silva, TE Dick, PR Larsen 1978 Endocrinology 103:1196 JE Silva, PR Larsen 1978 7 Clin Invest 61:1247 AC Bianco, JE Silva 1987 Endocrinology 120:55 MM Baqui et al 2000 Endocrinology 141:4309 FW Wassen et al 2002 Endocrinology 143:2812 K Bruck, 1998 In Fetal neonatal physiology RA Polin, WW Fox (eds) Philadelphia WB Saunders Co 676 D Salvatore, T Bartha, JW Harney, PR Larsen 1996 Endocrinology 137:3308 W Croteau et al 1996 J Clin Invest 98:405 L de Meis 2001 fi/osci/Jep 21:113 YT Yang, MA McElligott 1989 Biochem J26l:\ SJ Roberts, RJ Summers 1998 Eur J Pharmacol 348:53 FD McCarter et al 2001 J Surg Res 99:235 LG Fryer et al 2002 Biochem J 363:167 O Ezaki 1997 Biochem Biophys Res Commun 241:1 JE Danforth et al 1979 J Clin Invest 64:1336 K Scheidegger et al 1984 J Clin Endocrinol Metab 58:895 T Bartha et al 2000 £Wocnno/ogv 141:229 H al-Adsani, LJ Hoffer, JE Silva 1997 J Clin Endocrinol Metab 82:1118
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E Ferrannini et al 1999 Eur J Clin Invest 29:842 D Mentuccia et al 2002 Diabetes 51:880 LH Canani et al 2005 J Clin Endocrinol Metab 90:3472 X Liu et al 2003 J Clin Invest 111:399 R Docter et al 1993 Clin Endocrinol 39:499 B Mclver, CA Gorman 1997 Tliyroid 7:125 LJ De Groot 1999 J Clin Endocrinol Metab 84:151 J Faber, K Siersbaek-Nielsen 1996 Clin Chim Acta 256:115 IJ Chopra 1998 Uyroid 8:249 MM Kaplan et al 1982 Am J Med 72:9 DF Gardner, MM Kaplan, CA Stanley et al. 1979 N Engl J Med 300:579 JD Klemperer et al 1995 N EnglJ Med 333:1522 E Bennett-Guerrero et al 1996 JAMA 275:687 Z Eisenstein et al \978 J Clin Endocrinol Metab 47:889 EM Kaptein et al 1983 7 Clin Endocrinol Metab 57:181 JTM van der Heyden et al \9&6 Am J Physiol 25\:E156 RP Peelers et al 2003 J Clin Endocrinol Metab 88:3202 SA Huang 2005 Thyroid 15:875 SA Huang et al 2000 N EnglJ Med 343:185 S Pallud et al 1999 Endocrinology 140:2917 SA Huang et al 2005 Mol Endocrinol 19:3126 J Abuid, PR Larsen 1974 J Clin Invest 54:201 H Ishii et al 1981 7 Clin Endocrinol Metab 52:1211 M Sugawara et al 1984 Metabolism 33:332 M Nishikawa et al 1998 Biochem Biophys Res Commun 250:642 SY Wu, TP Shyh, I Chopra et al 1982 J Clin Endocrinol Metab 54:630 SY Wu et al 1982 J Clin Endocrinol Metab 54:630 H Burgi et al 1976 J Clin Endocrinol Metab 43:1203 MS Croxson, TD Hall, JT Nicoloff 1977 J Clin Endocrinol Metab 45:623 D Salvatore, H Tu, JW Harney, PR Larsen 1996 J Clin Invest 98:962
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Chapter 20. Biotechnology of selenocysteine Linda Johansson and Elias S. J. Amer Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden
Summary: In this chapter we describe strategies to produce synthetic selenoproteins, with a focus on recombinant selenoprotein production in E. coli. We further discuss the possible use of selenocysteine (Sec) in proteins for biotechnological applications. Such applications are based upon either the introduction of a selenium isotope, with specific characteristics such as highenergy radioactivity (as for ^'Se and ^^Se) or nuclear spin (^^Se), or on the high reactivity of the nucleophilic Sec residue enabling site-specific conjugation-based applications with electrophilic ligands. Utilization of Sec insertion for protein purification or detection purposes has recently been demonstrated in a number of different experimental systems and we envision that further such applications will be developed in the near future. Introduction Production of synthetic selenoproteins, although far fi-om trivial, is possible by utilizing various chemical or genetic approaches. Such production may be highly useful in enabling detailed studies of selenoproteins in general. Features of selenocysteine (Sec) not shared by any of the other 20 common amino acids, such as the selenium atom chemistry, isotope abundance and high nucleophilicity, make it tempting to introduce Sec into nonselenoproteins for use in Sec-targeted biotechnological applications. For that purpose, we developed a tetrapeptide motif for recombinant proteins expressed in E. coli containing a Sec-residue, named a Sel-tag [1], which will be described at the end of this chapter. Methods to obtain selenoproteins and numerous theoretically possible applications based upon the presence of a Sec residue in protein are summarized in Figure 1, illustrating the true biotechnological potential unleashed by the use of Sec. Production of selenoproteins In order to use Sec in proteins for Sec-based biotechnological applications, the first step is, naturally, to obtain the selenoprotein to serve as the basis for such applications. Purifying native selenoproteins fi-om natural sources (Step A, Figure 1) is ofl;en laborious, time-consuming and results in low product
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yields. Transfection of eukaryotic cells for eukaryotic selenoprotein overproduction is an alternative, but has hitherto also resulted in low yields [2,3]. One technique for producing selenoproteins is to synthetically incorporate a Sec residue into a target protein using chemical substitution reactions (Step B, Figure 1). Different methods for chemical synthesis of selenoproteins have been described, involving either native chemical ligation or expressed protein ligation [4-7]. In general, those methods utilize chemical synthesis of free Sec, chemical synthesis of peptides containing the Sec residue and, finally, ligation of the selenopeptide with a target protein. The reactivity of free Sec makes these methods chemically demanding and they require many steps in the synthesis of Sec-containing polypeptides. Nonetheless, it is likely that both protein ligation methods and recombinant selenoprotein production in E. coli (described next) will become useful techniques for the production of selenoproteins, the choice of which will be dictated by the requirements of each specific application.
Overproducing ducing systems
Natural sources |
Chemical synthesis of selenoproteins
Purification of native selenoproteins
^
Recombinant synthetic selenoprotein production
• /
Radical trapping in EPR studies
Labeling w. selenium radionuclides
Directed folding bydiseienide formation NMR studies
Sec-targeted fluorescence labeling Sec-targeted labeling w. §-emitters
PET studies
Phase determination in X-ray crystallography Sec as source for dehydroalanlne for DHA-targeting with nucleophlllc agents Sec-targeted chemical ligation with electrophlles
Figure 1. Strategies to obtain selenoproteins (A-C) and the different possible Sec-based applications discussed in this chapter [1-11].
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In 1992, it was shown for the first time that E. coli had the capacity to overexpress recombinant selenoproteins in a study involving bacterial formate dehydrogenase H [8]. However, the direct expression of heterologous recombinant proteins in E. coli was not considered possible, due to the fact that most selenoprotein genes would not be compatible with the bacterial Sec incorporation machinery (see Chapter 2 on prokaryotic selenoprotein synthesis machinery). One possibility to circumvent this problem for incorporating Sec into a recombinant protein expressed in E. coli is to use a cysteine (Cys) auxotrophic strain, which allows substitution of Cys with Sec by adding a selenium source to the sulfur-deficient growth medium [9]. This method is, however, not suitable if the protein of interest contains several Cys residues and the aim is to substitute only one residue. In 1999, we succeeded in the site-specific incorporation of Sec into mammalian selenoprotein thioredoxin reductase (TrxR) in high yields in E. coli (Step C, Figure 1) [10]. The open reading frame of rat TrxR was fused with an engineered variant of the bacterial SECIS element, enabling targeted co-translational Sec-incorporation using the bacterial selenoprotein synthesis machinery. This was possible to achieve with a maintained TrxR amino acid sequence due to a penultimate position of the Sec residue in TrxR. That allowed engineering of a functional SelB binding motif of the SECIS element to be positioned outside of the open reading fi-ame, thereby not interfering with the encoded amino acid sequence of the expressed gene. To achieve this a termination codon (TAA, or UAA in the mRNA) was inserted in the lower stem of the SECIS element downstream of the Sec-encoding TGA [10]. That approach proved to be a successful strategy and furthermore showed that the entire bacterial SECIS element did not need to be translated to be functional. We also showed that co-overexpression of the selA, selB, and selC genes, which are key components of the bacterial Sec-incorporation machinery, resulted in even higher selenoprotein yields. We achieved a production yield of 20 mg TrxR per liter of bacterial culture having approximately 25% of the native enzyme activity [80]. We have later shown that the specific activity of the recombinantly produced TrxR directly correlated to the ratio of full-length and UGA-truncated proteins [11]. The efficiency of Sec insertion in E. coli is known to be dependent upon several factors. Because the SelB elongation factor must form a quaternary complex with GTP, selenocystenyl-tRNA^^*^ and the SECIS-element, the stoichiometry between these factors is of major importance [12]. Furthermore, there is a competition between the SelB elongation factor and the bacterial release factor 2 (RF2, the prfB gene product) terminating translation at UGA codons [13]. By assessing different production conditions for the synthesis of mammalian TrxR in E. coli, we found that when expression was induced in late exponential phase, an unexpectedly large upregulation of the Sec incorporation efficiency increased total yield to 40 mg TrxR per liter of
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Selenium: Its molecular biology and role in human health
bacterial culture and to about 50% Sec content [11]. We suggested that this may have been due to a more efficient SelB function in comparison to RF2 in eariy stationary phase, but this proposition has not yet been experimentally verified. The Sec-containing full-length TrxR can easily be obtained in a pure form by a single-step purification over phenylarsine oxide (PAO) sepharose [1], with a final yield of about 15-20 mg selenoprotein obtained from a liter of bacterial culture. Production of recombinant selenoproteins in E. coli that carry an internal Sec residue requires engineering of a bacterial-type SECIS element within the open reading frame of the recombinant selenoprotein gene. In most cases, this necessitates introduction of point mutations in the protein to have the mRNA compatible with a functional SECIS element. In essence, the sequence of four to seven amino acid residues starting four positions downstream of the Sec must be restricted in such approach for selenoprotein production [10,14]. In spite of these limitations, the strategy has been used for production of a GPx variant [15], a methionine sulfoxide reductase B [16,17] and a Sec-containing glutathione S-transferase [18]. The results firom these studies show, in spite of the limitations of the technique, that recombinant production of selenoproteins with internal Sec-residues in E. coli is technically possible and may indeed become useful for certain applications. Recently an intriguing possibiUty was presented by Gladyshev and coworkers, showing that a distant SECIS element present in the 3'untranslated region can guide Sec-incorporation in bacteria as long as the SECIS element is structurally close to the UGA codon, albeit the efficiency was very low [19]. For further details on the general principle of expressing recombinant selenoproteins in E. coli see earlier reviews on the subject [14,20]. Applications based upon radiolabeling or detection of the selenium atom The Sec-residue can be utilized for introduction of selenium radionuclides, resulting in residue-specific biologically controlled radiolabeling of selenoproteins (Appl. 1, Figure 1). For recombinant selenoproteins expressed in E. coli this is achieved simply by adding the radionuclide to the bacterial growth medium. Provided that excess Cys is added to block nonspecific incorporation into Cys or Met residues, this results in a highly residuespecific labeling of the Sec moiety [21]. The selenium radionuclide most commonly used for this purpose is the gamma emitter ^^Se, which is currently commercially available in the form of [^^Se]selenite with high specific radioactivity (~2500 mCi/g) from the Research Reactor Center, University of Missouri-Columbia, USA. The ^^Se isotope has a high energy (0.86 MeV) and a half-life of 120 days and it is suitable for analysis by autoradiography, liquid scintillation or detection with gamma counters. Consequently, selenoproteins with ^^Se-labeled Sec can serve the basis for
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many different applications in basic science based upon detection of radioactive proteins, such as metabolic tracking and turnover studies or as radiolabeled antigens in Radioimmuno Assays. Detection of incorporated ^^Se also constitutes a useful validation method for demonstration of successful expression of recombinant selenoproteins mE. coli [10,16,18]. Another selenium radionuclide with potential biomedical importance is the positron emitter ^^Se (ti/2=7.1 hour), which can be produced in good yields [22] and which has been postulated to be suitable for use in positron emission tomography (PET) studies in humans [23]. PET is a non-invasive clinical method for detection of trace amounts of compounds labeled with positron emitters, which can be used to localize and quantify positron decays over time and thereby enables studies of biochemical and physiological processes in real-time in humans. The clinically used positron emitters exhibit short half-times and thus it is important to have fast labeling techniques of the ligand to be employed. For production of synthetic selenoproteins labeled with ^^Se, recombinant production in E. coli would likely be too slow. Possibly chemical synthesis of ^^Se-labeled selenoprotein ligands could be developed. A different approach to produce positron emitting protein ligands for PET is, however, to use the reactivity of a Sec residue for a Sec-targeted reaction with electrophilic agents containing positron emitting radionuclides. There are also naturally occurring stable selenium isotopes of biotechnological importance, e.g., the ^^Se isotope having a nuclear spin of 1/2, which makes it possible to be used for high-resolution NMR spectroscopy (Appl. 9, Figure 1) (reviewed in [24]). ''^Se has therefore been introduced into selenomethionine (SeMet) residues and subsequently used for NMR determinations of proteins expressed in E. coli [25]. A ^^Se-GPX could also be enriched from lamb having been fed a 5 months diet with ^^Se and the protein was then used for NMR studies [26]. Recently, synthetic methods to produce L-[^^Se]-Sec have been developed, which could expand the further use of ^^Se-labeled selenoproteins for NMR determinations [27]. The selenium atom can also be utilized in X-ray crystallography (Appl. 8, Figure 1), and SeMet introduction into proteins for multiwavelength anomalous diffraction (MAD) to solve the phasing problem is a well established method [28]. The development of methods for introducing Sec residues into proteins has recently been used for double labeling techniques infroducing both SeMet and Sec [29], or Sec alone [30], for further facilitated phase determinations. The different methods to produce synthetic selenoproteins as discussed above should hence be possible to use as an aid for protein structure determinations with X-ray crystallography.
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Selenium: Its molecular biology and role in human health
Applications based on tlie cliemical reactivity of Sec A Sec residue exhibits quite different properties compared to its sulfur containing analogue, Cys [31]. The most obvious difference is that the pKa for Sec is much lower than for Cys (5.2 vs. 8.3) [32]. Consequently, at physiological pH the selenol of Sec is mainly in its anionic selenolate form, while the thiol of a Cys residue is typically protonated, making Sec significantly more reactive than Cys. Sec is also a stronger nucleophile than Cys. The majority of the hitherto characterized selenoproteins are enzymes, where the Sec residue is essential for the catalytic activity since its reactivity is generally employed in the catalysis. An illustration of the qualitative differences between Sec-containing oxidoreductases and their Cyscontaining counterparts is the significantly lower activity of the latter [33,34], although changes in an active site microenvironment may activate Cys residues, in certain cases, to approach the reactivity of Sec [35]. The high reactivity of Sec can form the basis for numerous technological applications. By studying synthetic Cys- or Sec-containing peptides, the redox potentials of the Sec-Sec, Sec-Cys and Cys-Cys couples have been determined at pH 7 to -381 mV, -326 mV and -180 mV, respectively [36]. These redox potentials result in a preferential diselenide formation if two Sec residues are present among additional cysteines and it was therefore postulated that a pair of Sec residues could be introduced in place of two Cys residues in a protein, to allow for targeted diselenide generation and thereby directed correct folding of a protein [36] (Appl. 10, Figure 1). This has indeed been demonstrated with the synthetically produced endothelin-1 peptide where the introduction of two Sec-residues in place of Cys directed the correct oxidative folding of the peptide, although it contained two additional Cys residues [37]. Alternatively, for studies of folding intermediates or transition states during catalysis of redox reactions, introduction of a single Sec residue at specific sites in proteins or enzymes could be utilized for the trapping of otherwise transient disulfides or thiolate-targeted intermediates. Electron paramagnetic resonance (EPR) spin trapping [38] is a technique for the direct detection of radical species and could be used to detect formation of selenenyl radical formation in a selenoprotein (Appl. 11, Figure 1). By introducing a Sec residue into an enzyme, it may perhaps be possible to change the specificity or function of the enzyme, thus constituting a potential for a directed evolution of enzymatic activity (Appl. 5, Figure 1). This has indeed been demonstrated by the production of the artificial selenoenzyme selenosubtilisin, where the change of an active site Ser residue to a Sec residue in the serine protease subtilisin, resulted in conversion of the enzyme to a glutathione peroxidase mimic with peroxidase activity [39,40]. A similar approach has been reported in a study that changed a Ser to a Sec in a monoclonal antibody resulting in GPX activity [41,42], although in that case
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it is not clear why the targeted Ser residue should have been extraordinarily active as compared to other serine residues in the protein. A Cys residue in the active site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), when changed to a Sec residue, also revealed a novel peroxidase activity [43]. It is clear that not all cases of Sec introduction into the active sites of enzymes must yield peroxidase activity, e.g., as shown in the production of a Sec-containing variant of GST [18]. However, another GST isoform that was substituted with Sec indeed seemed to possess novel peroxidase activity [44]. Considering the different types of reactions catalyzed by native selenoproteins it is possible that production of synthetic selenoproteins may also be utilized for engineering of other types of reaction catalysts than those supporting peroxidase reactions, such as halogen transfer reactions (cf, the thyroid hormone deodinases). It was reported that the selenium atom can be lost from Sec during purification of selenoproteins, forming dehydroalanine [45]. This type of conversion can also be catalyzed deliberately by a chemoselective oxidation of Sec. The thereby formed electrophilic dehydroalanine moiety can then be used as precursor for peptide conjugation reactions with nucleophilic ligands (Appl.7,Figurel)[4]. Conjugation-based applications The nucleophilic properties of a Sec residue can be utilized for selective selenolate-targeting using electrophilic compounds (Appl. 2,3,6, Figure 1). For instance, there are many commercially available electrophilic thiolate-, and thereby selenolate-reactive probes, which can be used for fluorescence labeling preferentially at a Sec residue. A Sec-specific reaction that avoids targeting of less reactive Cys residues becomes possible when using short reaction times and low pH (described in more detail below in the discussion of Sel-tagged based applications). Analogously, it is possible to use electrophilic iodoacetamido-biotin reagents for specific Sec-labeling and thereby subsequent use of anti-biotin antibodies for detection [46]. We also recently demonstrated the use of electrophilic compounds containing short-lived positron emitters, such as "C, for labeling of Sec residues [1]. This is promising for use in the field of PET studies and is discussed further in the next section describing applications based upon a Sel-tag. Applications based on a Sel-tag We have recently developed a Sel-tag, which is a Sec-containing multifunctional tetrapeptide motif for recombinant proteins expressed in E. coli [1]. By fusing the C-terminal tetrapeptide of TrxR, -Gly-Cys-Sec-Gly, as a Sel-tag for non-selenoproteins we could take advantage of selenium biochemistry, which could, in theory, be utilized for all Sec-based
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applications discussed above. In addition, this motif, corresponding to the four last amino acids of the natural mammalian selenoprotein TrxR [47-49], is redox active. When reduced, the Sec residue becomes easily targeted by electrophilic compounds, but when oxidized it forms a selenenylsulfide with its neighboring Cys residue and thereby becomes essentially inert to alkylating agents [50]. Thus, due to presence of the Cys residue in the Seltag, the normally highly reactive selenium atom of Sec could be protected in the oxidized state of the protein as a result of the selenenylsulfide bond formed between the Sec and Cys residues within the Sel-tag. The selenolthiol motif, when reduced, e.g., by DTT, may furthermore serve the basis for purification of Sel-tagged proteins using a one-step purification technique with PAO sepharose. The PAO-sepharose had previously been utilized for purification of proteins containing vicinal dithiols [51-53]. We found that the affinity of a selenolthiol to PAO was much stronger than that of a dithiol and proteins bound to PAO sepharose through dithiols can thereby be eluted using DTT while the selenolthiol-containing Sel-tagged protein can not. The highly specific arsine oxide chelator dimercaptopropanol sulfonic acid (DMPS) could be used to elute the selenolthiol containing proteins [1]. In fact, for several Sel-tagged proteins, we have found the PAO sepharose purification procedure to be essentially equivalent in both yield and specificity to the commonly used purification of His-tagged proteins over Nickel columns. The Sel-tag has already been utilized for a number of biotechnological Sec-based applications. We have labeled a Sel-tagged mite allergen, Der p 2, with the gamma-emitting isotope ^^Se and utilized it for an in vivo study, tracking the ^^Se-labeled allergen in a mouse model for allergy [54]. We have also demonstrated that a selective selenolate-targeting of the Sec residue in Sel-tagged Der p 2 can be achieved with an electrophilic fluorescent probe for fluorescence labeling, in spite of the presence of six additional Cys residues in the protein [1]. This could be accomplished by incubating the reduced Sel-tagged protein for a short duration with the electrophilic fluorescent compound at a low pH in the presence of excess DTT, thereby scavenging labeling of Cys residues while allowing the highly reactive selenolate to become labeled. In the same study we showed that the Seltagged human vasoactive intestinal peptide (VIP) can be labeled with a fluorescent compound without interfering with its ability to bind to its native VIP-receptor[l]. We reasoned that a similar principle as for residue-specific fluorescent labeling could be used to label a Sec residue with electrophilic compounds containing short-lived positron emitters. This was indeed possible utilizing ["C]-methyl iodide, which gave an efficient "C-labeling of Sel-tagged proteins [1]. To label proteins or peptides with short-lived isotopes is generally a difficult task and this particular Sel-tag application could thereby
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become highly useful as a general method for generating radiolabeled protein ligands suitable for PET imaging studies. Concluding remarks The recent development of efficient production methods for synthesis of selenoproteins has opened new possibilities to use these proteins for Secbased biotechnological applications. As outlined in this chapter, such applications employ the unique features of Sec, the only natural seleniumcontaining amino acid. The biotechnological potential of these techniques is vast and we trust that the biomedical field involving biotechnology of Sec will expand rapidly in the near future. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
L Johansson, C Chen, J Thorell, A Fredriksson, S Stone-Elander, G Gafvelin, ESJ Amer 2004 Nature Methods 1 ;61 MJ Berry, GW Martin 3rd, R Tujebajeva et al 2002 Methods Enzymol 347:17 A Mehta, CM Rebsch, SA Kinzy, JE Fletcher, PR Copeland 2004 J Biol Chem 279:37852 MD Gieselman, Y Zhu, H Zhou, D Galenic, WA van der Donk 2002 Chembiochem 3:709 RJ Hondal, RT Raines 2002 Methods Enzymol ZAl-.lQ L Moroder 2005 J Pept Sci 11:187 R Quaderer, A Sewing, D Hilvert 2001 Helvetica ChimicaActa 84:1197 GT Chen, MJ Axley, J Hacia, M Inouye 1992 Mol Microbiol 6:781 S Muller, H Senn, B Gsell, W Vetter, C Baron, A Bock 1994 Biochemistry 33:3404 ESJ Am6r, H Sarioglu, F Lottspeich, A Holmgren, A B6ck 1999 J Mol Biol 292:1003 O Rengby, L Johansson, LA Carlson et al 2004 Appl Environ Microbiol 70:5159 P Tormay, A Sawers, A Bock 1996 Mol Microbiol 21:1253 JB Mansell, D Guevremont, ES Poole, WP Tate 2001 EMBO J20:7284 ESJ Amer 2002 Methods Enzymol 347:226 S Hazebrouck, L Camoin, Z Faltin, AD Strosberg, Y Eshdat 2000 J Biol Chem 275: 28715 S Bar-Noy, J Moskovitz 2002 Biochem Biophys Res Commun 297:956 HY Kim, VN Gladyshev 2004 Mol Biol Cell 15:1055 Z Jiang, ESJ Amer, Y Mu et al 2004 Biochem Biophys Res Commun 321:94 D Su, Y Li, VN Gladyshev 2005 Nucleic Acids Res 33:2486 L Johansson, G Gafvelin, ESJ Amer 2005 Biochim Biophys Acta, in press S Muller, J Heider, A B6ck 1997 Arch Microbiol 168:421 M Fassbender, D de Villiers, M Nortier, N van der Walt 2001 Appl Radiat hot 54:905 R Bergmann, P Brust, G Kampf, HH Coenen, G Stocklin 1995 Nucl Med Biol 22:475 H Duddeck 1995 Progress in NMR Spectroscopy 27:1 JO Boles, WH ToUeson, JC Schmidt et al 1992 J Biol Chem 267:22217 P Gettins, BC Crews 1991 J Biol Chem 266:4804 E Stocking, J Schwarz, H Senn, M Salzmann, L Silks 1997 J Chem Soc Perkin Trans 1:2442 WA Hendrickson, JR Horton, DM LeMaster 1990 EMBO J 9:1665 MP Strub, F Hoh, JF Sanchez, JM Strub, A Bock, A Aumelas, C Dumas 2003 Structure (Camb) \ 1:1359 JF Sanchez, F Hoh, MP Strub, A Aumelas, C Dumas 2002 Structure 10:1363 C Jacob, GI Giles, NM Giles, H Sies 2003 Angew Chem Int Ed Engl 42:4742 RE Huber, RS Criddle 1967 Arch Biochem Biophys 122:164
230 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.
Selenium: Its molecular biology and role in human health S Bar-Noy, SN Gorlatov, TC Stadtman 2001 Free Radio Biol Med 30:51 L Zhong, A Holmgren 2000 J Biol Chem 215:\S121 S Gromer, L Johansson, H Bauer et al 2003 Proc Natl Acad Sci USA 100:12618 D Besse, N Budisa, W Kambrock et al 1997 Biol Chem 378:211 S Pegoraro, S Fiori, S Rudolph-Bohner, TX Watanabe, L Moroder 1992, J Mol Biol 284:779 MJ Davies, CL Hawkins 2004 Free Radic Biol Med 36:1072 IM Bell, ML Fisher, ZP Wu, D Hilvert 1993 Biochemistry 32:3754 ZP Wu, D Hilvert 1989 J Am Chem Soc Wl :4513 L Ding, Z Liu, Z Zhu, G Luo, D Zhao, J Ni 1998 Biochem J 332 (Pt 1):251 GM Luo, ZQ Zhu, L Ding, G Gao, QA Sun, Z Liu, TS Yang, JC Shen 1994 Biochem BiophysRes Commun 198:1240 S Boschi-Muller, S Muller, A Van Dorsselaer, A Bock, G Branlant 1998 FEBS Lett 439:241 HJ Yu, JQ Liu, A Bock, J Li, GM Luo, JC Shen 2005 J Biol Chem 280:11930 S Ma, RM Caprioli, KE Hill, RF Burk 2003 J Am Soc Mass Spectrom 14:593 KE Sandman, CJ Noren 2000 Nucleic Acids Res 28:755 VN Gladyshev, KT Jeang, TC Stadtman 1996 Proc Natl Acad Sci USA 93:6146 L Zhong, ESJ Amer, A Holmgren 2000 Proc Natl Acad Sci USA 97:5854 L Zhong, ESJ Am6r, J Ljung, F Aslund, A Holmgren 1998 J Biol Chem 273:8581 J Nordberg, L Zhong, A Holmgren, ESJ Am6r 1998 J Biol Chem 273:10835 RD Hoffman, MD Lane \992 J Biol Chem 267:14005 E Kalef, PG Walfish, C Gitler 1993 Anal Biochem 212:325 GY Zhou, M Jauhiainen, K Stevenson, PJ Dolphin 1991 JChromatogr 568:69 L Johansson, L Svensson, U Bergstr5m et al 2005 FEBS J 272:3449
Part III
Selenium and human health
Chapter 21. Selenium, selenoproteins and brain function Ulrich Schweizer Neurobiology of Selenium, Neuroscience Research Center and Institute for Experimental Endocrinology, Charite-Universitatsmedizin Berlin, Charite Campus Mitte, D-WJJ7 Berlin, Germany
Lutz Schomburg Institute for Experimental Endocrinology, Charite-Universitatsmedizin, Berlin, Charite Campus Mitte, D-10JJ7 Berlin, Germany
Summary: After the discovery of selenium (Se) as an essential trace element, direct evidence that Se plays a role in brain function remained relatively scarce for many years. This was probably due to the remarkable stability of brain Se levels during times of dietary Se restriction in experimental animals. In these experiments, activities of the first known Sedependent enzymes, e.g. glutathione peroxidase (GPX), thioredoxin reductase (TrxR), and deiodinase (Dio), were also little changed in the brains of rodents fed Se-deficient diets for extended periods of time. Thus, the lack of spontaneous neurological deficits seemed to exclude an important role for Se in brain function. This notion remained largely unchanged despite the purification of selenoprotein P (SePP) from serum as a neurotrophic factor for cultured neurons and the finding that selenite is an essential component of media for in vitro culture of central neurons. Only later experiments revealed that Se-deficiency exacerbated the outcome of neurological disease in certain animal models. Oxidative stress is considered to play a role in neurodegenerative processes, and GPX 1-transgenic mice provided the first molecular proof for an involvement of selenoproteins in such conditions. Then, gene targeting of SePP led to clear-cut spontaneous neurological deficits in Se-deficient animals and placed SePP at center stage for the priviledged Se supply to the brain. Whether impaired expression of selenoproteins in human brain contributes to the incidence or severity of neurodegenerative disease remains to be established. Still, available evidence already suggests that selenoproteins are playing important roles for brain development, function, and disease in mice - and also most likely in humans.
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Introduction In 1957, Schwarz and Foltz identified Se as an essential trace element for rats. When maintained on a Se-deficient diet, animals developed liver necrosis, but could be rescued with a preparation called "factor 3" which was shown to contain Se [1]. Although subsequent studies revealed that the initial Se-deficient diet was also vitamin E-deficient, Se was now recognized as an essential trace element and no longer simply regarded as a potential environmental toxin. Unlike in most other organs, brain Se levels remained quite stable during dietary Se restriction [2-4]. Only one report demonstrated spontaneous neurological symptoms in Se-deficient mammals, i.e. "leg crossing", in Balb/c mice maintained on a Se-deficient diet [5], a finding which may be strain-specific since similar observations were not reported in other strains of mice or rats. Oxidative stress, i.e. a disproportionate increase of reactive oxygen species leading to the oxidation of cellular constituents like proteins, DNA, and lipids, is thought to contribute to the cellular damage during excitotoxicity and pathogenesis of neurodegenerative disorders [6-8]. Given the reactions catalyzed by known selenoenzymes, it is conceivable that selenoproteins modulate the outcome of neurological disease in animal models. GPxl degrades hydrogen peroxide, the product of superoxide dismutase (SOD). GPx4 degrades phospholipid hydroperoxides thereby potentially protecting cellular membranes fi-om oxidative damage. Methionine sulfoxides are formed fi-om protein-bound methionine during oxidative stress and can be reduced by methionine sulfoxide reductase (Msr) A and MsrB; the latter being also known as selenoprotein R. Mammalian TrxR accepts a wide range of substrates including hydrogen peroxide modulated redox-sensitive transcription factors. In fact, in animal models of neurodegenerative disease Se-deficiency generally exacerbated the neurological and histological damage and a simple Se-supplementation most often proved beneficial. Stroke During stroke or hypoxia/ischemia-reperfusion (HI), a dramatic increase in reactive oxygen species occurs that is believed to trigger molecular events culminating in increased apoptosis, necrosis, and neuroinflammation that may further increase neuronal cell loss and subsequently lead to memory impairment and motor incoordination [8-11]. Since stroke is among the leading causes of disabilities in aging Western societies, efficient treatments are urgently needed. Superoxide radicals (O2") liberated during HI are degraded by SOD. The role of SOD in tissue protection is clearly illustrated after HI by increased cell death in mice with reduced SODl activity [12,13] and in mice protected from HI by over-expression of SODl [14]. During catalysis, SOD consumes O2" and produces the less reactive hydrogen peroxide (H2O2). However, in the presence of Fe^^, H2O2 may decompose
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forming the highly reactive hydroxyl radical (Fenton reaction). Thus, GPX enzymes represent the second line of defense against reactive oxygen species. Based on its ubiquitous expression and ease of assay, GPXl is the best-studied member of the family of Se-dependent GPX isozymes. Neurons and astrocytes express GPXl, but most cytosolic GPX activity is localized in astrocytes [15,16]. In addition, an immunohistochemical study revealed GPX induction in astrocytes surrounding the infarcted area [17]. As expected, transgenic mice over-expressing GPXl are protected against HI in the brain. After HI, indices of cell death are reduced in GPXl-transgenic (tg) animals and infarct volume is significantly reduced. Accordingly, neurological deficits are mitigated in GPXl-tg animals as compared to wild-type controls [18,19]. In contrast, mice made genetically deficient for GPXl exhibit a drastically increased infarct volume, increased neurological deficits, and more pronounced neuronal cell death [20]. Similar observations have been made in myocardial HI models (reviewed in [21]). Thus, GPXl definitely acts as a protective enzyme during HI in vivo. In vitro. Furling et al. have shown that GPXl over-expression protects hippocampal slices from transient hypoxia and maintains electrophysiological properties, e.g., LTP induction [22]. These findings therefore underline the detrimental role played by H2O2 during HI and suggest that anti-oxidative therapy might represent a rational treatment for stroke. It should be stressed that cellular damage after stroke occurs not only in the immediate center of the infarcted area due to shortage of oxygen and energy substrates, but the damaged area grows for several days - even after the thrombus has been dissolved. The underlying mechanisms and contributions of vascular and immune cells, microglia, astrocytes, neurons and oligodendrocytes are not within the scope of this chapter, but the temporal delay of cellular demise has been regarded as an open window of opportunity in which anti-oxidative treatment may still be beneficial. It is known that GPX activity is induced while serum Se falls after stroke [23]. Interestingly, among men free of stroke at the outset, low serum Se was associated significantly with stroke mortality [24], but a larger study could not support this finding [25]. In some cases of rare familial childhood stroke, plasma GPX activity was reportedly only 50% in affected children as compared to their siblings. Interestingly, plasma GPX activity correlated inversely with platelet P-selectin expression and NO-induced aggregation in vitro suggesting a mechanistic link between thrombotic stroke and plasma GPX [26]. In animal models of HI, Se supplementation appeared protective [27,28]. The organoselenium compound ebselen exhibits weak GPX activity and also interacts with and possibly potentiates the Trx-TrxR system. The drug has therefore been tested in preclinical and clinical trials for the treatment of stroke. However, despite early encouraging results in rodent models, ebselen has not met these expectations in clinical trials [29].
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Unfortunately, less is known on the role of GPX4, the phospholipid hydroperoxide-specifc GPX, in HI in the brain. But cardiomyocytes infected with GPX4-expressing adenovirus showed protection of lipids, electron transport chain complex IV function and cellular damage after HI [30]. The Trx-TrxR system is likely involved in protection against HI. Overexpression of Trx in mice protected them against focal cerebral ischemia [31]. Recently, conditional knock-out mice for TrxRl and TrxR2 have been established [32,33] and it will be interesting to define the roles of these enzymes during HI. Interestingly, there seem to exist functional differences among the individual isoenzymes in vivo, since targeted disruption of TrxR2, but not of TrxRl, in the heart leads to dilated cardiomyopathy, a condition in which oxidative stress has also been implicated. Parkinson's Disease Oxidative stress is also believed to be involved in the pathogenesis of sporadic Parkinson's disease (PD) [7]. In this neurodegenerative disorder dopaminergic neurons located in the substantia nigra perish and thus the striatum is deafferented fi-om its main dopaminergic input. The resulting progressive dysfunction of the basal ganglia leads to a characteristic movement disorder which can pharmacologically be treated for some time, but ultimately has devastating consequences. While in some rare cases of familial PD the underlying gene defects have been identified, the cause of idiopathic PD which constitutes the vast majority of cases is still unknown. Some investigators suggest that endogenous or exogenous neurotoxins may contribute to nigral cell death. For example, hydrogen peroxide can be formed fi"om dopamine by the action of monoamine oxidase or by autoxidation of dopamine [34]. Signs of increased oxidative stress have been reported in substantia nigra fi'om PD brains, but due to the delayed tissue processing normally occuring after death of a patient, some of the oxidative damage may have taken place after death complicating interpretation of the results. However, consistent with a crucial role for oxidative stress, an increased amount of iron in substantia nigra was demonstrated in PD [35,36]. Moreover, modulation of iron availability in animal models of PD attenuated the susceptibility of nigral neurons to cell death [37] suggesting that the Fenton reaction contributes to dopaminergic neuron degeneration. Thus, a reduction of H2O2 levels by selenoenzymes might help reduce nigral cell demise. Several pharmacological models for PD in rodents and primates have been established, among them methamphetamine- (MA), 6-hydroxydopamine- (6-OHDA), methyl-phenyl-tetrahydropyridine- (MPTP) and diiminoproprionitrile (DIPN)-induced dopaminergic neurodegeneration. Cellular lesions in 6-OHDA, MA, and MPTP models of PD are increased in Se-deficient rats and can be reduced by Se administration [38,39]. Pretreatment of mice with Se in the DIPN model resulted in reduced lipid
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peroxidation products and inhibited neurobehavioural alterations in a dosedependent manner [40]. Striatal dopamine depletion was prevented by Se supplementation in the MA model [41]. Induction of Se-dependent enzymes like GPXl via Se administration dose-dependently attenuated neurodegeneration in 6-OHDA treated rats [42] and MA-treated rats and mice [39,43]. These Se-dependent protective effects are likely, at least partially, mediated via modulation of GPXl activity, because mice that over-express GPXl are protected against 6-OHDA induced nigral degeneration [44]. In addition, although brain Se levels are only mildly changed by Se-deficient diet in wild-type rodents, it has been observed that GPXl activity, a sensitive marker for Se status in other systems, is changed during Se-deficiency in the brain including substantia nigra and striatum [38,45]. In the MPTP model, GPXl-knockout mice showed increased sensitivity [46] providing further evidence for a protective role of GPXl in PD-like neurodegeneration - and for the involvement of H2O2 in this process. Thus, results from animal models for PD suggest that Se and Se-dependent enzymes, likely GPXl, protect nigral neurons. It should be noted that two conceptually different situations have been discussed: Decreased GPXl activity (or decreased Se levels) increase the susceptibility against neurotoxins, while transgenic overexpression of GPXl protects against neurodegeneration even in the presence of normal Se levels. Simply increasing dietary Se levels over the recommended dietary requirement was not reported to afford protection. Several studies aimed to correlate serum Se levels or blood cell GPX activity in patients with PD, but the results were inconsistent [47-50]. Moreover, Se determination in cerebrospinal fluid did not reveal differences between PD patients and controls [47,51]. It should be noted, however, that there is no strict correlation between blood and brain Se levels (see below). Determination of Se protein expression in human brain samples using immunohistochemical or activity assays, therefore, should be more informative. Damier et al reported increased GPX immunoreactivity in nigral astrocytes in PD [52], but direct measurement of enzymic activity in one study [53] revealed a 20% reduction in several brain regions from PD patients including substantia nigra, basal ganglia, and cerebral cortex, while in another study [48] no changes were observed. Among the many biochemical measures of oxidative stress in PD brain samples, loss of GSH is the most reproducible [7]. Interestingly, the amount of GSSG is not considerably changed and it is unknown how and where GSH is lost. Thus, while the GPX/GSH system proved particularly protective in models of PD, and oxidative stress is likely involved in PD, there is no clear data whether this system is disturbed in the brains of PD patients and whether or not observed changes in GPX and GSH levels are the cause or consequence of the disease process [7].
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Epilepsy Induction of oxidative stress by local application of Fe^^ to the rodent cortex leads to epileptiform EEG discharges and was used as a model for epilepsy [54]. Using this model, Rubin and Willmore showed that antiperoxidant treatment, including selenite, reduced the histopathological tissue damage as well as epileptic discharges. [55,56]. Conversely, reduced dietary Se intake increased seizure frequency in rats treated with the excitotoxin kainic acid and increased hippocampal cell loss [4]. Thus, it appears as if Se status affects the response to experimentally induced seizures although it has never been shown that Se treatment increased brain Se directly. Rather, some investigators found that brain Se levels are stable during dietary Se depletion, although a reduction of brain GPXl activity has been noted [4,39,45]. While more studies are needed to clarify this issue, it should be noted that seizures and muscle weakness have been observed in patients that had become severely Se-deficient during total parenteral nutrition [57,58] at a time when Se was not sufficiently included into dietary formulations. Probably the best evidence to link Se with epilepsy is given by two unrelated clinical reports. In the first study, Weber [59] showed that children with a form of intractable seizures had low blood Se and low plasma GPx activity. Dietary Se supplementation led to normalization of plasma Se and seizure activity was subsequently reduced. The study was initially not well received [60]. Several years later an unrelated study reported similar findings with children suffering from infractable seizures [61]. Again, the patients manifested low plasma Se and low plasma GPx activity. Dietary supplementation with Se compounds normalized plasma Se levels and the seizures subsequently responded to anticonvulsant therapy. Most importantly, upon cessation of Se freatment in one patient, seizures resumed but responded again to Se supplementation. Unfortunately, when the patient was again referred to another hospital, Se freatment was interrupted and the patient died in status epilepticus. Some of the patients described in the two reports above suffered from classical Se-deficiency symptoms like brittle hair, white nails, weak muscle, and in one case the knee joint exhibited morphological changes reminiscent of Kashin-Beck osteopathy. At present it is not clear whether Se deficiency was the primary reason for the seizures or, probably more likely, Se deficiency mediated the refractoriness of the seizures towards anticonvulsant therapy. In addition, the cause for the observed Se deficiency is not known. We hypothesised that mutations in genes involved in Se metabolism may underlie an impaired capacity to accumulate dietary Se or to retain Se in the body. Respective novel poljmiorphisms are currently under investigation (U. Schweizer, unpublished). Along these lines it should be noted that mice made genetically deficient for SePP display spontaneous seizures and a movement phenotype that depends on dietary Se availability [62-65] (see below and Chapter 10).
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Since Se-deficiency rendered rats more susceptible to the neurotoxin kainic acid [4], it was expected that transgenic over-expression of GPXl in mice protects them from seizures induced by kainic acid injection. Surprisingly it was found that GPXl over-expression increased hippocampal cell death and seizure intensity in transgenic animals [66]. The authors showed that increased GPXl activity led to increased extracellular GSSG levels after kainic acid treatment and speculated that this may activate the NMDA receptor. This explanation is conceivable as GSSG application is known to alter NMDA-R responses. GPX activity is increased in pilocarpine-treated rats before status epilepticus [67] and GSH/GSSG decreased in seizure prone mice [68], but it remains to be determined whether this is a protective adaptation or cause of the seizure. Clearly, this finding implicates that even generally protective enzyme systems need to be well-balanced and finelytuned into the physiological context. It should be noted that epilepsy is a frequent disorder probably afflicting almost 1 in 100 persons during their lifetime. The diverse manifestations of epilepsy are reflected by a plethora of possible causes as far as they have been identified. We do not believe that Se deficiency is a major cause of epilepsy in humans. However, there seems to exist a subset of Se-deficient patients who may respond favorably to Se administration. Other neurodegenerative disorders As in many other neurodegenerative disorders, oxidative stress has been implicated in the pathology of amyofrophic lateral sclerosis (ALS), which is known in the US as Lou Gehrig's disease'. Li this invariably fatal condition, spinal and cranial motomeurons, which innervate muscle fibers degenerate leading to weakness of the limbs, loss of speech, and respiratory disfress. In many cases there is also involvement of principal neurons in the primary motor cortex. Similar as in other major neurodegenerative disorders, most cases occur sporadically, but there are also cases of familial ALS (FALS) that helped identify some underlying gene defects in genes encoding SODl, alsin, and the androgen receptor. At first, oxidative stress seemed to cause ALS, since most patients suffering from FALS carry missense mutations in the SODl gene [69]. Accordingly, transgenic mice carrying human SODl mutant genes develop an ALS-like disease. Although questioned from the beginning, the popular notion that a loss-of-function mutation of SODl occurs in FALS has not been substantiated and more recent evidence rather established an unrelated gain-of-function mechanism. Still, markers of oxidative sfress are reportedly elevated in man and mice carrying SODl mutations. Regarding Se or selenoenzymes in ALS, however, we are not
' Famous US sportsman. New York Yankees 1925-1939, died in 1941 from ALS
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aware of data from animal models. The few clinical studies performed did not show a correlation of Se or plasma GPX with disease. Alzheimer's disease (AD) is a relatively common neurodegenerative disorder that develops at an advanced age. Cortical atrophy affects principal neurons and cholinergic neurons in the cortex and hippocampus. The disease is characterized by memory loss and diminished higher brain fimctions [70]. So called amyloid plaques are a pathological hallmark of the disease. These plaques consist of misfolded/aggregated protein. A fragment, Ap, of APP (amyloid precursor protein) is proteolytically generated and a major constituent of amyloid plaques. Certain alleles of APP, the E4 allele of apolipoprotein E, and the presenilin genes which encode polytopic fransmembrane proteins of the y-secretase complex are associated with AD. Since AD is such a common and regrettably devastating disease, an enormous amount of data have been accumulated regarding AD which cannot be reasonably dealt with here. It should be noted that only very recently a physiological function of APP has been elucidated in detail [71]. With respect to Se or selenoproteins, not many studies have been undertaken and the published ones did not yield a consistent picture. Several authors tested Se levels in AD brain [72-74] or plasma samples [75]. Two studies reported plasma and/or erythrocyte GPX measurements in AD patients [75,76]. Again, the resulting picture was not clear. Given the large variations of plasma Se and plasma GPX activity in human populations, it may not be possible to find clear associations with AD in small studies. We may expect more insight from the PREADVISE study which accompanies the SELECT trial. Li this longitudinal study over 5-12 years, patients are treated with Se and/or vitamin E or placebo. Neurological investigations are directed to uncover any protective effects of Se on the occurence of AD in the study population. The rationale for this study, again, is the notion that oxidative stress may be involved in AD pathogenesis. Interestingly, levels of Trx fall while expression of TrxR increases in the AD brain [77]. It is, however, conceivable that oxidative stress is rather a consequence of AD pathology related to protein misfolding or activation of microglial cells [8] and not the reason - and thus selenoproteins may modulate disease progression without the need to postulate a causal role for Se in AD incidence. Huntington's disease (HD) results from the expansion of a CAG repeat (encoding the amino acid glutamine) in a gene whose inheritance was dominantly linked to HD. The encoded protein, huntingtin, is as yet of unknown function and aggregates in response to enlarged polyglutaminerepeats. Although huntingtin is expressed in many neuronal cell types, mutant huntingtin initially causes a remarkably specific degeneration of small spiny GABAergic neurons in the striatum, but later the patients succumb to a devastating neurodegenerative disease. We have found no clinical data linking HD and Se status or selenoproteins and only one such
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study in a pharmacological animal model. Injection of rats with quinolinic acid leads to excitotoxicity, lipid peroxidation, neuronal death, and neurological symptoms similar to HD. Se supplementation of rats in that model was protective [78]. Friedreich's ataxia (FA) is one of the most common forms of autosomal recessive ataxia [79]. About 97% being due to expansion of a GAA trinucleotide repeat in intron 1 of the FRDA gene which impairs its expression. FA is characterized by degeneration of large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence of diabetes. The disorder is usually manifest before adolescence and is generally characterized by uncoordination of limb movements, dysarthria, and impairment of position and vibratory senses. The FRDA gene encodes the small mitochondrial protein frataxin that is involved in iron import into mitochondria and antioxidant defense [80]. Frataxin deficiency leads to mitochondrial iron overload, respiratory chain imbalance and increased ROS generation leading to oxidative damage. Moreover, a direct involvement of frataxin in ROS detoxification, activation of GPx and elevation of reduced thiols has recently been demonsfrated [81]. Because of the overlap in the antioxidant biochemistry of Se and the increased iron-induced mitochondrial peroxidation, a treatment of FA patients with Se might prove successful, but no data are presently available, yet. There is substantial evidence implicating oxidative stress in the pathophysiology of neurodegenerative disorders and thus it is conceivable that Sedependent enzyme systems may be involved in protection against neurodegeneration. A major problem for clinical studies trying to link neurodegenerative disorders with Se metabolism lies in the recruitment of a sufficiently large homogenous study cohort. Since most cases of neurological disease occur sporadically with unclear etiology and since even in one given disease several underlying gene defects have been identified, it may be hard to find such correlations in small groups of patients with heterogeneous etiology of the disease. While we are still lacking suitable surrogate markers for Se or selenoenzyme levels in the human brain, we are left with post mortem analyses and nutritional and/or transgenic animal models. Transgenic selenoprotein-deficient mouse models with neurological disease Selenoprotein P (SePP) is the major Se containing protein in human and rodent plasma [82] (see Chapter 10). Among selenoproteins it is unique since it contains more than one Sec residue (10 in man and mouse, 12 in cattle, and 17 in zebrafish). SePP plasma levels closely respond to dietary Se intake (see Chapter 35). Because of these and other properties, it was hypothesized from early on that SePP is a Se fransport protein [83].
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We and the group of R. Burk and K. Hill have independently inactivated the gene encoding SePP in the mouse and shown that SePP-KO mice displayed a neurological phenotype including seizures, a movement disorder, and axonal degeneration [62,63,65,84]. The neurological phenotype of SePPKO mice depends on dietary Se supply and can completely be abrogated if SePP-KO mice are supplemented with sufficient amounts of Se [64,65]. This finding contrasts sharply with the stability of brain Se levels and brain function during dietary Se restriction in rodents [2,4]. Thus, it appears as if, in the absence of SePP, the brain becomes sensitive to circulating Se implicating SePP as a crucial part of the mechanism that maintains preferential supply of Se to the brain [85]. We wondered at which time during development the lack of SePP manifests itself in neurological dysfunction. In oder to study this question, we have supplemented heterozygous SePP breeder pairs at different time points with selenite in their drinking water and found that SePP-KO mice can be rescued if Se treatment started from mating or from birth onwards. When Se supplementation started from weaning, after the phenotype (movement disorder and seizures) had already occurred, the mice were stabilized at the status quo, but the phenotype was not ameliorated (Figure 1).
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Figure 1. Experimental design for selenite supplementation of SePP-KO mice during development. Complete rescue of Se-dependent neurological phenotypes was observed if Se supplementation was initiated early at mating or birth, but was not achieved if started after weaning. Phenotypes developed progressively if supplementation was terminated at any time.
These findings suggest that a Se-dependent developmental process is taking place between birth and weaning that underlies the neurological defects. Since in rodents the cerebellum develops mainly from birth until
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weaning and since it controls movement co-ordination, we hypothesized that cerebellar development may be impaired in SePP-KO mice. A hallmark of hypothyroidism is incomplete cerebellar development. Granule cells, the most abundant neuronal cell type in the brain, are produced in the external germinal layer and migrate along radial glial fibers through the Purkinje cell monolayer towards their final destination, the inner granule cell layer. During developmental hypothyroidism migration of granule cells is delayed and arborization of Purkinje cell dendritic trees impaired. Thus, we studied thyroid hormone levels and deiodinase activities in SePP-KO mice in brain and other tissues and analysed cerebellar development morphologically. To our surprise, we could neither demonstrate delayed cerebellar granule cell migration nor incomplete dendritic tree development of Purkinje cells in SePP-KO cerebellum. In addition, activity of type 2 deiodinase, the major T4 activating deiodinase in brain, was unaltered [86]. Thus, we could not support local hypothyroidism as the cause of the movement disorder of SePP-KO mice. In addition, mice which were first supplemented with Se and thus phenotypically rescued, developed the movement phenotype after withdrawal of supplemental Se (Figure 1). Thus, we conclude that the movement phenotype of SePP-KO mice is not a result of developmental disturbance of cerebellar development, but may rather be a consequence of functional impairment or neurodegeneration due to Se depletion of the brain. There is indeed evidence that SePP is required for neuronal survival. Following the identification of the nerve growth factor (NGF) as a survival factor for sympathetic and sensory neurons [87], the search started for factors that could promote the survival of spinal and cortical neurons. Since it was known that factors contained within fetal calf serum (FCS) helped to maintain primary central neurons in culture, Kaufman and Barrett fractionated FCS and partially purified a protein unlike other known neurotrophic factors [88]. In a follow-up paper, Yan and Barrett [89] purified the corresponding factor to homogeneity and identified it as SePP. Despite its extraordinary potency in promoting neuronal survival, the issue was not followed much further and the search for neurotrophic factors remained centered on homologs of NGF and ligands for receptor tyrosine kinases. The study of SePP as neuronal survival promoting factor was probably loosing attraction after the development of serum-fi'ee culture conditions [90]. Thus it remained largely unappreciated in the field that Se was found indispensible for serum-fi'ee neuron culture. In fact, culture supplements like "N2", "B24", and the nowadays commercially available "B27" contain selenite as a Se source (approx. 50 nM). In a series of elegant studies, Takahashi and coworkers have shown that SePP can act as a Se supply protein [91,92], and in retrospect, it seems conceivable that SePP in FCS acts as the main Se source for cultured neurons in the absence of inorganic selenite.
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These in vitro studies suggested that other Se sources, like selenite, can substitute for SePP in order to promoter cellular function. Since SePP is expressed in most tissues including the brain, but most abundantly in liver, we wanted to determine the role of hepatically secreted SePP in contrast to locally expressed SePP in the brain. To this end, we made use of a mouse model in which selenoprotein expression is abrogated in a hepatocytespecific manner [93]. When we compared these mice with our SePP-KO mice, we demonstrated that Alb-Cre/Trsp"'" mice, although containing similarly reduced amounts of Se in plasma as SePP-KO mice, did not exhibit the same neurological deficits, but rather appeared entirely normal [94]. Brain Se and GPX activity remained unaltered in Alb-Cre/Trsp*"" mice, while kidney GPX and plasma GPX (which is secreted from kidney) were reduced similarly as in SePP-KO mice. We concluded from this study that local SePP expression in the brain is necessary for normal brain function, possibly because of a local transport or storage role of SePP within the CNS, while hepatically derived SePP supplies Se to other organs like, e.g., the kidney. Since SePP-KO mice can be rescued with inorganic Se supplements, plasma SePP is likely not the only chemical form of Se taken up by the brain.
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Figure 2. Cerebellar development is deranged in Tal-Cre/Trsp*"" mice. A sagittal section through the cerebellum at postnatal day 12 was stained with an antibody directed against parvalbumin as a marker for Purkinje neurons. There is marked Purkinje cell loss (open arrow) and some Purkinje cells are found at ectopic locations (solid arrow). Purkinje cell dendrites are stunted, partially disordered, if present at all.
Since metabolic disorders sometimes result in seizures, we wanted to verify that the neurological deficits in SePP-KO mice are primarily related to brain Se deficiency. To probe the role of selenoproteins for neuronal development and function, we inactivated selenoprotein biosynthesis in a neuron-specific manner using mice expressing Cre recombinase under control of the tubulin-
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a l promoter (Tal-Cre) [95]. In this model, Cre-mediated recombination starts after the final mitosis and neuronal differentiation, such that the target gene, in our case Trsp, is inactivated early-on in the life of respective neurons. Tal-Cre/Trsp"'*' mice are bom at the expected frequency indicating that there is no appreciable embryonic lethality. After about one week of age, however, the knockouts appear growth retarded and stop gaining weight. They do not attain postural control and die before the age of two weeks. While the forebrain seemed morphologically normal, we detected severe cerebellar hypoplasia. The folding of the cerebellar cortex and most of the volume of the cerebellum derives from the massive generation of granule cells during postnatal cerebellar development. Thus it is conceivable that the hypoplasia is associated with a reduction of granule cell number. We are currently in the process of determining whether granule cell proliferation is reduced or whether a fraction of differentiated granule cells dies in the absence of functional selenoproteins. Granule cell proliferation depends, among other signals, on sonic hedgehog secretion from Purkinje cell dendrites. Given the striking patchy lack of Purkinje cells and the thinning of the external germinal layer overlying zones of Purkinje cell loss, one may conclude that reduced proliferation of granule cells partly derives from the lack of a growth signal (Figure 2). On the other hand, the selenoprotein TrxR is implicated in DNA synthesis, and the massive proliferation of granule cell precursors in the external germinal layer might be impaired because of the lack of TrxR. It will be interesting to compare Tal/Trsp*"" mice with neuron-specific mutants for essential selenoproteins and thus assign specific roles in brain development and function to individual selenoproteins. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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Selenium: Its molecular biology and role in human health G Trepanier, D Furling, J Puymirat, ME Mirault 1996 Neuroscience 75:231 S Takizawa, K Matsushima, Y Shinohara, S Ogawa, N Komatsu, H Utsunomiya, K Watanabe 1994 J Neurol Sci 122:66 N Ishibashi, O Prokopenko, M Weisbrot-Lefkowitz, KR Reuhl, OMirochnitchenko 2002 Brain Res Mol Brain Res 109:34 N Ishibashi, O Prokopenko, KR Reuhl, O Mirochnitchenko 2002 J Immunol 168:1926 PJ Crack, JM Taylor, NJ Flentjar, J de Haan, P Hertzog, RC lannello, I Kola 2001 J NeurochemlS:\3S9 U Schweizer, L Schomburg 2005 lUBMB Life In press. D Furling, O Ghribi, A Lahsaini, ME Mirault, G Massicotte 2000 Proc Natl Acad Sci USA 97:4351 C Zimmermann, K Winnefeld, S Streck, M Roskos, RL Haberl 2004 Eur Neurol 51:157 J Virtamo, E Valkeila, G Alfthan, S Punsar, JK Huttunen, MJ Karvonen 1985 Am J Epidemiol 122:276 WQ Wei, CC Abnet, YL Qiao, SM Dawsey, ZW Dong, XD Sun, JH Fan, EW Gunter, PR Taylor, SD Mark 2004 Am J Clin Nutr 79:80 G Kenet, J Freedman, B Shenkman, E Regina, F Brok-Simoni, et al 1999 Arterioscler Thromb Vase Biol 19:2017 MA Ansari, AS Ahmad, M Ahmad, S Salim, S Yousuf, T Ishrat, F Islam 2004 Biol Trace Elem Res 101:73 R Gupta, M Singh, A Sharma 2003 Pharmacol Res 48:209 AR Green, T Ashwood 2005 Curr Drug Targets CNS Neurol Disord 4:109 JM Hollander, KM Lin, BT Scott, WH Dillmann 2003 Free Radic Biol Med 35:742 Y Takagi, A Mitsui, A Nishiyama, K Nozaki, H Sono, Y Gon, N Hashimoto, J Yodoi 1999 Proc Natl Acad Sci USA 96:4131 M Conrad, C Jakupoglu, SG Moreno, S Lippl, A Banjac, M Schneider, H Beck, AK Hatzopoulos, U Just, F Sinowatz, W Schmahl, KR Chien, W Wurst, GW Bomkamm, M Brielmeier 2004 Mol Cell Biol 24:9414 C Jakupoglu, GK Przemeck, M Schneider, SG Moreno, N Mayr, AK Hatzopoulos, MH de Angelis, W Wurst, GW Bomkamm, M Brielmeier, M Conrad 2005 Mol Cell Biol 25:1980 J Sian, M Gerlach, MB Youdim, P Riederer 1999 J Neural Transm 106:443 E Sofic, P Riederer, H Heinsen, H Beckmann, GP Reynolds,G Hebenstreit, MB Youdim 1988 J Neural Transm 74:199 D Kaur, JK Andersen 2002 Aging Cell 1:17 D Kaur, F Yantiri, S Rajagopalan, J Kumar, JQ Mo, R Boonplueang, V Viswanath, R Jacobs, L Yang, MF Beal, D DiMonte, I Volitaskis, L EUerby, RA Chemy, AI Bush, JK Andersen 2003 Neuron 37:899 HC Kim, WK Jhoo, DY Choi, DH Im, EJ Shin, JH Suh, RA Floyd, G Bing 1999 Brain /?e* 851:76 H Kim, W Jhoo, E Shin, G Bing 2000 Brain Res 862:247 S al Deeb, K al Moutaery, GW Bruyn, M Tariq 1995 J Psychiatry Neurosci 20:189 SZ Imam, GD Newport, F Islam, W Slikker, SF Ali 1999 Brain /fes 818:575 KS Zafar, A Siddiqui, I Sayeed, M Ahmad, S Salim, F Islam 2003 JNeurochem 84:438 V Sanchez, J Camarero, E O'Shea, AR Green, MI Colado 2003 Neuropharmacology 44:449 JC Bensadoun, O Mirochnitchenko, M Inouye, P Aebischer, AD Zum 1998 Eur J Neurosci 10:3231 A Castano, J Cano, A Machado 1993 J Neurochem 61:1302 P Klivenyi, OA Andreassen, RJ Ferrante, A Dedeoglu, G Mueller, E Lancelot, M Bogdanov, JK Andersen, D Jiang, MF Beal 2000 J Neurosci 20:1
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MV Aguilar, FJ Jimenez-Jimenez, JA Molina, I Meseguer, C Mateos-Vega, MJ Gonzalez-Munoz, F de Bustos, C Gomez-Escalonilla, M Ort-Pareja, M Zurdo, MC Martinez-Para 1998 J Neural Transm 105:1245 RJ Marttila, M Roytta, H Lorentz, UK Rinne 1988 J Neural Transm 74:87 J Kalra, AH Rajput, SV Mantha, K Prasad 1992 MolCell Biochem 110:165 P Johannsen, G Velander, J Mai, EB Thorling, E Dupont 1991 7 Neurol Neurosurg Psychiatry 54:679 I Meseguer, JA Molina, FJ Jimenez-Jimenez, MV Aguilar, CJ Mateos-Vega, MJ Gonzalez-Munoz, F de Bustos, M Orti-Pareja, M Zurdo, A Berbel, E Barrios, MC Martinez-Para 1999 J Neural Transm 106:309 P Damier, EC Hirsch, P Zhang, Y Agid, F Javoy-Agid 1993 Neuroscience 52:1 SJ Kish, C Morito, O Homykiewicz 1985 Neurosci Lett 58:343 LJ Willmore 1990 Epilepsia 31 Suppl 3:S67 JJ Rubin, LJ Willmore 1980 Exp Neurol 67:472 LJ Willmore, JJ Rubin 1981 Neurology 31:63 CL Kien, HE Ganther 1983 ^m y Clin Nutr 37:319 KM Brown, JR Arthur 2001 Public Health Nutr 4:593 GF Weber, P Maertens, XZ Meng, CE Pippenger 1991 Lancet 337:1443 U Schweizer, AU BrSuer, J K6hrle, R Nitsch, NE Savaskan 2004 Brain Res Brain Res RevA5:\(A VT Ramaekers, M Calomme, D Vanden Berghe, W Makropoulos 1994 Neuropediatrics 25:217 L Schomburg, U Schweizer, B Holtmann, L Flohe, M Sendtaer, J Kohrle 2003 Biochem J110-391 KE Hill, J Zhou, WJ McMahan, AK Motley, JF Atkins, RF Gesteland, RF Burk 2003 J Biol Chem 218:13,640 U Schweizer, M Michaelis, J Kohrle, L Schomburg 2004 Biochem J21&:2\ KE Hill, J Zhou, WJ McMahan, AK Motley, RF Burk 2004 J Nutr 134:157 R Boonplueang, G Akopian, FF Stevenson, JF Kuhlenkamp, SC Lu, JP Walsh, JK Andersen 2005 Exp Neurol 192:203 MI Bellissimo, D Amado, DS Abdalla, EC Ferreira, EA Cavalheiro, MG NaffahMazzacoratti 2001 Epilepsy Res 46:121 M Hiramatsu, A Mori 1981 Neurochem Res 6:301 DR Rosen, T Siddique, D Patterson, DA Figlewicz, P Sapp, A Hentati, D Donaldson, J Goto, JP O'Regan, HX Deng 1993 Nature 362:59 N Durany, G Munch, T Michel, P Riederer 1999 Eur Arch Psychiatry Clin Neurosci 249 Suppl 3:68 P Soba, S Eggert, K Wagner, H Zentgraf, K Siehl, S Kreger, A Lower, A Langer, G Merdes, R Paro, CL Masters, U MuUer, S Kins, K Beyreuther 2005 EMBO J 24M24 CR Comett, WR Markesbery, WD Ehmann 1998 Neurotoxicology 19:339 CR Comett, WD Ehmann, DR Wekstein, WR Markesbery 1998 Biol Trace Elem Res 62:107 D Wenstrup, WD Ehmann, WR Markesbery 1990 Brain Res 533:125 I Ceballos-Picot, M Merad-Boudia, A Nicole, M Thevenin, G Hellier, S Legrain, C Berr 1996 Free Radic Biol Med 20:579 C Jeandel, MB Nicolas, F Dubois, F Nabet-Belleville, F Penin, G Cuny 1989 Gerontology 35:275 MA Lovell, C Xie, SP Gabbita, RW Markesbery 2000 Free Radic Biol Med 28:418 A Santamaria, R Salvatierra-Sanchez, B Vazquez-Roman, D Santiago-Lopez, J VilledaHemandez, S Galvan-Arzate, ME Jimenez-Capdeville, SF Ali 2003 J Neurochem 86:479 MB Delatycki, R Williamson, SM Forrest 2000 J Med Genet 37:1
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Selenium: Its molecular biology and role in human health M Babcock, D de Silva, R Oaks, S Davis-Kaplan, S Jiralerspong, L Montermini, M Pandolfo, J Kaplan 1997 Science 276:1709 SA Shoichet, AT Baumer, D Stamenkovic, H Sauer, AF Pfeiffer, CR Kahn, D MuUerWieland, C Richter, M Ristow 2002 Hum Mol Genet 11:815 RF Burk, KE Hill (2005) Annu Rev Nutr 25: 215 MA Motsenbocker, AL Tappel 1982 Biochim BiophysActa 719:147 WM Valentine, KE Hill, LM Austin, HL Valentine, D Goldowitz, RF Burk 2005 Toxicol Pathol 33:570 L Schomburg, U Schweizer, J K6hrle 2004 Cell Mol Life Sci 61:1988 L Schomburg, C Riese, M Michaelis, E Griebert, MO Klein, R Sapin, U Schweizer, J K6hrle 2005 Endocrinology In press. R Levi-Montalcini 1987 EMBO y 6:1145 LM Kaufman, JN Barrett 1983 Science 220:1394 J Yan, JN Barrett 1998 JNeurosci 18:8682 GJ Brewer, CW Cotman 1989 Brain Res 494:65 Y Saito, K Takahashi 2002 EurJBiochem 269:5746 Y Saito, N Sato, M Hirashima, G Takebe, S Nagasawa, K Takahashi 2004 Biochem J 381:841 BA Carlson, SV Novoselov, E Kumaraswamy, BJ Lee, MR Anver, V Gladyshev, DL Hatfield 2004 J Biol Chem 279:8011 U Schweizer, F Streckfuss, P Pelt, BA Carlson, DL Hatfield, J Kohrle, L Schomburg 2005 5/oc/iemJ386:221 V Coppola, CA Barrick, EA Southon, A Celeste, K Wang, B Chen, E Haddad, J Yin, A Nussenzweig, A Subramaniam, L TessaroUo 2004 Development 131:5185
Chapter 22. Selenium as a cancer preventive agent Gerald F. Combs, Jr. Grand Forks Human Nutrition Research Center, USDA-ARS, Grand Forks, ND 58202, USA
Junxuan Lii Hormel Institute, University of Minnesota, Austin, MN 55912, USA
Summary: Most epidemiological studies have shown inverse associations of selenium (Se) status and cancer risk; almost all experimental animal studies have shown that supranutritional exposures of Se can reduce tumor yield; and each of the limited number of clinical intervention trials conducted to date has found Se treatment to be associated with reductions in cancer risks. The known metabolic functions of Se, which appear to be discharged by a fairly small number of selenoproteins may not fully explain these effects, particularly those observed in response to Se-supplementation of non-deficient subjects. Emerging evidence indicates anticarcinogenic roles of at least some selenoproteins, namely, those involved in antioxidant protection (the glutathione peroxidases), redox regulation (the thioredoxin reductases) and hormonal regulation of metabolism (iodothyronine 5'deiodinases). The fact that abundant empirical evidence has shown anticarcinogenic effects of Se in individuals with apparently full selenoenzyme expression suggests other mechanisms with relevance to non-deficient populations. Certain Se-metabolites (hydrogen selenide, methylselenol, seleno-diglutathione) can be anti-carcinogenic by inhibiting cell proliferation, stimulating cell death by apoptosis, and inhibiting neoangiogenesis. Therefore, while the hypothesis remains plausible that Sedeprivation may increase cancer risk by compromising selenoprotein expression, there is strong support for the hypothesis that supranutritional exposures to Se can reduce cancer risk. These hypotheses are not mutually exclusive, and it is likely that Se can function as a cancer preventive agent through both nutritional and supranutritional mechanisms. Emergence of a selenium-cancer link The nutritional essentiality of Se was recognized in the late 1950's when the element was found to be the active principle in liver that could replace vitamin E in the diets of rats and chicks for the prevention of vascular,
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muscular and/or hepatic lesions [1]. The first suggestion that Se may be anticarcinogenic came a decade later and was based on empirical observation of an inverse relationship of cancer mortality rates and forage crop Se contents in the United States [2,3]. The body of scientific evidence that has subsequently been developed indicates that, indeed, Se can play a role in cancer prevention. Epidemiological Evidence The epidemiological literature on Se and cancer has been reviewed [4-7]. Most, but not all, of this literature has found Se status to be inversely associated with cancer risk. Prospective cohort studies in several countries have all shown cancer cases to have significantly lower mean pre-diagnostic serum Se levels than controls [8-15]. Negative associations have been found for various parameters of Se status and risks to cancers or pre-cancerous lesions of the bladder [8], brain [16], esophagus [17], lung [18-20], head and neck [21], ovary [9], pancreas [22], thyroid [23], stomach [24,25], melanoma [26], prostate [27] and colon [28]. Animal Model Evidence Studies with animal tumor models have shown that Se treatment can reduce tumor yields. Some years ago. Combs [29] estimated that, of what was then more than 100 studies in which tumor production and/or preneoplastic endpoints had been measured, two-thirds showed that supranutritional Se doses reduced the incidences of such outcomes, with half showing reductions of 50% or more. Further studies have demonstrated similar reductions in tumor yields (see [30]) or experimental metastases [31,32]. Four studies have found selenite treatment to enhance tumorigenesis; but the interpretation of these is not straightforward, as three [33-35] found increases in tumors at one site to be accompanied by reductions at another site, and one [36] found such enhancement only when the carcinogen was administered in a certain way. Clinical Trial Evidence Several clinical trials have been conducted to determine the efficacy of Se in reducing cancer risk in humans. Yu et al [37,38] reported that, after 8 yrs, the incidence of primary liver cancer (PLC) was 35% lower in a community using selenite-enriched table salt compared to non-treated communities which showed no changes in PLC incidence; however, their data were not analyzed statistically. They also reported lower PLC incidence among hepatitis surface antigen-positive subjects randomized to a Se-enriched yeast treatment (200 meg Se per day) in comparison to a placebo group (0 vs. 5 cases, Plprotein synthesis tapoptosis, 4'AP-l, >lNF-kB
NazSeO.
•-GPXsJRs —•• ^ROS, tredox control SeMet SeCys
t02", tH202, tDNA SSBs, S/G2-arrest, >lpolyamines, tapoptosis iPKC, i endothelial MMP, ^epithelial VEGF
CHjSeOjH CHjSeCN CHjSeCys
Gi arrest, tcaspase-mediated apoptosis (CH3)2Se (breath)
(w/o genotoxicity)
I. (CH3)3Se^ (urine) Figure 1. Se-metabolites apparently active in cancer prevention (after [92]). Abbreviations: SeMet, selenomethionine; SeCys, selenocyeteine; CH3Se02H, methyl-seleninic acid; CHsSeCN, methylselenocyanate; CHsSeCys, Se-methylseleno-cysteine; GPXs, glutathione peroxidases; TRs, thioredoxin reductases; ROS, reactive oxygen species; SSBs, DNA single strand breaks; PKC, protein kinase C; MMP-2, matrix metalloproteinase-2; VEGF, vascular endothelial growth factor; PSA, prostate specific antigen; AR, androgen receptor.
Hydrogen selenide appears to be an important player in Se-anticarcinogenesis by way of its further metabolism. Its oxidative metabolism produces superoxide anion (O2 ) and H2O2, the formation of which induces DNA single-strand breaks leading to S phase/G2 cycle arrest and cell death by
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apoptosis [103-107]. This mechanism would appear to mediate seleniteinduced apoptosis, as the genotoxic and pro-apoptotic effects of selenite on leukemia, mammary or prostate cancer cells have been shown to be blocked by a superoxide dismutase or its mimetics [103,108,109], but not by an hydroxyl free radical scavenger [110]. Further, catalase added to the cell culture medium blocked the induction of cell death by selenite [111]. In addition, H2Se can be methylated to produce a string of metabolites that, although being readily excreted, include some that are anti-carcinogenic. Ip, Ganther and coworkers [112-120] have produced strong experimental evidence that the anti-tumorigenic effects of Se are mediated by methylselenol (CHsSeH) or its derivatives (see Figure 1). They found that the CHaSeH-precursors selenobetaine (CH3Se02H) and methyl-selenocysteine (CHsSeCys) are anti-carcinogenic in the 7,12-dimethylbenzanthracene (DMBA)-induced rat mammary tumor model, each being somewhat more efficacious than selenite. In contrast, dimethyl selenoxide, which is metabolized to dimethylselenide ([CH3]2Se) and very rapidly excreted in the breath, was very poorly chemo-preventive, and the rapidly excreted urinary metabolite trimethylselenonium ([CHsJsSe^) was completely ineffective. Further work has shown that the CHsSeH-precursors methylselenocyanate (CHsSeCN) and CHsSeCys can each inhibit mammary cell growth, arresting cells in the Gi or early S phase and inducing apoptosis [106,107,118-122], The latter effect is caspase-dependent [123], as methyl-Se induced apoptosis involves at least three caspase-dependent actions: mitochondrial release of cytochrome C, cleavage of poly(ADP-ribose), and DNA nucleosomal fragmentation. Selenite-induced cell death, in contrast, is independent of these death proteases [109,121,123,124]. That methyl-Se can cause caspasedependent apoptosis in cell lines that do not contain functional p53 [124] suggests that its pro-apoptotic action is independent of p53. This was also evident in a recent study in which methyl-Se induced apoptosis of p53positive, LNCaP cells was found not to involve a change in p53 activation [125]. Se-Methylselenocysteine has been shown to inhibit the cell cycle regulatory enzymes CDK2 and protein kinase C (PKC) [126,127]. Unlike the proximal H2Se-precursors, CHaSeH-precursors potently inhibit the expression of matrix metalloproteinase (MMP-2) in vascular endothelial cells and of vascular endothelial growth factor (VEGF) in cancer cells [121,122,128,129], critical components of the angiogenic response, suggesting that methyl-Se inhibits cellular proliferation and survival of activated endothelial cells by inhibiting neo-angiogenesis. Sub-apoptotic concentrations of methyl-Se have been shown to reduce androgen receptor protein expression [130] and to inhibit androgen-stimulated PSA promoter transcription [130-132], to reduce PSA expression and secretion [130], and to cause rapid PSA degradation [130]. These findings suggest a unique
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Selenium: Its molecular biology and role in human health
mechanistic basis for the apparent sensitivity of the prostate to Seanticarcinogenesis [46,47]. Because methyl-Se compounds can be demethylated ultimately to feed the H2Se-exchangeable metabolic pool (see Figure 1), both CHsSeCys and dimethylselenoxide can support GPX expression [116]. Despite that phenomenon, evidence indicates that CHaSeH and its precursors have anticarcinogenic actions independent of those associated with the H2Se pool. Ip et al [112-117] found that arsenic, which competitively inhibits both the methylation of H2Se and the demethylation of CHsSeH (and the analogous di- and tri-methylated species) greatly reduced the anti-tumorigenic effects of selenite while enhancing those of selenobetaine or methylselenocysteine (CHjSeCys) which yields CHjSeH metabolically. Specifically, CHaSeHprecursors were shown to lack the genotoxic (DNA single-strand breaks [106,107,118,133] or DNA-oxidative damaging [134]) effects of selenite or selenide. The anti-carcinogenic activities of the methylated Se-metabolites and synthetic Se-compounds are likely related to reactions with critical proteins as well as to redox cycling, which effects may selectively impact the transformed phenotype. Ganther [128] described ways in which Secompounds may affect cellular proteins: through the formation of selenotrisulfide (-S-Se-S-) and selenylsulfide (-S-Se-) bonds and the catalysis of disulfide bonds formation/ dissolution, which would affect the activities of many enzymes with critical sulfhydryl groups; and through the formation of diselenide bonds (-Se-Se-) affecting the activities of selenoproteins which have SeCys residues at their active centers. Selenium-induced inhibition, presumably due to one or more of these reactions, has been demonstrated for a variety of relevant enzymes: ribonuclease [135], Na,K-ATPase [136], PKC [127,137,138]. Inhibition of PKC would be particularly important, as that enzyme system is known both to activate nuclear transcriptional factors and to bind phorbol ester-type tumor promoters. The inhibition of PKC by a Semetabolite such as CHsSeH would be expected to trigger a number of downstream effects including cell cycle arrest, apoptosis and angiogenic switch regulation. Evidence for at least some of these effects has been reported in response to the CHsSeH-precursors: decreased cdk2 kinase activity [126]; decreased DNA synthesis and elevated gadd gene expression [107]; inhibition of vascular endothelial MMPs and VEGF expression [139]. Thus, it appears that Se- doses large enough to support high, steady-state concentrations of CHsSeH can effect anti-carcinogenesis by inhibiting critical redox-sensitive factors including PKC and, probably, NF-kB [140] and AP-1, thus, impairing tumor cell metabolism and transformation. These effects would appear to be fairly targeted to certain factors, rather than involving wider perturbations in cellular redox control. After all, Semetabolites are typically present in tissues in much lower (nano- to micro-
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molar) concentrations than those (miUimolar) of thiols. In fact, susceptibility to redox modification by Se-attack seems to be limited to structures containing clustered cysteinyl residues [137,138]. Many of the effects of Se-compounds on cell proliferation may result from their abilities to form catalytically active, redox-cycling intermediates. Selenite, diselenides and the oxidation product of H2Se, selenium dioxide, for example, can each react with GSH to produce the selenolate ion (RSe) [141-143]. In the presence of GSH and molecular oxygen, RSe" can cycle continuously to generate Oi' and H2O2. This redox cycling is thought to be the basis of Se-toxicity, and it is possible that it may also contribute to anticarcinogenesis. Spallholz et al (144) found that dimethyldiselenide ([CH3Se]2) was the most catalytically active of a series of 19 Se-compounds^ in its ability to generate in vitro O2" in the presence of GSH and O2. They attributed this activity to CHsSeH produced by the reduction of ([CH3]Se)2 presumably generating the radical anion CHsSe'; however, it remains to be determined whether such catalytically active species can be generated intracellularly as the result of the metabolism of proximal (e.g., CHsSeCys, CH3Se02H) and/or upstream (e.g., SeMet, SeCys) precursors. There is no evidence that the common forms of Se in foods and feedstuffs, the selenoamino acids selenomethionine (SeMet) and selenocysteine (SeCys), are directly anticarinogenic. However, each can be metabolized first to H2Se and, then, to CHsSeH (see Figure 1). That conversion occurs directly for SeCys, which cannot be used directly in general protein synthesis; it is catabolized by a lyase to yield H2Se. The process is not direct for SeMet, which can enter the general protein pool as a mimic of methionine (Met). In fact, the conversion of SeMet from either dietary or proteinturnover sources necessarily involves its first being converted to SeCys by the Met-transsulfuration pathway. For this reason, most studies have found SeMet to be generally less anti-carcinogenically efficacious than SeCys or selenite [145-149], as would be expected in short-term studies and, particularly, under conditions of limiting Met supply. However, under steady-state conditions effected by long-term use, and particularly with highMet diets, one would expect the anti-tumorigenic efficacy of SeMet to approach that of SeCys and selenite. A number of synthetic Se-compounds have also been found to be anticarcinogenic. Ip et al [149] tested a series of alkylselenocyanates (H[CH2]xSeCN) using the DMBA-induced murine mammary carcinogenesis model, finding that anti-carcinogenic efficacy varied directly with increasing ^In addition to ([CH3]Se)2, these included nine other catalytically active Se-compounds: selenite, selenium dioxide, selenocystine, selenocystamine, diselenopropionic acid, diphenyldiselenide, dibenzyldiselenide, pXSC and 6-propylselenouracil; and nine Se-compounds that were not catalytically active: elemental Se, selenate, SeMet, CHaSeCys, selenobetaine, dimethylselenoxide, selenopyridine, TPSe and potassium selenocyante.
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Selenium: Its molecular biology and role in human health
chain length up to five carbons. The same group [150] also showed that allyl-selenocysteine, which is expected to yield allylselenol, a fairly hydrophobic metabolite, is more anti-carcinogenic than the corresponding alkylseleno-cysteine. Several aryl selenocyanates have also been found to be anti-tumorigenic. The more effective of these are benzylselenocyanate [151153], p-methoxybenzyl-selenocyate [152],/;-phenylselenocyanate [152-156]. These compoimds are thought to undergo initial metabolism through arylselenol, which may explain their similar responses to the alkylselenocyanates and other CHsSeH-precursors. Each induces apoptosis of cancer cells in vitro without inducing DNA single strand breaks. When compared to selenite on a molar basis, these forms are not only less effective in supporting GPX expression but also less toxic; yet, they offer comparable anti-tumorigenic efficacy [157,158]. It would appear that the anticarcinogenic efficacies of these synthetic Se-compounds are related to their relative lipophilicities and, thus, to uptake/retention by transformed cells. Accordingly, their anti-tumorigenic efficacies would appear to be affected by dietary fat intakes, being enhanced by the use of low-fat diets [159]. That anti-carcinogenicity need not involve selenoprotein expression is again evidenced, this time by triphenylselenonium chloride (TPSe), which is antitumorigenic at fairly high levels of exposure (dietary EC5o=15 ppm for preventing DMBA-induced mammary cancer [158]). The Se in TPSe is tightly bonded to three unsubstituted benzene rings rendering it unavailable to metabolism, ineffective in supporting GPX expression in the Se-deficient rat, and without adverse effects on rat growth at dietary levels as high as 200 ppm [160]. Conclusion Increasing evidence shows that Se-compounds can inhibit and/or delay carcinogenesis in animal models and reduce the risks for at least some kinds of cancer in humans. These effects may involve the protective, nutritional functions of Se as an essential constituent of a number of metabolically important selenoenzymes; such functions may be compromised in Sedeficient individuals. Recent evidence suggests that allelic variants of some selenoproteins may be related to cancer risk. In addition to such effects, certain Se-metabolites, notably methyl-Se compounds, appear to inhibit carcinogenesis through mechanisms unrelated to the nutritional functions of Se and at doses greater than necessary for such functions, i.e, at supranutritional levels of exposures. Thus, the emerging picture is of Se as a nutrient that functions in anti-carcinogenesis in two ways: as an essential constituent of metabolically important selenoproteins, and as a source of anticarcinogenic metabolites. Because selenoprotein expression appears to be optimized at lower levels of Se exposure than are necessary to support the
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262 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.
Selenium: Its molecular biology and role in human health WJ Blot, JY Li, PR Taylor, W Guo, SM Dawsey, B Li 1995 Am J Clin Nutr 62:14248 WJ Blot et al 1993 J Nat Cancer Inst 85:1483 WJ Blot 1997 Proc Soc Exp Biol Med 216:291 K Krishnaswamy, MP Prasad, TP Krishna, VV Annapuma, GA Reddy 1995 Eur J Cancer 31:41 MP Prasad, MA Makunda, K Krishnawamy 1995 Eur J Cancer 31B:155 LCClarketal 1996y.4/«iWe^^i5oc 276:1957 LC Clark et al 1998 Brit J Urol 81:730 AJ Duffield-Lillico, et al 2002 Cancer Epidem Biomarkers Prev 11:630 ME Reid et al 2002 Cancer Epidem Biomarkers Prev 11:1285 AJ Duffield-Lillico et al 2003 Br J Urol 91:608 AJ Duffield-Lillico et al 2003 J Nat Cancer Inst 95:1477 J N^ve 1995 J Trace Elements Med Biol 9:65 Panel on Dietary Antioxidants and Related Compounds 2000 Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Beta-Carotene and other Carotenoids. National Academy Press Washington DC KE Burke, GF Combs Jr, EG Gross, KC Bhuyan, H Abu-Libdeh 1992 Nutr Cancer 17:123 BC Pence, E Pelier, DM Dunn 1994 J Invest Dermatol 102:759 AM Diamond, P Dale, JL Murray, DJ Grdina 1996 Mutat Res 356:147 Y Kise et al 1991 Nutr Cancer 16:153 GF Combs Jr, LC Clark, BW Tumbull 2001 Proc 7'* Internat Symp Selenium Biol Med 152 J Lii, C Jiang 2005 Antioxidants Redox Signaling 7:1715 M Berggren et al 1996 Anticancer Res 16:3459 GPowisetal 1996^nricancerZ)n.001)
The immune response to influenza is characterized by 2 stages, an innate stage and a cell mediated stage. The initial, innate response, involving natural killer (NK) cells, dendritic cells and macrophages is essential for directing the subsequent cell-mediated response. We examined key aspects of the innate response in Se-deficient mice. Specifically, interferon (IFN)-a and IFN-P mRNA, which help control viral replication and activate a host of anti-viral genes, were reduced in Se-deficient influenza infected mice (Figure 3). IFN-y, which is produced by NK cells early in infection, was also reduced in Se-deficient infected mice (Figure 3). Interestingly, NK cell activity is unaffected in Se-deficient mice (data not shown). Together, these
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data indicate that Se-deficient mice have impaired, early anti-viral cytokine responses.
IFN-a
IFN-P
IFN-Y
Figure 3. Quantitative RT-PCR was performed for the cytokines and normalized to G3PDH. The data are expressed as arbitrary units +/- SEM. Se deficiency resulted in 2-4 fold decrease in production of IFN-a, IFN-P and IFN-y, at 24h p.i.
In order for the lung inflammation to occur in the infected influenza mice, a co-ordinate production of chemokines must occur. This process was altered in the Se-deficient mice. Chemokine mRNA levels for RANTES, MlP-la, MIP-P and MCP-1 were highest on days 4 and 5 post infection for the Se-adequate mice, and then began to decrease, whereas these chemokines were highest at later time points for the Se-deficient mice [19]. Clearly, Se deficiency leads to an increase in influenza-induced histopathology which is associated in part with altered chemokine and IFN expression. Because host Se deficiency induced changes in the coxsackievirus genome, we reasoned that the increased virulence of the influenza virus in the Sedeficient mice may also be due to changes in the viral genome. Virus was recovered from the lungs of Se-deficient and Se-adequate mice and all 8 viral RNA segments were sequenced and compared with the sequence of the input strain. Surprisingly, few changes were found in the HA and NA segments of the virus, which are associated with a high mutation rate. Changes in the HA and NA segment were random, and found in viruses obtained from both Seadequate and Se-deficient mice. In stark contrast, however, the M gene
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contained multiple mutations [20]. As shown in Table 1, three separate isolates from 3 individual Se-deficient mice all had identical mutations in 29 positions. One of the 3 isolates from a Se-deficient mouse had an additional 6 mutations. None of these changes were seen in viruses obtained from the Se-adequate mice. Thus, as for coxsackievirus, influenza virus replicating in a Se-deficient host undergoes rapid genetic change, resulting in a more virulent virus which can now cause disease even in a host with normal Se status. Poliovirus and Se deficiency A recent study has reported that subjects in the United Kingdom with low Se status (< 1 i^Mol/L) have a decreased immune response to poliovirus vaccination [21]. Of particular note, they found an increased mutation rate of the vaccine strain of vaccine virus which had been shed in the feces. Supplementation with Se of the low Se status population enhanced the immune response and lowered the number of mutations found in the shed vaccine strain of the virus. Thus, low Se status was associated with increased mutation rate of the live attenuated poliovirus vaccine strain when compared with vaccinated individuals supplemented with Se. To assess the mutation rates, the investigators utilized temporal temperature gradient electrophoresis (TTGE). Although this technique can identify mutations occurring in the genome, it does not provide information on which specific nucleotides were altered. The Broome et al. study supports the hypothesis that polio vaccination of individuals with low Se status may lead to increased mutations in the vaccine strain of virus. This area of research is particularly relevant in view of recent findings that attenuated poliovirus vaccine sfrains have circulated and reverted to virulence in several areas of the world where undernutrition is prevalent [22]. Selenium and other viruses Infection with human immunodeficiency virus (HIV) results in a loss of CD4+ helper T cells and subsequent immune dysfunction leading to increased opportunistic infections. In addition, oxidative stress increases during an HIV infection. A number of studies have examined the relationship between specific nutritional factors and disease progression and survival of HIV infected individuals. Se status of HIV infected individuals has also been studied. In developed countries (France and the US), 3 studies have demonstrated that lower serum Se levels are associated with an increased risk of mortality from HIV [23-25]. In a study of HFV infected pregnant women in Tanzania, low selenium status was found to be associated with accelerated HIV disease progression [26].
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Table 1. Comparison of nucleotide sequences of influenza A/Bangkok/1/79 M gene of the infecting virus and of virus isolated from Se-adequate (Se+) and Se-deficient (Se-) mice.
Nucleotide Position 136 205 238 309 322 325 328 331 334 370 371 406 439 454 455 502 503 524 525 544 566 567 568 610 619 652 655 667 669 670 677
Infecting I Virus A G G G A C A A T A G C A C C
cA G G A C C G A G C G G G A G
Se status of host virus isolated from: Se± Se+ Se+ Se; Soz Sez A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G
A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G
A G G G A C A A T A G C A C C C A G G A C C G A G C G G G A G
C A G A C T G C C C T T G A C T C A G C T C A G A T A A G G A
C A G A C T G C C C T T G A C T C A G C T C A G A T A A G G A
C A A A C T G C C C T T G A A T C A A C T T A G A T A A A G A
AA Change
RtoK
AtoS
AtoT TtoA
AtoT
AtoT
Se levels have also been inversely correlated with hepatitis B virus infection. Lifection with hepatitis B virus is a major health problem throughout the world. In addition, chronic hepatitis B infection is thought to
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be a significant factor in most hepatocellular carcinomas, a highly malignant neoplasm with a high mortality rate. A study from Taiwan [27] demonstrated that mean Se plasma levels were significantly lower in hepatocellular carcinoma patients, as compared with individuals testing positive for hepatitis B virus. A further study from Qidong county in China [28] demonstrated a protective effect of Se supplementation in a population at high risk of developing primary liver cancer due to a high prevalence of hepatitis positive individuals. Conclusion Low host selenium status has been shown to be important in driving viral mutations. This increase in viral mutations in a Se-deficient host may be due to an increase in oxidative stress status, as virus which replicated in GPX-1 knockout mice also mutated. Emerging viruses are either newly arisen viruses or are viruses that are rapidly expanding their range. Understanding the mechanisms underlying the evolution of emerging viruses is critical to predicting new viral outbreaks and devising new strategies to limit the emergence and spread of these new pathogenic forms. Data from the Se studies demonsfrates that host Se status is a driving force for emergence of new viral variants. These observations suggest a new area for research, namely the interaction of host nufrition and viral evolutionary processes. The precise mechanism(s) by which a deficiency in Se leads to mutations in a viral genome remains to be determined. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BQ Gu 1983 Chin Med J 96:251 C Su, C Gong, J Li, L Chen, D Zhou, Q Jin 1979 Chin MedJ59:466 LQ Ren, XJ Li, GS Li, ZT Zhao, B Sun, F Sun 2004 World J Gastroenterol 10:3299 JF Woodruff 1980 Am J Pathol 101:427 J Bai, S Wu, K Ge, X Deng, C Su 1980 Acta Acad Med Sin 2:29 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander \994 J Infect Dis 170:351 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander 1994 J Med Virol 43:166 JK Reffett, JW Spears, TT Brown Jr 1988 J Animal Sci 66:1520 JF Reffett, JW Spears, TT Brown Jr 1988 JNutr 118:229 DN Cook, MA Beck, T Coffman, SL Kirby, JF Sheridan, IB Pragnell, O Smithies 1995 5«e«ce 269:1583 MA Beck, CC Matthews 2000 Proc Nutr Soc 59:1 MA Beck, Q Shi, VC Morris, OA Levander 1995 Nat Med 1:433 MA Beck, RS Esworthy, Y-S Ho, F-F Chu \99SFASEBJ \2:l\43 MA Beck, PC Kolbeck, LH Rohr, Q Shi, VC Morris, OA Levander 1993 J Nutr 124:345 MA Beck, Q Shi, VC Morris, OA Levander 2005 Free Radic Biol Med 38:112 CB Bridges, SA Harper, K Fukuda, TM Uyeki, NJ Cox, JA Singleton 2003 Morb Mortal Wkly Rep 52:1 BR Murphy, RG Webster 1996 Fields Virology BN Fields (ed) Lippincott-Raven Philadelphia PA pi 397
298 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Selenium: Its molecular biology and role in human health AC Ward 1997 Virus Genes 14:187 MA Beck, HK Nelson, Q Shi, P Van Dael, EJ Schiffrin, S Blum, D Barclay, OA Levander 2001 F^SESy 10.1096/fj.00-072ige HK Nelson, Q Shi, P Van Dael, EJ Schriffrin, S Blum, D Barclay, OA Levander, MA Beck. 2001 FASEBJ\0M9m].Q\-Q\\5f)e CS Broome, F McArdle, J A Kyle, F Andrews, NM Lowe, CA Hart, JR Arthur 2004 J Clin Nutr m:\54 OM Kew, RW Sutter, EM de Gourville, WR Dowdle, MA Pallansch 2005 Ann Rev Microbiol 59:5S7 MK Baum, G Shor-Posner, S Lai, G Zhang, H Lai, MA Fletcher, H Sauberlich, JB Page 1997 y.4/D5 15:370 A Campa, MK Shor-Posner, F Indacochea, G Zhang, H Lai, D Asthana, GB Scott, MK Baum 1999 y^/DS 20:508 J Constans, JL Pellegrin, C Sergeant, M Simonoff, L Pelegrin, H Fleury, B Leng, C Conri 1995 y4/DS 10:392 R Kupka, GI Msamanga, D Spiegelman, S Morris, F Mugusi, DJ Hunter, WW Fawzi 2004 yiVMfr 134:2556 M-W Yu, I-S Homg, K-H Hsu, Y-C Chiang, Y-F Liaw, C-J Chen 1999 Am J Epidemiol 150:367 SY Yu, YJ Zhu, WG Li 1997 Biol TraceElem Res5(,:\\l
Chapter 26. Role of selenium in HIV/AIDS Marianna K. Baum and Adriana Campa Florida International University, Stempel School of Public Health, Department of Dietetics and Nutrition, U200SW8th Street, Miami, Florida 33199, USA
The advent of Highly Active Antiretroviral Therapy (HAART) in the late 90s has transformed HIV infection from a deadly condition into a chronic, manageable viral infection in developed countries [1]. The developing world, however, accounts for 96% of the global HIV-l infections, and in most of these countries, antiretrovirals are not yet widely available. The number of persons living with Human Immuno-Deficiency Virus (HIV) infection and Acquired Immuno-deficiency Syndrome (AIDS) worldwide has been estimated to be approximately 40 million [2], and this figure includes approximately 5 million people who acquired HIV in 2004. In the same period, approximately 3.1 million adults and children died from AIDS, and 14,000 new individuals are still infected daily, a number that lessens hopes for a rapid solution to this pandemic [2]. The gap between developed and developing countries in the control of the pandemic and treatment of infected persons is growing, and one of the factors fueling the epidemic in poor countries is malnufrition. Moreover, protein-energy malnutrition (PEM), and the accompanying and aggravating micronufrient deficiencies, are already an overwhelming health problem and still the main cause for immune disturbances in poor countries [3,4]. Sub-Saharan Africa, where the greatest growth in severe and generalized malnutrition has occurred in the last two decades [5], is also the region in which 12 out of 44 countries have more than 10% prevalence of HIV in the adult population [6]. Numerous studies have demonstrated that nutritional deficiencies accelerate HIV disease progression and decrease survival [7-16]. Moreover, nutrient deficits interfere with the effectiveness of antiretrovirals by delaying the recuperation of the immune system and aggravating side-effects attributed to treatment [17-20]. Selenium appears to have a multifactorial role in HIV-l infection. Selenium status affects HIV disease progression and mortality [14-16] through various potential mechanisms. In two recent studies, deficiency of selenium has been associated with elevated measures of HIV infectivity [21,22], and therefore, with increased potential to transfer the infection.
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Selenium is required for the function of gluthatione peroxidase, a biological antioxidant that protects against oxidative stress. Other selenoproteins may also act as antioxidants by the incorporation of selenocysteine in their molecules [23]. In HIV-infected persons, dietary selenium intake was strongly associated with reduced measures of oxidative stress [24]. Adequate selenium status may also be essential for controlling viral emergence and evolution [25,26]. In addition, selenium may enhance resistance to infection through modulation of both cellular and humoral immunity. Plasma selenium levels are associated with interleukin production and subsequent changes in Thl/Th2 cytokine responses [27,28]. Other nutritional factors interact with selenium status and are important in HIV-1 disease progression and mortality. These factors include disease stage, nutritional status at the onset of the disease, types of treatment and compliance, and secondary infections that may act independently or in combination. Treatment of malnutrition, and the accompanying micronutrient deficiencies, thus, requires a carefully individualized approach. This chapter will review the role of selenium in HFV-l disease progression, morbidity and mortality, as well as the factors that may affect these relationships. Selenium and immunity in HIV Selenium has been shown to affect the immune process [29]. In vivo and in vitro studies suggest that selenium may act at different levels of immune function. In animal models, selenium deficiency was shown to impair the ability of phagocytic neutrophils and macrophages to destroy antigens, and selenium status was associated with humoral immune response [30]. In humans, Broome and colleagues [31] found that in a population of sixty-six healthy participants who were marginally deficient in selenium ( 25 selenoprotein genes identified in mammalian systems (see Chapter 9). Seleno-enzymes as peroxynitrite reductases Protection against oxidative damage is the mechanism by which selenium is likely to exert some protective effects on immunity. Nitric oxide (NO) has
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microbiocidal effects, yet under oxidative conditions in which superoxide (O2") is produced concomitantly (by neutrophils and mononuclear phagocytes), the highly reactive and destructive oxidant, peroxynitrite (ONOO) is produced. Experiments performed in vitro show that the addition of selenocysteine and selenomethionine protects plasmid DNA from ONOOmediated damage [6]. Selenomethionine, selenocysteine and the GPx mimic ebselen were more effective protectants than selenite, showing specificity in the reaction [7]. However, selenoenzymes including GPx and TR also protect from ONOO-mediated damage [8-9]. The roles of the GPx and TR families as a peroxynitrite reductase need to be further elucidated by studies in intact cells. Selenium and eicosanoid metabolism The eicosanoids include the leukotrienes, the thromboxanes, the prostaglandins and the lipoxins (see [10] for review). Selenium (mediated through GPxl and GPx4) probably has anti-inflammatory effects, preventing the release of inflammatory mediators through reduction of organoperoxides which mediate production of active leukotrienes. The conversion of arachidonic acid to prostaglandin GG2 is catalysed by cyclo-oxygenase. This enzyme requires a minimal level of peroxide to function. However, if peroxide levels in the cell are high, cyclo-oxygenase activity is also inhibited [11,12]. Despite the fact that reduction of the hydroperoxyeicosatetraenoic acids to hydroxyeicosatefraenoic acids requires the reductive power of peroxidases, the resultant products are generally proinflammatory. Anti-inflammatory activity of selenium may be explained by the ability of selenoenzymes to inhibit the 5- and 15-lipoxygenase enzymes, which convert arachidonic acid to the 5-hydroperoxyeicosatetraenoic acid precursor of the leukotrienes [13,14]. The conversion of selenite to selenide (which inhibits lipoxygenase) appears to be catalysed by a reaction of NADPH with TR [15]. Selenium deficiency also leads to decreased leukotriene B4 synthesis, this leukotriene impairs the functions and mobility of phagocyctes [16]. Another important effect of selenium deficiency is the disturbance in the balance of production of the pro-coagulant thromboxanes and the anticlotting prostacyclins [17,18]. This could underlie the prevalence of atherosclerosis in populations which have low dietary selenium intake (see [19] for review). Evidence suggests that selenium supplementation can only protect against atherosclerosis in populations whose selenium intake is below the recommended daily allowance [20]. Platelet GPxl activity is sensitive to the effects of selenium deficiency in humans, this being associated with increased aggregation, thromboxane B2 production and the synthesis of lipoxygenase-derived products. In such people, selenium supplementation
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increases platelet GPxl activity and decreases hyperaggregation (see [11] for review). Effect of selenium on adhesion molecules and cytokines Pro-inflammatory cytokines, such as tumor necrosis factor-a and interleukin1, induce many of the adhesion molecules which are upregulated in inflammation. Existing evidence is more consistent with a selenium effect on adhesion molecule expression through regulation of cytokine release. In general, selenium-deprived cells or endothelium from selenium-low individuals have a higher constitutive expression of adhesion molecules, and selenium supplementation decreases their expression. Endothelial cells obtained from asthmatic patients, had significantly higher constitutive expression of P-selectin, vascular adhesion molecule-1, E-selectin and intercellular adhesion molecule than cells from normal subjects. However, after 3 months of selenium supplements a significant decrease in vascular adhesion molecule-1 and E-selectin expression was observed [21]. This was also confirmed by treatment of cultured endothelial cells with 6 nM to 48 nM selenium. Similarly, bovine endothelial cells grown under selenium-deficient conditions and stimulated with tumor necrosis factor-a, had higher levels of E-selectin, P-selectin expression and intercellular adhesion molecule-1, which was manifested by greater adherence of neutrophils [22]. A GPx mimic inhibited the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 and GPx analogs prevented tumor necrosis factor-a-stimulated expression of P-selectin and E-selectin, as well as tumor necrosis factor-a and interleukin-1-stimulated interleukin-8 release in human endothelial cells [23, 24]. Oxidative stress induces several pro-inflammatory cytokines including: interleukin-1, interleukin-6, interleukin-8 and tumor necrosis factor-a possibly through the activation of the transcription factors AP-1 and N F - K B [25]. Pre-incubation of keratinocytes with selenium abrogated upregulation of the mRNAs for interleukin-6 and interleukin-8 [26] in response to ultraviolet radiation B, a potent environmental oxidative stress. IL-10 immunostaining in murine keratinocytes was also suppressed by pretreatment with selenomethionine before UVB treatment [27]. The speciation of selenium is probably important for the frace element to exert optimal effects. For example, selenomethionine seems a better protectant than selenite against ultraviolet radiation B-induction of cytokines. Since GPxl depletes reduced glutathione, cytokine release into the culture fluid of endothelial cells increased after treatment with selenite [28]. Also, selenite (but not selenomethionine) supplementation of BALB/c mice increased the release of interleukin-1 and tumor necrosis factor-a from phytohaemagluttinin-P stimulated splenic macrophages [29]. This could result from the pro-oxidant effects of high doses of selenite. Moreover
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selenite and selenocystamine, but not selenomethionine, increased oxidative stress, oxidative DNA damage and apoptosis in keratinocytes [30]. There are few reported studies on the effects of cytokines on selenium metabolism. Treatment of the liver cell line HepG2 with interleukin-ip, tumor necrosis factor-a, or interferon-y had no effect on selenoprotein P expression. However, transforming growth factor-P (100 pM for 48 h) led to a 21% decrease in expression of selenoprotein P mRNA [33]. Incubation with transforming growth factor-P also down regulated mRNA and activities of GPxl and catalase [33]. Effects of selenium on cell-mediated and humoral immunity Studies of the effects of selenium on cell-mediated immune cells and on antibody production have been reviewed up to 1990 [5]. Many of these studies were performed by veterinary researchers and used simultaneous supplements of selenium and vitamin E, since the nutrients can act synergistically, and may substitute for each other. This is a factor that needs to be borne in mind when interpreting these early studies. In rats, selenium deficiency decreased IgG production slightly, but had no effects on IgA production. However, IgM production was greatly decreased and lowered further by vitamin E deficiency. Selenium supplementation partially ameliorated the vitamin E deficiency-induced decreases in IgA and IgG [34]. IgG levels were higher in cows given 120 ng/kg selenium and calves fi"om these cows had higher post suckle serum IgG levels. Thus, maintaining optimal selenium intake may promote health of offspring as well as of the mothers. The effect of vitamin E and selenium supplements on the immune responses of domestic animals has been reviewed by Finch and Turner in 1996 [35]. Selenium-enriched diets given to poultry improve their antibody responses to Salmonella and aflatoxin vaccination [36]. A combination of vitamin E and selenium supplements in the diet increased both antibody titres to Newcastle disease virus and gave maximum gain in body weight [37]. In sheep vaccinated against Chlamydia psittaci, which causes abortion, injection of selenium (0.1 mg/kg) alone increased the Chlamydia antibody response. However, this was decreased if coadministered with vitamin E [38]. Selenium supplementation improves responses in most studies of cellmediated immunity. Selenium deficiency in rats results in decreased candidiacidal activity in neutrophils and impairs survival after Staphylococcus aureus infection [39]. Polymorphonuclear cells also provide defense against mastitis in cattle. Supplementation of such cells in vitro with selenium and vitamin E increased superoxide production and migration after stimulation with phorbol esters [40 Selenite enhances chemotaxis of macrophages. Murine infection with the parasite Trypanosoma cruzi was treated with selenium at 0 ppm, 2 ppm, 4
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ppm, 8 ppm, or 16 ppm as sodium selenate in drinking water [41]. Sixty four days after infection, the mice without selenium supplements had all died. But 60% of the animals in groups supplemented with 4 and 8 ppm selenium survived. Survival was much less in the group fed 16 ppm, indicating an optimal dose is important for selenium protection. Studies with experimental and agricultural animals all support a role for selenium, albeit not always consistent, in maintaining components of the immune system and the ability to resist parasitic infection [42-44]. The importance of selenium and other nutrients in maintaining resistance to viral infection has been proved in a number of studies mainly with mice [44]. As well as decreasing selenium status, knockout of glutathione peroxidase 1 also enhances the negative effects of viruses in mice [44]. Effects of selenium on interleukin-2 receptor and lymphocytes Selenium augments the performance of both T- and B-lymphocytes and perhaps the common effect is through the up-regulation of the interleukin-2 receptor a and p subunits which results in a greater number of high affinity interleukin-2 receptors in mice [45] and humans [46]. This is accompanied by enhanced proliferation and differentiation into cytotoxic effector cells [47]. Selenium supplementation in humans (200 )J.g/day for eight weeks) also up-regulates the activity of cytotoxic T-cells (118%), natural killer cells (82%) [47] and down-regulates the activity of suppressor T-cells. The lytic capabilities of natural- and lymphokine activated-killer cells in humans, were increased, purportedly by up-regulation of interleukin-2 receptors [48]. In rats given selenite (0.5 ppm, 2.0 ppm or 5.0 ppm) in the water supply, the response of natural killer cells was boosted in the group receiving 0.5 and 2.0 ppm selenite, but cell activity in the 5.0 ppm group was similar to that of unsupplemented animals. Antibody synthesis was not significantly increased, but fell in the group given 5.0 ppm selenium. Production of prostaglandin E2 was decreased at all selenium doses [49]. Similarly, an inhibitory effect of selenite on natural killer cell activity and lymphokine activated killer cell activity was seen in human lymphocytes supplemented in culture with 0.8 fig/ml selenite. However, this is a very large dose and may be toxic. In the same study, lymphocyte proliferation to T-cell mitogens was suppressed by selenium in the range of 0.5-1.0 fig/ml [50]. Activated T-cells have increased activity of the enayme selenophosphate synthetase, which is essential for the synthesis of selenocysteine, an obligatory step in selenoprotein synthesis [51]. This is consistent with higher concentrations of selenium found in immune-active tissues such as spleen, lymph nodes and liver [4]. The importance of selenium in maintaining cellular immunity was further supported by studies in uremic patients [52]. Patients had lower plasma selenium concentrations than controls. Supplementation with 500 ^g of selenium thrice weekly for three months
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was followed by 200 ng/day for the next three months. Although no change in lymphocyte numbers or subpopulations was observed, delayed-type hypersensitivity responses (to phytohaemoagluttinin) were significantly higher in the selenium-supplemented group after 6 months, compared with their own pre-experimental levels and a placebo group. The augmented responses dropped to pre-supplementation values 3 months after ceasing selenium supplementation. The overall conclusion was that selenium supplementation could be beneficial in uremic patients. On the other hand, there was no improvement with selenium and zinc supplements on the delayed-type hypersensitivity responses in elderly patients, despite an improved humoral immune response to influenza vaccination [53]. Selenium and immune-mediated disease in humans From the findings listed above, it may be expected that selenium would have beneficial effects on inflammatory conditions such as rheumatoid arthritis. However, there are few blinded, controlled trials exploring such possibilities. Epidemiological studies indicate that serum selenium levels are correlated with disease state. However, reports do not agree on association.s of disease with GPx activities in rheumatoid arthritis patients. This could be because sub-forms of the disease were not categorized in some studies. Moreover, in some variants of the disease neutrophil GPx activity was not increased by dietary selenium supplementation. Thus lack of effect of selenium on arthritic symptoms in some studies may reflect an already adequate selenium status. The results of several studies have been summarized (see [19,54] for review). Nevertheless, some studies have shown that low selenium status may be a risk factor for rheumatoid factor-negative (but not rheumatoid factor-positive) arthritis [55]. Because selenoproteins can be acute phase reactants, a decrease in plasma selenium is not necessarily associated with loss of antioxidant function. In Crohn's disease patients -in which immune activation may be mediated by reactive oxygen species- there was a negative correlation between plasma selenium and soluble interleukin-2 receptor and erythrocyte sedimentation rate. The soluble interleukin-2 receptor concentration is positively correlated with the degree of immune activation [56]. Oxidative stress and micronutrient deficiencies have been identified along with selenium deficiency and decreased red cell GPx activity as risk factors for the development of asthma (see [57] for review). In juvenile asthma patients with intrinsic disease, 100 \x.g selenite/day improved their clinical symptoms [58]. Protection against asthmatic wheeze has been found in adult asthma patients in England [19] and in asthmatic children in New Zealand [59]. The reasons for this are not clear, but atopic asthmatics have low platelet and red blood cell GPx activities. However, GPx activity was higher in eosinophils in both normal and asthmatic subjects (but was not different
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between these groups) than in neutrophils from both groups. The higher GPx activity of eosinophils may have prolonged their survival at inflammatory sites, thus driving the inflammatory process [60]. An increase in the incidence of asthma has recently been noted in the Western World and more studies into potential benefits of increased selenium intake in asthmatics are warranted. Clinical trials on sepsis and systemic inflammatory response syndrome patients suggest that these patients have low plasma selenium and GPx activity. Two independent prospective studies showed a beneficial therapeutic response in patients given selenium supplements [61]. Selenium supplementation of otherwise healthy human subjects can also have beneficial effects on immune related processes. In UK subjects with blood selenium levels of approximately 1 (iM, pre-treatment with selenium has improved polio virus handling after vaccination. This was associated with an augmented cellular immune response manifest as increased interferon-y gamma and IL-2 and IL-10 production [62]. Other studies that have increased selenium intake in human volunteers have resulted in improved activation and proliferation of B lymphocytes [63]. Lipoxygenase activity in lymphocytes is also associated with a small nucleotide polj^norphism in GPx4 [64]. In the macrophage cell line J774.1 selenium in vitro enhances phagocytosis, degranulation and production of superoxide after stimulation. Additionally, TNFa, IL 1 and IL 6 release was enhanced when compared with selenium-deficient cells [65]. Selenium and the skin The skin is the body's largest organ and as well as its main interface with the environment. Skin is continually exposed to many stresses due to the products of commensal organisms on the surface as well as the oxidative stress and cell damage caused by exposure to ultraviolet radiation. Both selenomethionine and selenite at nanomolar concentrations can protect keratinocytes, melanocytes and fibroblasts from UV-induced cell death and apoptosis. The processes behind these effects include inhibition of oxidative DNA damage, lipid peroxidation, apoptosis, suppression of inflammatory and immune suppressive cytokine release, and modulation of p53 activity (see [66] for review). Selenium can prevent UVB-induced skin tumors in hairless mice, (see [66] for discussion); it remains to be seen if the protective effect against skin cancer in mice also operates in humans. In skin, selenium deficiency in vivo may impair immunity by decreasing infiltration of Langerhans cells after stimulation by UV irradiation [67]. Selenium and aging Throughout life, cells accumulate oxidative damage -"the oxidative theory of aging" - (see [68] for review) and the aging lymphocyte population fails to
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expand as effectively on antigenic challenge, resulting in damage to both mitochondrial and nuclear DNA. Apart from lipid peroxidation, there is an accumulation of carbonyl moieties on protein, both types of lesions being produced by oxidative stress. Due to oxidative metabolism, mitochondria accumulate damage, which helps release more reactive oxygen species, exacerbating the process [68]. For example, treatment of fibroblasts with non-lethal doses of hydrogen peroxide activates a senescence program, which leads to growth cessation. Thus, a role for GPxl and other selenoproteins in slowing cellular damage and the aging process is possible. The efficiency of the immune system declines with age and the elderly are more prone to infections than young or middle aged adults. Generally, the response to antigen challenge and the ratio of effector to naive T-cells decreases, along with a decrease in the ratio of CD4 to CDS T-cells and decreased ratio of CDS" to CD5^ B-cells [69]. There is also decreased ability of macrophages and monocytes to destroy microbes. For example, aged mice produced weak interferon-y and interleukin-2 responses to the parasite Trypanosoma musculi [70]. Decreased proliferation of spleen lymphocytes to allogeneic or mitogen stimulation from aged mice was restored by dietary selenium supplements [71]. The mechanism appeared to be via upregulation of the interleukin receptor. Selenium supplementation in vitro enhanced the previously depressed chemotactic and cytokine release capabilities of polymorphonuclear cells from elderly donors [72]. In elderly humans, low blood selenium and erythrocyte GPxl activity was correlated with lower triiodothjmnine (T3) to thyroxine (T4) ratios, mainly due to raised T4 concenfrations, and was seen with advancing age [73,74]. Selenium supplementation decreased the serum T4 concentration. The agerelated decline in T3:T4 ratios was ascribed to impaired iodothyronine-5'deiodinase activity. Impaired T4 to T3 conversion will affect general metabolism, including immunity. Longevity in areas of the world which had selenium-rich soils was noted by Foster and Zhang in 1995 [75]. Less people over 80 years of age were found in areas where the selenium-deficiency diseases Kashin-Beck and Keshan disease were endemic [75]. A hypothesis has been put forward which proposes that the areas of the world which have higher lifespan than (national) average are areas where soil selenium is high, but mercury content, which sequesters selenium, is lowest [76]. In humans, cancer is a disease associated principally with old age, pointing to an age-dependent decrease in the efficiency of the immune system to detect and desfroy tumors. This may be due to a decrease in the effectiveness of natural killer cells allied to nutritional deficiencies. In a free-living elderly Italian women (ages 90-106 years of age), the percentage of natural killer cells in the circulation was related to serum selenium content [77]. An adequate selenium intake may maintain GPx activities in aging cells. Increasing the intracellular hydrogen peroxide content by blocking GPx
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activity with buthionine sulfoximine and inhibiting catalase activity by aminotriazol treatment raised the levels of collagenase mRNA [78]. Collagenase-1 activity contributes to connective tissue damage, which is a feature of tumor expansion, inflammatory disease and photo-aging. GPxl activity is lower in neutrophils from human volunteers over 65 years of age compared with cells from younger volunteers [79]. The enzyme from the elderly group had a decreased V max compared with that in neutrophils from younger donors (21-34 years of age). Furthermore, in the cells from the young group, the affinity (Km) of the enzyme for its substrate increased on neufrophil activation which, did not occur in the elderly group. Finally, telomere length decreases with age in peripheral leukocytes and this is accelerated by oxidative stress in fibroblasts [80]. A role has been proposed for GPxl in the maintenance of telomere length. The rate of telomere shortening and carbonyl group accumulation was inversely correlated with GPxl activity in fibroblasts [80]. Furthermore, experiments with human breast cells which were fransfected with DNA constructs to produce lines that had differing GPxl expression, revealed an important role for the enzyme in the protection against oxidative-induced mitochondrial DNA damage. Lines contained 100 times differing GPx activities. Exposure to 25 nM menadione for 1 hour caused approximately three-fold more single-sfrand breaks and 8-oxo-deoxyguanosine residues in the low GPx lines [81]. As well as GPx, the mitochondrial TR [82] may play an important role in limiting oxidative damage in immune cells. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
JC Fantone, PA Ward 1982.4m J Pathol 107:397 JT Rotruck, AL Pope, HE Ganther, AB Swanson, DG Hafeman, WG Hoekstra 1973 Science 179:588 L Flohe, WA Gunzler, HH Schock 1973 FEBS Lett 32:132 RC Dickson, RH Tomlinson 1967 Clin Chim Acta 16:311 JE Spallholz, LM Boylan, HS Larsen 1990 Ann NY Acad Sci 587:123 I Roussyn, K Briviba, H Masumoto, H Sies 1996 Arch Biochem Biophys 330:216 K Briviba, I Roussyn, VS Sharov, H Sies 1996 Biochem 7319:1315 H Sies, VS Sharov, LO Klotz, K Briviba \997 J Biol Chem 272:27812 GE Arteel, K Briviba, H Sies 1999 Chem Res Toxicol 12:264 GR Davies, DS Rampton 1997 Euro J Gastroenterol Hepatol 9:1033 D Vitoux, P Chappuis, J Amaud, M Bost, M Accominotti, AM Roussel 1996 Annates De Biologic Clinique 54:181 MJ Pamham, E Graf 1987 Biochem Pharmacol 36:3095 O Werz, D Steinhilber 1996 Eur J Biochem 242:90 C Schewe, T Schewe, A Wendel 1994 Biochem Pharmacol 48:65 M Bjomstedt, B Odlander, S Kuprin, HE Claesson, A Holmgren 1996 Biochem 35:8511 C Gairola, HH Tai 1985 Biochem Biophys Res Commun 132:397 YZ Cao, CC Reddy, LM Sordillo 2000 Free Radical Biol Med 28:381 M Meydani 1992 Biol Trace Element Res 33:79 MP Rayman 2000 Lancet 356:233
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27. 28. 29. 30. 31. 32. 33. 34.
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JK Huttenen 1997 Biomed Environment Sci 10:220 M Horvathova, E Jahnova, F Gazdik 1999 Biol Trace Element Res 69:15 JF Maddox, KM Aheme, CC Reddy, LM Sordillo 1999 JLeuco Biol 65:658 P D'Alessio, M Moutet, E Coudrier, S Darquenne, J Chaudiere 1998 Free Radical Biol Med 24:979 M Moutet, P D'Alessio, P Malette, V Devaux, J Chaudiere 1998 FreeRadical Biol Med 25:270 G Powis, JR Gasdaska, A Baker 1997 Adv Pharmacol 38:329 RC McKenzie, TS Rafferty, GJ Beckett, JR Arthur 2001 in Selenium: Its molecular biology and role in human health DL Hatfield (ed) Kluwer Academic Publishers Chapter 21:257 TS Rafferty, C Walker, JA Hunter, GJ Beckett, RC McKenzie 2002 Br J Dermatol 146:485 R Tolando, A Jovanovic, R Brigelius-Flohe, F Ursini, M Maiorino 2000 Free Radical Biol Med 2^:919 VJ Johnson, M Tsunoda, RP Sharma 2000 Arch Environ Contamination Toxicol 39:243 MS Stewart, JE Spallholz, KH Neldner, BC Pence 1999 Free Radical Biol Med 26:42 GN Schrauzer 2000 JNutr 130:1653 JE Spallholz 1994 Free Radical Biol Med 17:45 V Mostert, I Dreher, J Kohrle, J Abel 1999 FEBS Letts 460:23 S Bauersachs, M Kirchgessner, BR Paulicks 1993 J Trace Elements Electrolytes Health
&Disl:Ul 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.
JM Finch, RJ Turner 1996 Res Vet Sci 60:97 SM Hegazy, Y Adachi 2000 Poultry Sci 79:331 BK Swain, TS Johri, S Majumdar 2000 Brit Poultry Sci 41:287 N Giadinis, G Koptopoulos, N Roubles, V Siarkou, A Papasteriades 2000 Comp Immunol Microbiol Infec Dis 23:129 R Boyne, JR Arthur, AB Wilson 1986 J Comp Pathol 96:379 N Ndiweni, JM Finch 1996 Vet Immunol Immunopathol 51:67 CD Davis, L Brooks, C Calisi, BJ Bennett, DM McElroy 1998 J Parasitol 84:1274 JA Rooke, JJ Robinson, JR Arthur 2004 J Agric Sci 142:253 A Smith, KB Madden, KJ Au Yeung, A Zhao, J Elfrey, F Finkelman, O Levander, T Shea-Donohue, JF Urban 2005 JNutr 135:830 MA Beck, J Handy, OA Levander 2004 Trends in Microbiol 12:417 M Roy, L Kiremidjianschumacher, Hi Wishe, MW Cohen, G Stotzky 1992 Proc Soc Exp Biol Med 200:26 M Roy, L Kiremidjianschumacher, Hi Wishe, MW Cohen, G Stotzky 1994 Biol Trace Element Res 41:103 L Kiremidjianschumacher, M Roy, HI Wishe, MW Cohen, G Stotzky 1996 Biol Trace Element Res 4l:\\5 L Kiremidjianschumacher, M Roy, HI Wishe, MW Cohen, G Stotzky 1996 Biol Trace Element Res 52:227 LD KoUer, JH Exon, PA Talcott, CA Osboume, GM Heningsen 1986 Clin Exp Immunol 63:570 MP Nair, SA Schwartz 1990 Immunopharmacol 19:177 MJ Guimaraes, D Peterson, A Vicari et al 1996 Proc Natl Acad Sci USA 93:15986 M Bonomini, S Forster, F Derisio et al 1995 Nephrol Dialysis Transpl 10:1654 F Girodon, P Galan, AL Monget et al. 1999 Arch Internal Med 159:784 U Tarp 1995 Analyst 120:877 P Knekt, M Heliovaara, K Aho, G Alfthan, J Mamiemi, A Aromaa 2000 Epidemiology 11:402 JM Reimund, C Hirth, C Koehl, R Baumann, B Duclos 2000 Clinical Nutr 19:43 LS Greene 1995 J Am Coll Nutr 14:317
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L Hasslemark, R Malgren, U Zetterstorm 1993 Allergy 48:30 R Shaw, K Woodman, J Crane et al. \994 N Zealand J Med 107:387 NLA Misso, DJ Peroni, DN Watkins, GA Stewart, PJ Thompson 1998 J Leuk Biol 63:124 R Gartner, M Angstwurm 1999 Medizinische Klinik 94:54 CS Broome, F McArdle, JAM Kyle, F Andrews, NM Lowe, CA Hart, JR Arthur, MJ Jackson 2004 Am J Clin Nutr 80:154 WC Hawkes, DS Kelley, PC Taylor 2001 Biol Trace Element /Jes 81:189 S Villette, JAM Kyle, KM Brown, K Pickard, JS Milne, F Nicol, JR Arthur, JE Hesketh 2002 Blood Cells Molecules & Diseases 29:174 N Safir, A Wendel, R Saile, L Chabraoui 2003 Clin Chem Lab Med 41:1005 RC McKenzie 2000 Clin Exp Dermatol 25:1 TS Rafferty, M Norval, A El-Ghorr, GJ Beckett, JR Arthur, F Nicol, JAA Hunter, RC McKenzie 2003 Biol Trace Elem Res 92:161 T Finkel, NJ Holbrook 2000 Nature 408:239 BM Lesourd 1997 Medizinische Klinik 66:S478 JW Albright, JF Albright 1998 Exp Gerontol 33:13 M Roy, L Kiremidjianschumacher, Hi Wishe, MW Cohen, G Stotzky 1995 Proc Soc Exp Biol Med 209:369 MT Ventura, E Serlenga, C Tortorella, S Antonaci 1994 Cytobios 11:115 Olivieri, D Girelli, M Azzini et al 1995 Clinical Sci 89:637 Olivieri, D Girelli, AM Stanzial, L Rossi, A Bassi, R Corrocher 1996 Biol Trace Element Res 51:31 HD Foster, LP Zhang 1995 Sci of Total Environ 170:133 HD Foster 1997 Medical Hypoth 48:355 G Ravaglia, P Forti, F Maioli et al 2000 Am J Clin Nutr 71:590 P Brenneisen, K Briviba, M Wlashek, J Wenk, K ScharfetterKochanek 1997 Free Radical Biol Med 22:515 Y Ito, O Kajkenova, RJ Feuers et al 199?, J Gerontol Series A-Biol Sci Med Sci 53:M169 V Serra, T Grune, N Sitte, G Saretski, T Von Zglinicki 2000 Ann NY Acad Sci 908:327 J Legault, C Carrier, P Petrov et al 2000 Biochem Biophys Res Com 272:416 S Watabe, Y Makino, K Ogawa et al 1999 Eur J Biochem 264:74
Chapter 28. Selenium and male reproduction Matilde Maiorino, Antonella Roveri, Fulvio Ursini Department of Biological Chemistry, University of Padova, Viale G. Colombo, 3, 1-35121 Padova, Italy
Regina Brigelius-Flohe Department Biochemistry of Micronutrients, Gennan Institute of Human Nutrition PotsdamRehbruecke (DIfE), Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany
Leopold Flohe MOLISA GmbH, Universitatsplatz 2, D-39106 Magdeburg, Germany
Summary: Selenium deficiency has long been documented to result in impaired male fertility of rats, mice and boars. The prominent feature of selenium-deficient spermatozoa is a distorted architecture of the mid piece, where normally the mitochondria are embedded into a keratinous matrix called the mitochondrial capsule. This material, which contains most of the selenium of sperm, is composed of oxidatively cross-linked proteins, a major component being the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx). PHGPx is abundantly synthesized in round spermatids under indirect control of testosterone. In late phase of spermatogenesis, the active soluble peroxidase is transformed into an enzymatically inactive structural protein by an oxidative process that is not understood in detail. Likely, it involves oligomerization of PHGPx itself, cross-linking of PHGPx with the sperm mitochondrion-associated cysteine-rich protein (SMCP) and other cysteine-rich proteins and selenadisulfide reshuffling with or without the aid of thioredoxin-glutathione reductase. Introduction The potential relevance of selenium to the reproductive system in livestock, laboratory animals and humans has been considered for at least five decades [1]. Impaired reproductive abilities due to selenium deficiency were reported for both sexes. In cows, cystic ovarian disease [2] and retained placenta [3-5] appear to respond to selenium supplementation; infertility of ewes may be associated with selenium deficiency [6,7]; and a selenium-deficient diet
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resulted in reduced egg production and embryonic survival in hens that could be normalized by selenium supplementation [8]. The biochemical basis of these disturbances in female reproductive ability remains elusive. In contrast, a molecular basis for the impaired spermatogenesis, as was first reported for rats [9-12], mice [13,14] and boars [15], is emerging. Function and morphology of sperm in selenium deficiency Data on the influence of selenium on human reproduction are scarce and contradictory (compiled in [1,16]). Thus, the role of selenium in human fertility must be largely inferred from studies in laboratory animals. Interestingly, testes have the ability to accumulate selenium and to retain this trace element even during substantial selenium deficiency [17,18]. Specific alterations of sperm were first seen in rats depleted of selenium for two generations [9,10]. In mice, these alterations increased through successive generations of selenium deprivation [13,14]. Therefore, impairment of male fertility carmot reasonably be expected to result fi-om transient variations of selenium supply. In severe and prolonged selenium deficiency, male rats and mice become sterile as spermatogenesis is arrested. The seminiferous epithelium is degenerated and the lumen of the testicular tubules has the appearance of being more or less devoid of sperm [13, 19]. Clearly, this kind of azoospermia or aspermia mimics a block in cell division. The functional and morphological alterations of spermatozoa, observed in less severe selenium deprivation, are more discrete. In rats, the prominent feature is reduced sperm motility leading to impaired fertilization capacity [11]. Sperm motility is less affected by selenium deprivation in mice [13]. In both species, abnormal sperm morphology is observed [11,12,14]. Characteristically, the mid-piece of the spermatozoon, that harbours the helix of mitochondria embedded in a keratin-like matrix, appears structurally disturbed, fuzzy or broken. The sperm tail, consequently, appears distorted, and isolated sperm heads and tails are often seen. A particularly weak point in the seleniumdeficient rat sperm mid-piece appears to be an impaired fusion of the annulus with the mitochondrial sheath, which leads to flagellar disorganization and disruption [20]. Interestingly, the mid-piece is precisely the part of the spermatozoon where Brown and Burk [17] found most of the selenium accumulated when ^^Se was injected into rats. Within the midpiece, selenium proved to be primarily associated with a cysteine- and proline-rich protein in rodents [21,22] and bulls [23]. Cloning of this 'mitochondrial capsule selenoprotein (MCS)', howev er, revealed that it was not a selenoprotein [24,25]. It therefore was re-named "sperm mitochondrion-associated cysteine-rich protein (SMCP) [24]. After this investigation, the search for the real selenoprotein(s) in sperm became revitalized.
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Selenoproteins in the male genital system Pulse-labeling experiments with ^^Se in selenium-deprived rats show specific selenium incorporation into a variety of testicular and epididymal proteins. They cover a wide range of apparent molecular masses when separated on SDS-polyacrylamide gels [26,27]. Some of bands on gels could be tentatively identified as their molecular weights correspond to those of cytosolic glutathione peroxidase (cGPx), phospholipid hydroperoxide GPx (PHGPx), mitochondrial and cytosolic thioredoxin reductases [28], and selenoprotein P (SelP). A band with an apparent MW of 34 KDa, which was only seen in testis [27] and contained in sperm nuclei [29], could be attributed to a PHGPx variant (snGPx or nPHGPx) with a chemically distinct N-terminus [30] that results from the use of an alternative transcription start, representing a nuclear targeting sequence [30], and it is driven by an alternative promoter within the first intron in gpx-4 [31]. More recently, a thioredoxin reductase variant with a fused glutaredoxin sequence (TGR), that displays glutathione, thioredoxin reductase and protein isomerase activities, was shown to be particularly abundant in elongating spermatids at the site of the mitochondrial sheath formation [32]. A band corresponding to a 15 KDa selenoprotein is detected in prostate epithelium [29]. Selenoprotein P is a secreted extracellular protein with multiple selenocysteine residues. It was surprisingly found to be expressed in Leydig cells of mice by means of/« situ hybridization [33]. The promoter region of the SelP gene contains putative SRY sites that are presumed to bind the sexdetermining region product of the Y chromosome [33]. The specific role of SelP in testis is unknown. SelP (-/-) mice, however, have a low testicular selenium content that results in infertility due to impaired sperm motility [34,35]. The observations point to a role of testicular SelP in assuring selenium supply to the seminiferous epithelium. Glutathione peroxidase activities have been repeatedly measured in testis and epididymis. Yet most of the early investigations did not differentiate between the different types of GPx (reviewed in [36]) and thus have been practically useless in elucidating a role of selenium in fertility. The data may be compromised by summing up GPx activities of the selenoperoxidases, of the androgen-responsive cysteine homologs of extracellular GPx [37] and even of GSH-S-transferases [38]. The presence of cytosolic GPx in testis was shown by cGPx activity measurement after separation from PHGPx and by in situ hybridization [39,40]. cGPx has been implicated in antioxidant defence in Leydig cells that are presumed to produce H2O2 during steroid hormone synthesis [41]. There appears to be a general feeling that the seminiferous and mature sperm also require a particularly efficient protection against oxidative stress [42-44]. cGPx, as the selenoperoxidase most efficient in H2O2 reduction, would indeed be the enzyme of choice to meet this demand [36]. There is, however,
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no indication of any predominance of cGPx in particular sites of the genital system. In situ hybridization studies display an uncharacteristic low level of cGPx mRNA in rat testis [39] and cGPx activities are accordingly low [39,45]. Finally, a specific role of cGPx in male reproduction can be ruled out as cGPx knock-out mice develop and reproduce normally [46]. In contrast, PHGPx is abundantly present in rat testis [43,47-49], but only after puberty [47,48]. The peripuberal increase in testicular PHGPx can be prevented by hypophysectomy and restored by application of chorion gonadotropin [47] indicating a hormonal control of PHGPx gene expression. Attempts to verify the hormonal control of PHGPx gene transcription by means of reporter gene constructs, however, have been unsuccessful [39]. In addition, testosterone or forskolin did not directly activate transcription and inhibition by 17-(3-estradiol could not be detected in hormone-responsive T47D and MCF7 cells [39]. Testosterone and forskolin also did not enhance PHGPx activity when added to decapsulated testes [39]. An explanation for these seemingly contradictory results was provided by in situ hybridization: PHGPx mRNA was seen to be predominantly expressed in a cell layer of the seminiferous epithelium representing the round spermatids [39]. Later studies with isolated spermatogenic cells confirmed the preferential expression in round spermatids [50]. The thickness of the spermatid layer reflecting proliferation of the germ epithelium is controlled by testosterone that is provided by gonadotropin-stimulated Leydig cells. When the Leydig cells are selectively destroyed, for example, by ethane dimethane sulfonate, the spermatid layer shrinks with some delay, and the PHGPx content of whole testis decreases in parallel to the disappearance of spermatids [39]. The hormonal control of PHGPx in testis, thus, is an indirect one. The PHGPx gene is not regulated by hormone action, but the transcribing cell type depends on testosterone. The burst of PHGPx gene transcription is reflected by a high content of PHGPx protein detected by immune histochemistry [47] and high PHGPx activity measured with the specific substrate phosphatidylcholine hydroperoxide [39,47,51,52]. PHGPx mRNA declines with elongation of spermatids and it is no longer detectable in spermatozoa. Immunostained PHGPx declines similarly, but remains faintly visible in spermatozoa. In contrast, PHGPx activity becomes almost undetectable in mature epididymal spermatozoa [51]. The PHGPx protein, however, is still present in spermatozoa, but as an enzymatically inactive, densely packed material contributing to constitute a considerable portion of the mitochondrial capsule of spermatozoa [51, 52]. The PHGPx protein can be solubilized out of this keratinous material by strong reduction and chaotropic agents and detected by MALDI-TOF mass spectrometry or Westem blotting [51]. Prolonged preincubation with 0.1 M DTT or mercaptoethanol even leads to the recovery of enzymatic activity [51].
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The nuclear form of PHGPx was originally considered to be a spermspecific protein ('sperm nuclei GPx', snGPx) but its messenger was later shown to be present in various somatic cells [31,53]. It was believed to be essential for chromatin condensation and thus pivotal for spermatogenesis [30]. Targeted deletion of nPHGPx, in fact, led to a defective chromatin condensation and head instability. Surprisingly, however, these defects were limited to spermatozoa collected from the epididymis, and nPHGPx -/- mice remained fully fertile [54]. A pivotal function of nPHGPx in male reproduction can therefore be ruled out. Since cytosolic PHGPx is also detectable in spermatid nuclei [55], the latter may compensate for the nPHGPx missing in the knockout mice. Impact of PHGPx moonlighting on sperm maturation The puzzling switch of PHGPx from an active peroxidase in spermatogenic cells to an enzymatically inactive protein in spermatozoa raises the question of what this new example of 'moonlighting' [56-58] might mean in the context of male fertility. The chemical process leading to the transformation of the soluble active peroxidase to a structural protein, although unknown in detail, is an oxidative one. The keratinous capsule material resists solubilizers like guanidine or sodium dodecyl sulfate, unless disulfides are reduced. Only upon the reductive treatment, monomeric PHGPx can be recovered. Inversely, when total sperm proteins are reductively solubilized and exposed to H2O2 in the absence of low molecular weight thiols, they readily form high molecular weight aggregates containing PHGPx. Purified PHGPx is also polymerized by H2O2 in the absence of GSH [51]. However, the product thus formed is a linear oligomer of PHGPx molecules having the active site selenium reacted with cysteine 148 on the back side of another PHGPx molecule [59]. These PHGPx oligomers easily dissolve with physiological concentrations of GSH. Taken together, the observations indicate that PHGPx, when oxidized by hydroperoxides in the absence of GSH, not only reacts with its exposed thiols but also with other protein thiols, and thereby becomes cross-linked probably via Se-S bonds. In enzymological terms, the proposed reaction simply means that the selenolate function of the ground state PHGPx is oxidised by ROOH to a first intermediate, which is a selenenic acid derivative (E-SeOH) (see Chapter 15]. The first intermediate then reacts with protein -SH instead of GSH to form the second intermediate (E-Se-S-Prot). Regeneration of the ground state enzyme (E-Se) that is commonly carried out by a second GSH, proceeds only slowly with this intermediate. The inactive, insoluble oxidation products of PHGPx might be thus considered as alternate substrate dead-end intermediates. Such reactions likely occur in late spermatogenesis. The transition from round to elongated spermatids is paralleled by a decrease of GSH and protein thiols [42,60-62].
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Evidently, the loss of GSH in this phase of spermatogenesis forces PHGPx into the alternate substrate pathway. Beyond these basics, little else can be said with certainty about the transformation process. The mechanism leading to the pivotal disappearance of GSH in late spermatogenesis is obscure. Interestingly, not only GSH, but also GSSG and mixed disulfides derived from GSH become undetectable. This could result from increased GSH metabolism or, more likely, from GSH oxidation followed by the release of GSSG [63,64] and subsequent extracellular degradation by y-glutamyl transpeptidase, which is absent in spermatogenic cells, but abundant in adjacent testicular and epididymal tissue [65]. Again, the source of the required oxidation equivalents that becomes up regulated at the specific point of sperm differentiation remains elusive. Some of the reaction partners that are required to build up the mitochondrial capsule have been recently identified in the mitochondrial capsule by HPLC-ESI-MS/MS: SMCP fragments, voltage dependent anion channel (VDAC2) and three types of keratins (complex I, acidic, kbl type II, and k5) [66]. Among those, SMCP with its 30% cysteine residues is the most likely candidate to react with PHGPx. This assumption is corroborated by strict co-localization of SMCP and PHGPx in the mitochondrial midpiece [67]. Moreover, peptides with adjacent cysteine motifs, which are abundant in SMCP, proved to be excellent substrates of PHGPx [66]. The cys-cys motifs of SMCP classifies this protein as a "high sulfur keratin-associated protein' ' (KAP), thus offering the intriguing possibility to bridge PHGPx/SMCP copolymers with keratins in the mid piece architecture. VDAC-2 finally has been shown to be linked to the outer dense fibers and to the mitochondria in bovine sperm [68]. The interplay of these capsule components is far from being clear, but a plausible sequence of reactions might be: i) oxidation of cys-cys motifs in SMCP by PHGPx; ii) reshuffling of disulfide and (selena-) disulfide bonds with or without the aid of TGR, which can act as a protein disulfide isomerase [32]; and iii) fixation of crosslinked proteins to the fibrous sheath and the mitochondrial surfece via SMCP moieties and/or VDAC2. Attempts to corroborate the essentiality of the capsule components by inverse genetics yielded ambiguous results. Knock-out of PHGPx resulted in prenatal fatality [69,70]. Interim data obtained along this line, however, were revealing [71]. Nine male chimeric mice with genotype +/- having more than 50% PHGPx reached maturity. They were fertile, but among 190 offspring not a single homozygous or hemizygous mouse deficient in PHGPx could be identified. This observation suggested that not even hemizygous cells could contribute to the male germ line. The chimeric mice did not display any obvious phenotype apart from mosaic-like disturbance of the testes: While parts of the testis looked normal, others, obviously derived from hemizygous cells, were markedly altered. In the tubules of affected areas, only few
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morphologically disturbed spermatozoa were detectable. The alterations, e.g, distorted tails, fuzzy and broken mid pieces, and isolated heads strongly mimic pathologies seen in selenium deficiency. Thus far, the results comply with our presumption that PHGPx is indispensable for the integrity of the mid piece architecture. Another observation, however, suggests that PHGPx must indeed have a dual function in spermatogenesis. In the affected testicular tissue of chimeric mice, the seminiferous epithelium looked degenerated and severely disorganized. These results could, however, not be reproduced in another strain of mice in which hemizygous PHGPx +/- mice remained fertile (M. Conrad, personal communication). Similarly, targeted disruption of SMCP resulted in infertility and asthenozoospermia in some mice, but only in marginal disturbance of spermatogenesis in others [72]. The sensitivity of the SMCP knock-out to the genetic background clearly points to the presence of a back up system that may or may not substitute for its cross-linking function. Conclusions and outlook Of the known selenoproteins, cGPx appears dispensible to fertility. One or the other thioredoxin reductase, because of the link of thioredoxin to nucleic acid metabolism, may be indispensable to sustain proliferation of the seminiferous epithelium. Thioredoxin reductase deficiency may therefore account for the complete arrest of spermatogenesis observed in rodents deprived of selenium for several generations. PHGPx proved to be pivotal for rodent spermatogenesis. In early spermatogenic cells, it is present as an active peroxidase and may regulate proliferation and/or differentiation. During maturation, it builds up the capsular architecture by oxidizing SMCP adjacent cysteine residues to cystine that finally undergo reshuffling and possibly proofreading by TGR, which also is a selenoprotein [32]. In mature spermatozoa, PHGPx represents a structural component of the mitochondrial capsule. The relevance of other selenoproteins to male fertility remains to be established. The dual role of PHGPx during sperm maturation appears to apply to other mammals including man, but not to non-mammalian vertebrates or metazoa [73]. The clinical relevance of sperm PHGPx content to fertility is supported by preliminary studies on subjects with fertility problems. The PHGPx activity of the sperm samples, as measured after reactivation, correlated positively with functional parameters indicative of fertiUty [74,75]. PHGPx genes of infertile subjects showed a trend towards a higher content of single nucleotide polymorphisms (SNPs) than in the controls, but most of the SNPs were not associated with infertility [76]. Some multiple mutations of gpx-4 were only observed, however, in infertiles. Taken together, mutations in gpx-4 can at best be considered a rare cause of infertility. Large scale and well designed studies will be required to determine how fi-equently PHGPx deficiency accounts for impaired fertility
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and whether such deficiencies are due to insufficient selenium supply, defect of PHGPx, defects in the PHGPx gene, or an altered regulation of PHGPx biosynthesis. References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
Subcommittee on Selenium, Committee on Animal Nutrition, Board on Agricolture, National Research Council 1983 Selenium in nutrition. National Academy Press, Washington, DC JA Harrison, DD Hancock, HR Conrad 1984 J Dietary Sci 61:12,1 N Trinder, CD Woodhouse, C Renton 1969 Vet Rec 85:550 EC Segerson, GJ Riviere, HL Dalton, MD Whitacre 1981 J Dairy Sci 64:1833 WR Julien, HR Conrad, JE Jones, AL Moxon 1976 J Dairy Sci 64:1833 ED Andrews, WJ Hartley, AB Grant 1968 AT Z Vet J16:3 WJ Hartley, AB Grant 1961 Fed Proc 20:679 AH Cantor, ML Scott 1974 Poult Sci 53:1670 KE McCoy, PH Weswig 1969 JNutr 98:383 ASH Wu, JE Oldfield, OH Muth, PD Whanger, et al 1969 Proc West Soc Am Soc An Sci 20:85 SH Wu, JE Oldfield, PD Whanger, PH Weswig 1973 BiolReprod 8:625 AS Wu, JE Oldfield, LR Shull, PR Cheeke 1979 Biol Reprod 20:793 E Wallace, HI Calvin, GW Cooper 1983 Gamete Research 4:377 E Wallace, GW Cooper, HI Calvin 1983 Gamete Research 4:389 CH Liu, YM Chen, JZ Zhang, MY Huang, et al 1982 Acta Vet Zootech Sinica 13:73 GN Schrauzer 1998 Selen: neue Entwicklungen aus Biologic, Biochemie und Medizin, Johann Ambrosius Barth Verlag, Huethig GmbH, Heidelberg, Leipzig DG Brown, RF Burk 1973 J Nutr 103:102 DBehne.THofer-Bosse 1984 y^«7 KO** trsp in various tissues and organs using promoters that are tissue and organ specific SP'^ rescue in KO"* mice [6,7]
G37 trsp'/Atrsp trsp'/Atrsp''
Partial SP'^ rescue in KO'' mice [6,7] SP'^ replacement in liver^
G31 trsp'/Atrsp/^
SP*^ replacement in liver^
"Genotype designations used for mouse models (see text). *Uses - the various uses of the mouse models with accompanying references (see also text). "SP - selenoprotein(s). ''KO - knockout. *AM Diamond, personal communication. -'R Irons, BA Carlson, DL Hatfield, C Davis, submitted. ^ A Carlson, ME Moustafa, R Shrimali, M Rao, N Zhong, S Wang, L Feigenbaum, BJ Lee, VN Gladyshev and DL Hatfield, submitted.
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Initially, the resulting transgenic mice encoding multiple copies of trsp' and G37trsp' (see Table 1) were examined [8]. This study provided the first example of transgenic mice engineered to contain functional tRNA transgenes. The fact that over-expression of wild type Sec tRNA^^^'^'^^*' due to the extra copies of trsp' did not appear to influence selenoprotein synthesis in the organs and tissues examined suggested that the levels of the Sec tRNA^^'^^^'^' isoforms were not limiting in protein synthesis (reviewed in [4,5]). However, mice carrying G37trsp' had a pronounced effect on selenoprotein expression and the effect occurred in a protein- and tissuespecific manner [8]. The most and least affected selenoproteins were glutathione peroxidase 1 (GPxl) and thioredoxin reductase 1 (TRl), respectively, and the organs which manifested the most and least affect on selenoprotein synthesis were liver and testes, respectively. The mutant Sec tRNA'^''^'" product from G37trsp' lacked the i*A base modification and altered the levels of the two host Sec tRNA'^"^^^'' isoforms is such a manner that the Um34 species was reduced and the mcm^U species was enriched. As the amount of the mutant tRNA increased with increasing numbers of transgenes, the amount of the Um34 species and the amount of some selenoproteins, and in particular, GPxl also decreased. The correlation in reduction of the Um34 isoform and certain selenoproteins led us to propose that the Um34 modification is responsible for the expression of several selenoproteins that are involved in the lower echelon of selenoprotein hierarchy expression [5-7]. Interestingly, many of the selenoproteins that are expressed in the lower echelon of selenoprotein hierarchy and are sensitive to selenium status, such as GPxl, serve largely stress-related functions, while those that are expressed in the upper echelon of selenoprotein hierarchy and are less sensitive to selenium status, such as TRl, serve largely housekeeping functions. Those members associated with stress-related phenomena are the ones dependent on the Sec tRNA'^"'^^° Um34 modification for their expression (see also Chapter 3 and [5-7]). As shown in Table 1, the G37trsp' mice have been used in several different studies. The studies have shown that these mice, which are deficient in selenoproteins involved in stress-related phenomena, have an enhanced 1) skeletal muscle adaptation after synergist ablation and following exercise [9], 2) incidence of prostate malignancy when the mice also carry an oncogene directed to this tissue (see legend to Table 1), and 3) incidence of breast malignancy when the mice also carry an oncogene directed to this tissue (see legend to Table 1). In addition, we have observed that G'i7trsp' mice a significantly greater number of azoxymethane (AOM)-induced aberrant crypt formations (preneoplastic lesions in the colon) than wild type mice (R Irons, BA Carlson, DL Hatfield and CD Davis, submitted). Supplementing the diets of the AOM treated G37trsp' and wild type mice with 0.1 and 2.0 i^g/g selenium significantly reduced the incidence of preneoplastic lesions in both
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mouse lines. These observations are the first to provide evidence that both selenoproteins and low molecular weight selenocompounds play a role in the cancer protective effects of selenium. In another study involving Sec tRNA^^^''^^'^ mutant transgenic mice, we examined the effect of A34trsp', which also lacked Um34, on selenoprotein synthesis (B.A. Carlson, M.E. Moustafa, R. Shrimali, M. Rao, N. Zhong, S. Wang, L. Feigenbaum, B.J. Lee, V.N. Gladyshev and D.L. Hatfield, submitted). The effects of A34trsp' on down regulating stress-related selenoprotein expression were similar to those of G31trsp'. Since both these mutants lack Um34, these observations provided further evidence that Um34 is responsible governing the expression of those selenoproteins involved in stress-related phenomena. Transgenic/knockout mouse models Since the standard knockout of trsp is embryonic lethal [9,11], it appeared that this mutant could not be used for further study of selenoprotein expression. However, we devised a means of rescuing selenoprotein expression by crossing heterozygous trsp knockout mice with homozygous trsp' transgenic mice and breeding the offspring to obtain a line of mice lacking trsp (Atrsp) that was dependent on the trsp' for survival [6,7]. One advantage of rescuing a knockout mouse with a wild type or mutant transgene is that the number of transgenes, and therefore, the levels of the corresponding gene product can be maintained at normal or elevated amounts depending on the transgene copy number. Rescuing with 20 copies of the wild type transgene enriched the Sec tRNA'^"^^''' population several fold, but little or no effect on selenoprotein expression in various tissues or organs was observed [6,7]. These observations provided further evidence that Sec tRNA^^"^^*^ is not limiting in selenoprotein biosynthesis (reviewed in [4,5]). Rescue of the Atrsp mice with the mutant transgene, G37trsp' afforded us with an opportunity of obtaining a mouse line with a mutant transgene wherein there is no background of host Sec tRNA'^'^'^^^*'' and selenoprotein expression is therefore totally dependent on the mutant tRNA. Gil trsp yielded a tRNA that lacked two base modifications, i*A37 and Um34 (see above, Chapter 3 and [6,10]) and mice rescued with G37trsp' lacked several selenoproteins including glutathione peroxidases 1 and 3, SelR and SelT [6,7]. Interestingly, we were not successful in rescuing selenoprotein synthesis in Atrsp mice with A34trsp' (see reference to Carlson et al above). As discussed in greater detail in Chapter 3, the novel regulation of several selenoproteins involved in stress-related functions occurs at the level of translation.
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Conditional knockout mouse models Removal of trsp from the mouse genome is embryonic lethal [9,11] which prevented further study of the standard trsp knockout per se in selenoprotein expression. We therefore prepared the conditional knockout of trsp using loxP-Cre technology [9]. The removal of floxed trsp in mouse mammary epithelium [9] and in liver hepatocytes [12] was examined, trsp was targeted for removal in mammary epithelium using transgenic mice carrying the Cre recombinase gene under the control of the mouse mammary tumor virus long terminal repeat promoter or the whey acidic protein promoter. Neither Cre promoter was effective in complete removal of trsp in mammary epithelial cells, but the Sec tRNA^^^"^^^^*^ population was substantially reduced to alter selenoprotein expression in a protein specific manner [9]. In liver, however, the targeted removal of floxed trsp with transgenic mice carrying the Cre recombinase under the control of the albumin promoter was virtually complete [12]. Surprisingly, the mice survived without selenoprotein expression in hepatocytes which comprise about 85% of the liver cell mass. Selenoprotein P (SelP), which is the only known selenoprotein with multiple Sec residues (see Chapters 9, 10 and 21), would seem to be largely made in the liver and transported to other organs and tissues as its level was reduced about 75% in plasma of the selenoproteinless liver knockout mice. These mice appeared phenotypically normal until about 24 hours before death and death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat [52]. Although most animals lacking selenoprotein expression in their liver died within two to three months in this initial study, these animals may be kept alive for extended periods of time on a diet enriched in other nutrients (U. Schweizer, L. Schomburg and J. Kohrle, personal communication). This is an important observation since these animals live much longer on a different diet and can be subjected to various environmental agents to study the role of selenoproteins in liver function and health. As selenium has been implicated in heart disease (see Chapter 25) and immune function (Chapter 27), we examined the role of selenoproteins in cardiovascular disease and the immune system. By targeting the removal of trsp in either endothelial cells or myocytes in skeletal and heart muscle, we have elucidated the role of selenoproteins in cardiovascular disease (R. Shrimali, J.A. Weaver, G.R. Miller, B.A. Carlson, S.V. Novoselov, E. Kumaraswamy, V.N. Gladyshev and D.L. Hatfield, submitted). Removal of selenoprotein expression in endothelial cells was embryonic lethal. 14.5-dayold embryos had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes, however, manifested no apparent phenotype until about day 12 after birth, when affected mice developed decreased mobility and an increased respiratory rate, followed by death
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within a few hoxirs. Although there was no evidence of inflammation of the skeletal muscle in mice that lacked trsp in myocytes, they had moderate to severe myocarditis with inflammation extending into the mediastinum.. Targeted removal of trsp in endothelial cells demonstrated an essential role of selenoproteins in their development, while targeted removal of trsp in myocytes demonstrated an essential role of selenoproteins in proper function of cardiac muscle. These studies also showed a direct connection between the loss of selenoprotein expression and cardiovascular disease (R. Shrimali, J. A. Weaver, G.R. Miller, B.A. Carlson, S.V. Novoselov, E. Kumaraswamy, V.N. Gladyshev and D.L. Hatfield, submitted). The conditional knockout of trsp is an important mouse model as it provides a means of elucidating the roles of selenoproteins in health, development and/or function in tissues or organs for which there is a specific promoter. Promoters that function to express Cre early in development of a specific tissue or organ can be used to assess the role of selenoproteins in development of that tissue or organ. On the other hand, promoters that function to express Cre after the tissue or organ is developed can be used to assess the role of selenoproteins in proper function of that organ or tissue. Furthermore, promoters that function to express Cre either early or late in development of a specific tissue or organ, and provided the animal survives for a long period of time following trsp knockout, can be used to examine the animal's ability to handle various forms of stress (e.g., viral or bacterial infection, carcinogen(s), cancer driver gene(s), etc). Those animals that survive for long periods of time, while lacking selenoprotein expression in a specific tissue or organ, can also be used to assess the role of small molecular weight selenocompounds in health. Therefore, the conditional knockout of trsp in specific tissues and organs is an important tool for assessing the role of selenoproteins in health and development and this model can also be used to assess the role of small molecular weight selenocompounds in protecting the animal against environmental stresses. Transgenic/conditional knockout mouse models Although the rescue of mice encoding a A/rs/j mice with G37trsp' provides a novel model for studying the role of housekeeping and stress-related selenoproteins in health and the role of the two Sec tRNA'^"^'^*^ isoforms in governing selenoprotein expression [6,7], this mouse model permit us to focus on the animal as a whole and not on not on the roles of these components in individual organs and tissues. However, removal of selenoprotein expression by targeting floxed trsp with a specific promoter Cre and replacing selenoprotein expression with a mutant trsp' permits us to assess the role of housekeeping and stress-related selenoproteins, as well as the role of both isoforms, in health and/or proper function of specific organs or tissues. Selenoprotein removal was targeted in liver of the floxed trsp
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mouse using the albumin Cre promoter and selenoprotein expression was replace with either G37trsp' or A34trsp' (B.A. Carlson, M.E. Moustafa, R. Shrimali, M. Rao, N. Zhong, S. Wang, L. Feigenbaum, B.J. Lee, V.N. Gladyshev and D.L. Hatfield, submitted). The pattern of replacing selenoprotein expression, wherein several selenoproteins associated with stress were not recovered, was similar with either the G37 and A34 mutant transgenes. The fact that the two mutant Sec tRNA^^'"^'^'^ isoforms govern selenoprotein expression in virtually an identical manner without the influence of host wild type Sec tRNA^^''^*'' demonstrates that Um34 is responsible for the synthesis of stress-related selenoproteins. Importantly, this mouse model will most certainly provide insights into one of the central questions in the selenium field which is "What are the contribution of selenoproteins versus low molecular weight selenocompounds in the cancer chemopreventive effects of selenium and other health benefits of this trace element?" Both these mutant transgenes replaced housekeeping selenoprotein expression, but not stress-related selenoprotein expression. Acknowledgements This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. References 1. 2. 3. 4. 5. 6. 7. 8.
9.
10.
11. 12. 13.
LC Clark etal 1996 y^M^ 276:1957 http://www.crab.org/select/ http://www.cancer.gov/clinicaltrials/ft-ECOG-5597 DL Hatfield, VN Gladyshev 2002 Mol Cell Biol 22:3565 DL Hatfield, BA Carlson, XM Xu, H Mix, VN Gladyshev 2006 Prog Nucl Acids Res Mol Biol (In press) BA Carlson, XM Xu, VN Gladyshev, DL Hatfield 2005 J Biol Chem 280:5542 BA Carlson, XM Xu, VN Gladyshev, DL Hatfield 2005 In Grosjean (Ed) Fine-Tuning of RNA Functions by Modification and Editing. Topics in Current Genetics Vol 12, p 431 ME Moustafa, BA Carlson, MA El-Saadani, GV Kryukov, QA Sun, JW Harney, KE Hill, GF Combs, L Feigenbaum, DB Mansur, RF Burk, MJ Berry, AM Diamond, BJ Lee, VN Gladyshev, DL Hatfield 2001 Mol Cell Biol 21:3840 TA Homberger, TJ McLoughlin, JK Leszczynske, DD Armstrong, RR Jameson, PE Bowen, ES Hwang, H Hou, ME Moustafa, BA Carlson, DL Hatfield, AM Diamond, KA Esser 2003 J Nutrition 133:3091 E Kumaraswamy, BA Carlson, F Morgan, K Miyoshi, GW Robinson, D Su, S Wang, E Southon, L TessaroUo, BJ Lee, VN Gladyshev, L Hennighausen, DL Hatfield 2003 Mol Cell Biol 23:1477 LK Kim, T Matsufuji, S Matsufuji, BA Carlson, SS Kim, DL Hatfield, BJ Lee. 2000 RNA 6:1306 MR Bosl, K Takaku, M Oshima, S Nishimura, MM Taketo 1997 Proc Natl Acad Sci USA 94:5531 BA Carlson, SV Novoselov, E Kumaraswamy, BJ Lee, MR Anver, VN Gladyshev, DL Hatfield 2004 J Biol Chem 279:8011
Chapter 30. Drosophila as a tool for studying selenium metabolism and role of selenoproteins Cristina Pallares, Florenci Serras and Montserrat Corominas Departament de Geneica, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain
Summary: The synthesis of selenoproteins is conserved in organisms ranging from bacteria to humans, including flies, and involves the differential decoding of the UGA stop codon as a selenocysteine (Sec). Drosophila genetics provides an excellent tool with which to investigate the synthesis machinery as well as the functions of specific selenoproteins. Mutations in some components of the selenoprotein synthesis machinery have opened new questions regarding their function. Only three selenoproteins have been identified so far in the fly genome, including sps2. Although mutants that lack the translation elongation factor selB/eEFsec are viable and fertile, the effects of mutations in the selD/spsl gene demonstrate that it is required for development and cell proliferation, hi this chapter, we review the recent advances on fly selenoproteins and their machinery of synthesis. Introduction For more than a century Drosophila melanogaster has been one of the most important genetic models used in biology. Few systems are so easy to manipulate or offer the powerful advantage of allowing researchers to approach any biological question at various points in the development of the organism using tools ranging from genetics to genomics or biochemistry. Due to the remarkable similarities between invertebrates and vertebrates at many levels, Drosophila biology has come to serve as a source of information for human biology and can be expected to have a direct impact on our understanding of human health. Analysis of selenium (Se) metabolism has been shown in recent years to be no exception. As an example, it is worth mentioning that the first mutation in a Drosophila gene involved in Se metabolism was isolated in a P-ZacFF transposon insertion mutant collection screen aimed at identifying genes involved in imaginal disc morphogenesis, a process with no a priori link with Se [1,2]. hi this screen, it was found that mutant flies for an enzyme involved in Se metabolism are lethal in larval/pupal stages, demonstrating the importance of Se in life. Studies on Se metabolism undertaken in a variety of systems have shown that this trace element is both essential and potentially toxic [3-5]. Se occurs
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most commonly in the organism as a selenocysteine (Sec) residue in natural proteins referred to as selenoproteins [6]. Incorporation of Sec into selenoproteins involves an unusual translation step in which UGA—^normally a stop codon—specifies Sec insertion. This step requires a stem-loop element termed a Sec insertion sequence (SECIS) present in the 3' untranslated regions (3' UTRs) of mRNAs encoding eukaryotic selenoproteins [7,8]. While this represents the main route of specific incorporation, Se can also be found as selenomethionine (Sem) or Sec that has not been specifically incorporated [9]. As in other eukaryotes, the Sec synthesis machinery in Drosophila appears to be quite conserved when compared with that of prokaryotes, as shown by the identification of key homologous genes [1,1012]. In this chapter, we will review current understanding of the biology of selenoproteins and their synthesis in Drosophila, as well as addressing how flies have been used to approach functional studies. Sec synthesis machinery Se intake Se is mainly obtained fi-om the diet and, consequently, physiological levels are constrained by concentrations in the food source [13]. Selenocompounds are mainly obtained as selenoaminoacids, such as L-selenomethionine and LSec, and some inorganic compounds, mostly selenate. Also, a small proportion of the Se in the organism seems to comefi-omrecycling pathways [14]. Se coming fi-om diet or protein turnover can be metabolized by a Sec lyase present in some prokaryotes [15,16] and mammals [14,17], but this enzyme has not yet been identified in flies. Sec lyase catalyzes the conversion of Sec to L-alanine and elemental Se that can be reused for Sec synthesis. The strong dependence on Se coming from the diet raises questions about the role of Se metabolism. To address this, a simple defined medium was devised that supports the growth of adult Drosophila and requires Se supplementation for optimal survival [5]. Those experiments showed that a Se concentration in the fly medium of 10"*-10"^ M mimics normal culture and seems to be the most beneficial for life span. On the other hand, insufficient Se intake in Drosophila produces a clear reduction in survival and flies grown on a Se-depleted medium show more than 50% reduction in the numbers of eggs laid and, thus, reduced fertility. Furthermore, Se deficiency somehow results in down-regulation of the mRNAs encoding selenoproteins [5] as has been observed in mammals [18], revealing the role of Se in selenoprotein expression. Selenocystevl-tRNA The most remarkable feature of Sec synthesis is that it involves its own specific tRNA [19]. In flies, tRNA.SelC is present as a single-copy gene on chromosome 2 that generates a 90-nt tRNA, with some characteristic
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nucleosides that are also present in vertebrates, namely 5 methylcarboxymethyluridine, N6-isopentenyladenosine, and pseudouridine at positions 34, 37, and 55, respectively [10,20]. Like its counterpart in higher organisms, Drosophila tRNA.SelC, also designated Sec-tRNA^^*"^'^", is first aminoacylated with serine and then modified to selenocysteinyl-tRNA, in a reaction that uses selenophosphate as the Se donor. Sec synthetase, the enzyme that catalyses this reaction, has not yet been identified in Drosophila [21]. Electrophoresis of Sec tRNA'^^^^" reveals two major bands expressed throughout development in Drosophila. In mammals, there are also two isoforms of Sec-tRNAf^'^^"" that differ by methylation of mcm^U to form methylcarboxymethyl-5'-uridine-2'-0 methylribose (mcm^Um); this methylation is considered to be the final step of maturation. Furthermore, this reaction depends on Se content, increasing with Se concentration, and seems to have regulatory implications [22]. This may not be the case in Drosophila, since the methylation process is not described and both pools have identical structures, probably corresponding to two stable conformations [20]. Selenophosphate synthetase The biosynthesis of Sec requires monoselenophosphate, the activated form of Se, in the cytoplasm. Selenophosphate synthetase catalyzes a reaction involving ATP and hydrogen selenide coming from the diet or Se delivery from recycling pathways to generate selenophosphate (the Se donor) together with AMP and orthophosphate [14, 15, 23]. In prokaryotes, this reaction is performed by the selD gene product [19]. This gene can encode either a selenoprotein (H. influenzae, M. jannaschii) or an ortholog that contains Cys {E. coli with Cys-17). The presence of this Cys or its equivalent Sec in the catalytic domain of SelD has been considered essential for its enzymatic activity in vitro [23-24]. More recently, it was suggested that replacement of the Cys-17 residue with serine (Ser) might render the enzyme able to use only Se delivered from the recycling pathway [15]. Like in vertebrates, two orthologs of selD have been described in Drosophila [1,11,12]. The Sec-containing form, Sps2, was the first selenoprotein identified in flies [12]. The second selenophosphate synthetase, Spsl or SelD, was separately described following the isolation of a cDNA clone [11] and after screening of a P-element insertion mutant collection to identify genes that affect cell proliferation [1]. This mutation, known as patufet or selD'""^, is lethal in homozygous flies at larval and pupal stages and leads to a marked disruption in the size and morphology of the imaginal discs and brain hemispheres. Drosophila SelD/Spsl has an arginine residue (threonine in mouse and human) instead of Cys or Sec [1]. Northern blot analysis reveals that this gene, located at position 50E on the second chromosome, is expressed
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throughout development and in situ hybridization shows that low-level expression is ubiquitous up to early gastrulation, at which point expression is upregulated in the gut primordium and nervous system [1,11]. The expression in the brain and imaginal discs, the tissues that will contribute to adult structures, shows a dynamic pattern that appears to correlate with cell proliferation. Analysis of colocalization of selD/spsl expression with BrdU labeling reveals that expression levels are high in dividing cells and low or undetectable in non-dividing cells [25]. Interestingly, these highly proliferative tissues are the most damaged in patufet mutant flies [1]. More detailed studies of cell-cycle markers in the imaginal discs and brain of selD/spsI mutants have shown a reduction in the number of BrdUincorporating cells, fewer mitotic cells, as indicated by a reduction in histone H3 phosphorylation, and high levels of cyclin B expression, suggesting that cells could remain arrested in G2 phase [25]. Loss of function mutations in the selD/spsI gene trigger cell death, mostly through the caspase-dependent Dmp53/Rpr pathway, indicating that the gene is essential for cell viability [1,26]. Flies or cells that are homozygous for seliy""^ have an altered redox system, leading to a burst of reactive oxygen species (ROS) [25-26]. Genetic evidence shows that the initiator caspase DRONC is activated and the effector caspase DRICE is processed to commit mutant cells to die [26]. Moreover, in that study, it was also shown that ectopic expression of the inhibitor of apoptosis DIAPl rescues the viability of seHy""^ mutant cells. These observations indicate that ROS-induced apoptosis in Drosophila is mostly through the caspase-dependent Dmp53/Rpr pathway. Generation of genetic mosaics by mitotic recombination in flies is a powerful technique with which to address gene function, since it allows clones of homozygous mutant cells to be generated in a heterozygous background. This is especially useful in the case of lethal mutations such as seljy""^. Homozygous selDf""^ clones in the adult wing are rounded and smaller than control clones, they contain fewer cells and those cells are smaller, and they reveal some autonomous effects of the mutation, such as suppression of vein differentiation [1]. Non-autonomous effects were also observed in wild-type cells surrounding the clone, leading to abnormal differentiation of ectopic veins. Similar clonal phenotypes were observed in cells homozygous for loss-of-function mutations in genes involved in the Drosophila EGF receptor (DER) pathway [27]. These results, together with some evidence that ROS may play an important role in signal transduction [28], raise questions about how alteration of the redox balance caused by the mutation affects the Ras/MAPK signaling pathway. Both the rough eye phenotype and the ectopic wing veins induced in gain-of-function mutants of the Ras/MAPK pathway are clearly suppressed by the removal of one copy of the selenophosphate synthetase product [29]. The hypothesis that the
Drosophila
selenium
metabolism
and selenoproteins
2>A1
seliy""^ mutation selectively modulates the RAS/MAPK pathway through alteration of the redox balance is further supported by the finding that an increase in ROS caused by the amorphic catalase allele Caf', one of the main enzymes of the Drosophila antioxidant system, also reduces RAS/MAPK signaling [29]. The results of these experiments strongly suggest that accumulation of ROS should be substantially different between heterozygous flies for those mutations and wild-type organisms. Although haplo-insufficient selDf""^fliesdo not have an apparent phenotype when kept under normal laboratory conditions, a significant decrease in life span is observed when they are treated with oxidants [30]. In contrast, while increased amounts of superoxide dismutase (SOD) extend longevity [31], overexpression of spsl in motomeurons leads to a reduction in life span, possibly due to an accumulation of toxic precursors [30]. Finally, it is clear that selLf"^ causes an impairment of selenoprotein synthesis, as revealed by the failure to detect selenoproteins in protein extracts of mutant larvae grown in fly medium containing ^^Se [25]. Therefore, it is tempting to speculate that selenoproteins may be instrumental in maintaining a certain redox state in the cell, as has already been shown for several mammalian selenoproteins [9,32]. Despite the findings mentioned above, it should be noted that purified Drosophila selD/spsl expressed in E. coli does not catalyze selenidedependent ATP hydrolysis in vitro and does not complement a selD deficiency in bacteria [11]. Although at first sight these results appear to disagree with those of other studies using the human spsl gene, in which weak complementation of an E. coli mutation was observed [33], if we take into account the fact that organisms that possess one variant also contain the other we can consider the possibility that SelD/Spsl may have a different function in Se metabolism. It is possible that SelD/Spsl is only efficient in using Se delivered fi-om Sec recycling pathways, as seems to be the case in the human spsl gene fi-om human-lung adenocarcinoma cells [14] or in the E. coli selD(C17S) [15]. The other selenophosphate synthetase, Sps2, contains a Sec residue at the position equivalent to E. coli Cys-17, suggesting a possible autoregulatory role in Sec synthesis; this gene also carries a mammalian-type SECIS. Transgenic embryos expressing a luciferase reporter containing the 3'UTR of the sps2 gene showed significantly higher reporter activity than those lacking the sps2 3'UTR, demonstrating the functionality of the SECIS element present in this 3' terminal region [12]. The spsl gene, located at 2L/31D, is expressed as at least two transcripts: sps2-RA (1348 bp), which was described initially [12], and sps2-RB (1295bp), which is the product of an alternative splicing event in the 4rt exon that, according to FlyBase (www.flybase.org), generates a change in the open reading fi-ame. Both transcripts could encode putative selenoproteins due to
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an in-frame TGA codon and the SECIS element. Both proteins contain the ATP/GTP binding site required for ATP hydrolysis during selenophosphate production [12]. Expression of the different transcripts has been assessed by both Northern blot analysis and in situ hybridization [12]. Northern blot analyses at different developmental stages revealed the presence of at least the larger transcript throughout development. In situ hybridization studies show a strong accumulation of maternal transcripts produced by nurse cells during oogenesis; these remain present at high levels up to the blastoderm stage (maternal effect). During the remainder of embryogenesis, zygotic expression occurs in a more restricted pattern and is first detected in the embryonic midgut primordium and later in a variety of tissues and organs including the gut and nervous system. A regulatory element that is thought to regulate cell-proliferation-related genes in Drosophila has been identified in the sps2 gene [34,35]. This DNA replication-related element (DRE) located downstream of the initiation site of the gene is essential for its transcription [35]. A transcription factor that specifically binds the DRE sequence has also been isolated in flies and ablation of this factor by double-stranded RNA interference (dsRNAi) experiments shows a significant decrease in dsps2 promoter activity [35]. Sec translational machinery Although much progress has been made in resolving the machinery associated with Sec translation in prokaryotes and vertebrates, less is known about the particular features of the system in Drosophila. Nevertheless, the conservation of some of the elements that have been identified suggests that the basic machinery would be the same. Briefly, the model proposed for eukaryotes includes a requirement for cis and trans factors that form a ribonucleoprotein complex known as a selenosome that functions to incorporate Sec at a UGA codon and thereby prevents translation being stopped. This selenosome complex will consist of at least a SECIS element in the 3 'UTR of the selenoprotein mRNA as a cis factor and a SECIS binding protein 2 (SBP2), a Sec-elongation factor (SelB/eEFsec), a Sec-tRNA '^''^ ^^ and the ribosome itself as trans factors. SBP2 is thought to interact with a conserved region of the SECIS element known as the quartet, with the 28S ribosomal RNA, and with SelB/eEFsec, which will also specifically bind the Sec-tRNA^^'^'^'''^, leading to co-translational incorporation of a Sec when a UGA triplet is encountered [36]. In the fruit fly. Sec insertion is directed, like in other eukaryotes, by the presence of a SECIS in the 3'UTR of the selenoproteins [8]. The Drosophila SECIS element contains the canonical characteristics found mostly in eukaryotic SECIS: the core structure with the quartet of non-Watson-Crick interacting base pairs, and the unpaired adenosines in the apical loop.
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Moreover, this SECIS appears to belong to form 2 in all the identified selenoproteins because of the presence of an additional small stem-loop at the top of the SECIS element [5,12,38]. The trans factors that are known to be required include the Sec-tRNA gene [10] and SelB/eEFsec [37]. Drosophila selB/eEFsec, located at position 57E on Chromosome 2, was found by sequence homology in an in silico analysis of the genome [37]. Its single transcript seems to be expressed throughout development, at high levels during early embryogenesis, lower levels during larval stages, and then at increasing levels in pupae and adults. Because SelB/eEFsec is essential for selenoprotein biosynthesis, it is a suitable target to mutate as a model in which to study the effects of selenoprotein deficiency. selB/eEFsec knockout mutants have been generated by homologous recombination, giving rise to flies that are unable to synthesize SelB/eEFsec and consequently fail to decode the UGA codon as Sec. Moreover, in spite of the impairment of the Sec UGA-decoding mechanism, selB/eEFsec mutant animals develop into fertile flies. In addition, although most known selenoproteins in eukaryotes seem to be involved in antioxidative defense and redox metabolism, life-span studies in the selB/eEFsec mutant do not reveal a role in viability and the mutants do not show sensitivity to induced oxidative stress. These findings challenge the view that a Sec-based oxidative stress defense system was responsible for conserving the selenoprotein biosynthesis system over the course of evolution [37]. Selenoproteins In recent years, genome projects have become an extremely powerful tool through which to identify protein-coding genes. However, because of the non-standard use of the UGA codon, computational gene prediction methods were unable to identify selenoproteins in the sequence of eukaryotic genomes until recently. Only the identification of members of the synthesis machinery was possible based on homology with known genes, as mentioned earlier, for example, with Sps2, the first selenoprotein identified in flies [12]. Using a biochemical approach it was shown that metabolic labeling of flies with '^Se revealed three clear major bands [5,25]. According to the predicted molecular weight, the 42-43 kDa band could correspond to the Sps2 selenoprotein itself, but those experiments did not provide any information on the nature of the other bands. Two different in silico approaches have been used in an attempt to solve this problem. Briefly, one approach combined and improved existing gene prediction programs and developed a method that relies on the prediction of SECIS elements alongside the prediction of genes in which a strong codon bias characteristic of protein-coding regions extends beyond a TGA codon that interrupts the open reading fi-ame [38]. The other approach involved a computational screen to search for SECIS elements followed by
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selenoprotein gene signature analyses [5]. Both screens led to the identification of three selenoprotein genes in fruit flies: the previously identified sps2 and two new selenoproteins named SelM (also known as SelH or BthD) and SelG (SelK or G-rich). Both proteins incorporate ^*Se when transfected in mammalian cells [38] and all of them were detected after metabolic labeling of flies with ^^Se [5]. These findings indicate that the genes encode true selenoproteins. Furthermore, both of the newly identified genes have paralogs that employ Cys instead of Sec as well as orthologs that use Sec or Cys in other organisms, including vertebrates [38]. SelM/BthD/SelH Selenoprotein SelM/BthD was the first component identified [5,38] in a new family that currently includes several Cys or Sec ortholog representatives in eukaryotes, including the uncharacterized human selenoprotein H [39]. This gene maps to position 12A8 on the X chromosome and has two distant paralogs, CG13186 and CG15147, the latter containing Cys instead of Sec [38]. The selM/BthD gene encodes a 30 kDa protein containing 249 amino acids and a Sec residue belonging to the CXXU motif near the N-terminus [5]. This motif, which is also found in both bacteria and animals, including the mammalian selenoproteins SelT, SelW, and SelH, is similar to the redox motif CXXC [22], suggesting a redox function, with the Sec possibly forming a selenenylsulfide bridge. SelM/BthD shows a dynamic expression pattern. High levels of transcript are detected in adult females, with abundant expression in the developing ovary. In contrast, the expression in males is very weak [5,40]. During early embryogenesis, both transcript and protein seem to display abundant ubiquitous expression, especially in the blastoderm, suggesting that there is a strong maternal contribution [38,40]. At late stages of embryogenesis selM/BthD expression accumulates in the developing salivary gland [40]. Finally, although selM/BthD mRNA appears to be more weakly expressed during larval stages [5,40], in situ hybridization reveals that the transcript is ubiquitously distributed in imaginal disc and larval brain [38]. A dynamic subcellular distribution has been detected using a specific antibody against SelM/BthD [40]. The protein distribution in various Drosophila tissues is cytoplasmic, with a particulate pattern observed in salivary glands. Immunolocalization studies in SL2 cells reveal a colocalization with a Golgi marker, suggesting a possible role in protein secretion or processing [40]. Two different RNAi strategies for silencing selM/BthD expression have been employed to show that loss of selM/BthD reduces viability, although with differing penetrance. Hypomorphic mutants generated by dsRNA injection in embryos exhibit dramatically reduced embryonic viability [41]. Moreover, the use of inducible duplex RNAi under the control of Gal4 drivers revealed that loss of selM/BthD interferes with salivary gland
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morphogenesis and reduces animal viability [40]. SelM/BthD silencing decreases total anti-oxidant capacity in embryos and Schneider cells and increases lipid peroxidation. On the other hand, transient expression of selM/BthD in the cell line decreases lipid peroxidation, suggesting that this protein may have antioxidant functions [41]. SelG/SelK/G-rich Selenoprotein G/G-rich—^named on the basis of its 28% glycine residues—is a less well-known selenoprotein with a mass of 12 kDa. Although homologs of SelG/SelK containing Cys or Sec can be found in vertebrates [5,38,39], its function remains to be elucidated. Located at position 10F4 on Chromosome X, the selG/selK gene gives rise to a single transcript that encodes a 110amino acid selenoprotein, with a Sec residue at the C-terminal penultimate position, similar to some mammalian thioredoxin reductases [22]. SelG/SelK has a cysteine paralog, SelG-like [38]. The two genes appear in tandem, separated by only 320 bp, have the same exonic structure, and share 65% identity at the protein level. Northern blot analysis shows that it is expressed at all stages of fly development [5], while in situ hybridization reveals the distribution to be ubiquitous during embryonic stages [38]. One approach to elucidating selG/selK function has been the characterization of RNAi hypomorphic mutants in cells and embryos. Embryos microinjected with dsRNA corresponding to selG/selK display decreased viability, considered as a percentage of hatched larvae, in addition to morphological defects or developmental retardation [41]. On the other hand, studies in S2 cells have not revealed an effect of SelG/SelK on the redox system. Concluding remarks We would like to conclude this chapter by addressing some of the challenging questions that remain to be resolved. First, only three selenoproteins have been identified in Drosophila so far and it remains possible that the true number of selenoproteins will prove to be higher. Moreover, while interfering with mRNA encoding selG/selK and selM/BthD seems to reduce viability [39,40], the precise cellular function of these fly selenoproteins is unknown except in the case of Sps2, an enzyme involved in the synthesis of selenoproteins [12]. Second, it will be essential to elucidate the role of the various Cyscontaining paralog genes. Do they act as a functional backup? If so, to what extent are the selenoproteins and their paralog genes redundant? Also, it remains to be determined whether or not the two variants of selenophosphate synthetase, SelD/Spsl and Sps2, are redundant. Third, loss of function mutations in selD/spsl are lethal and result in a lack of selenoproteins [1,25]. However, eEFsec mutants, which lack
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Selenium: Its molecular biology and role in human health
selenoproteins, are viable [37]. This raises the question of whether or not selenoproteins are essential for insect viability and to what extent fly selenoproteins contribute to redox balance or have adopted other functions. It is possible that, in addition to its role in Se metabolism, selD/spsl is also involved in processes that are essential for viability. Alternatively, the accumulation of Se compounds due to the lack of enzymatic activity could account for reduced viability. Drosophila genetics provides an opportunity to approach these questions using a variety of tools to create alleles for those genes and, beyond that, to study their role in metabolism, proliferation, growth and development. Transposable P-elements are still widely used as mutagenesis reagents and form the backbone of projects that seek to generate mutant insertions in every predicted gene in the fly genome. Molecularly mapped deletions have been generated at both a genome-wide and a custom-made level using genetically engineered vectors based on the FLP/FRT system [42]. Moreover, elements have been developed for a wide range of transgenic applications, including enhancer trapping, gene tagging, targeted misexpression, RNA interference delivered by the Gal4AJAS system and homologous recombination. Further genetic experiments will be required to reconcile these issues. References 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
B Alsina, F Serras, J Bagufta, M Corominas 1998 Mol Gen Genet 151-.WZ F Roch, F Serras, FJ Cifuentes, M Corominas, B Alsina, M Amoros, A Lopez-Varea, R Hernandez, D Guerra, S Cavicchi, J Bagutia, A Garcia-Bellido 1998 Mol Gen Genet 257:103 K Schwarz, CM Foltz 1957 J Am Chem Soc 79:3292 OF Combs Jr, SB Combs 1986 The Role of Selenium in Nutrition Academic Press Inc New York pp 532 FJ Martin-Romero, GV Kryukov, AV Lobanov, BA Carlson, B J Lee, VN Gladyshev, DL Hatfield 2001 J Biol Chem 276:29798 DM DriscoU, PR Copeland 2003 Annu Rev Nutr 23:17 MJ Berry, L Banu, YY Chen, SJ Mandel, JD Kieffer, JW Harney, PR Larsen 1991 Nature 353:273 A Krol 2002 Biochimie 84:765 D Behne, A Kyriakopoulos 2001 Annu Rev Nutr 2\:453 BJ Lee, M Rajagopalan, YS Kim, KH You, KB Jacobson, DL Hatfield 1990 Mol Cell fi/o/10:1940 BC Persson, A Bock, H Jackie, G Vorbruggen 1997 J Mol Biol 274:174 M Hirosawa-Takamori, H Jackie, G Vorbruggen 2000 EMBO Rep 1:441 CB Allan, GM Lacourciere, TC Stadtman 1999 Annu Rev Nutr 19:1 T Tamura, S Yamamoto, M Takahata, H Sakaguchi, H Tanaka, TC Stadtman, K Inagaki 2004 Proc Natl Acad Sci USA 101:16162 GM Lacourciere, H Mihara, T Kurihara, N Esaki, TC Stadtman 2000 J Biol Chem 275:23769 GM Lacourciere, TC Stadtman 2001 Biofactors 14:69 H Mihara, T Kurihara, T Watanabe, T Yoshimura, N Esaki 2000 J Biol Chem 275:6195
Drosophila selenium metabolism and selenoproteins 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.
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PM Moriarty, CC Reddy, LE Maquat 1998 Mol Cell Biol 18:2932 TC Stadtman 1996 Amu Rev Biochem 65:83 X Zhou, SI Park, ME Moustafa, BA Carlson, PF Grain, AM Diamond, DL Hatfield, BJ Lee, 1999 J Biol Chem llA:mi9 H Romero, Y Zhang, VN Gladyshev, G Salinas 2005 Genome Biol 6: R66 DL Hatfield, VN Gladyshev 2002 Mol Cell Biol 22:3565 Z Veres, lY Kim, TD Scholz, TC Stadtman 1994 J Biol Chem 269:10597 lY Kim, Z Veres, TC Stadtman 1992 J Biol Chem 161:19650 B Alsina, M Corominas, MJ Berry, J Bagufla, F Serras 1999 J Cell Sci 112:2875 M Morey, M Corominas, F Serras 2003 J Cell Sci 116:4597 FJ Diaz-Benjumea, E Hafen 1994 Development 120:569 T Finkel 1998 Curr Opin Cell Biol 10:248 M Morey, F Serras, J Bagufla, E Hafen, M Corominas 2001 Dev Biol 238:145 M Morey, F Serras, M Corominas 2003 FEBS Lett 534:111 TL Parkes, A J Elia, D Dickinson, AJ Hilliker, JP Phillips, GL Boulianne, 1998 Nat Genet 19:171 S Gromer, JK Eubel, BL Lee, J Jacob 2005 Cell Mol Life Sci 61:1A\A SC Low, JW Harney, MJ Berry 1995 J Biol Chem 270:21659 A Matsukage, F Hirose, Y Hayashi, K Hamada, M Yamaguchi 1995 Gene 166:233 JS Jin, S Back, H Lee, MY Oh, YE Koo, MS Shim, SY Kwon, I Jeon, SY Park, K Back, MA Yoo, DL Hatfield, BJ Lee 2004 Nucleic Acids Res 32:2482 A Lescure, D Fagegaltier, P Carbon, A Krol 2002 Curr Protein Pept Sci 3:143 M Hirosawa-Takamori, HR Chung, H Jackie 2004 EMBO Rep 5:317 S Castellano, N Morozova, M Morey, M J Berry, F Serras, M Corominas, R Guigo 2001 EMBO Rep imi GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 &ie«ce 300:1439 SY Kwon, P Badenhorst, FJ Martin-Romero, BA Carlson, BM Paterson, VN Gladyshev, BJ Lee, DL Hatfield 2003 Mol Cell Biol 23:8495 N Morozova, EP Forry, E Shahid, AM Zavacki, JW Harney, Y Kraytsberg, MJ Berry, 2003 Genes Cells 8:963 E Ryder 2004 F Blows, M Ashbumer, R Bautista-Llacer, D Coulson, J Drummond, J Webster, D Gubb, N Gunton, G Johnson, CJ O'Kane, D Huen, P Sharma, Z Asztalos, H Baisch, J Schulze, M Kube, K Kittlaus, G Renter, P Maroy, J Szidonya, A RasmusonLestander, K Ekstrom, B Dickson, C Hugentobler, H Stocker, E Hafen, JA Lepesant, G Pflugfelder, M Heisenberg, B Mechler, F Serras, M Corominas, S Schneuwly, T Preat, J Roote, S Russell Genetics 167:797
Chapter 31. Selenoproteins in parasites Gustavo Salinas Catedra de Inmunologia, Facultad de Quimica-Facultad de Ciencias, Universidad de la Republica. Instituto de Higiene, Avda. A. Navarro 3051, Montevideo, CP 11600, Uruguay
Alexey V. Lobanov and Vadim N. Gladyshev Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA
Summary: Parasites, which cause an enormous burden in the population of the third world, are a diverse group of organisms, many of which are sensitive to oxidative stress imposed by their hosts. In recent years, several selenoprotein families, some with antioxidant properties, have been described and characterized in metazoan parasites. Glutathione peroxidase and thioredoxin glutathione reductase (TGR) appear to be essential selenoproteins in flatworms (phylum Platyhelminthes). TGR is the single enzyme that provides reducing equivalents to both thioredoxin and glutathione pathways, in contrast to hosts, which evolve parallel pathways. In roundworms (phylum Nematoda), selenoproteins have recently been described, revealing species differences in the Sec/Cys protein sets and the presence of an unusual SECIS element. Plasmodium sp, one of the most important protozoan parasites that affect humans, also decode Sec. The selenoprotein families encoded by Plasmodial genomes have neither Sec nor Cys homologs in their hosts, raising the possibility that targeting their selenoproteomes may provide new treatment strategies. Introduction Although significant research efforts have been made to study selenoproteins and selenocysteine insertion systems in humans and various model organisms, little has been reported in the literature regarding the utilization of selenium in eukaryotic parasitic organisms. This chapter focuses on the progress made in the characterization of selenoenzyme families in flatworms, the recent advances in the synthesis and utilization of selenoproteins in roundworms and protozoan parasites, and discusses why selenoproteins of platyhelminths and Plasmodia may represent interesting targets for chemo- or immune-prophylaxis.
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Parasites: diverse organisms that face similar oxidative stress challenges Parasites live at least part of their lifecycle inside another organism (the host), which they exploit for their own survival and reproduction. This definition includes different types of infectious agents (viruses, bacteria, fungi, protozoa, helminths). However, for historical reasons, the term is most often reserved for 'protozoa' and 'helminths' organisms. Indeed, parasitology was identified as a separate research field during the exploration of the tropics and the establishment of 'tropical medicine' [1]. Both 'protozoa' and 'helminths' also include free-living organisms, and neither 'protozoa' nor 'helminths' are monophyletic; on the contrary, both groups are represented by highly divergent phyla. Nonetheless, this historical classification is not useless. These two groups of parasites are very different: protozoan are unicellular protists, which multiply quickly within the host, and are, in most cases, intracellular in habitat; in contrast, helminths are metazoan organisms with complex multicellular organization (with nervous system and reproductive organs), which undergo complex metamorphoses and migrations within the host. Table 1 presents the main features of the major human parasitic infections. Table 1. Major human parasites (Source: [2])
Protozoan parasites'* Species (Disease) Plasmodium sp (Malaria) Trypanosoma brucei (sleeping sickness ) Trypanosoma cruzi (Chagas disease*) Leishmania sp (Leishmaniasis)
Helminths parasites^ Species/Disease Schistosoma sp (Schistosomiasis or bilharzia^) Onchocerca volvulus (Onchocerciasis or river blindness ) Filariidae family (Lymphatic filariasis )
Phylum Apicomplexa
Death per year/DALYs' 1,124,000/42,280,000
Kinetoplastida
50,000/1,590,000
Kinetoplastida
13,000/649,000
Kinetoplastida
59,000/2,357,000
Phylum Plathyhelminthes
Death per year/DALYs 15,000/1,760,000
Nematoda
0/987,000
Nematoda
0/5,644,000
"DALYs: DisabiUty Adjusted Life Years (the number of healthy years of life lost due to premature death and disabiUty). Protozoan parasites include many diverse phyla, among them Apicomplexa and Kinetoplastida.
Selenoproteins in parasites
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Distribution: mainly confined to poorer tropical areas of Africa, Asia and Latin America. More than 90% of malaria cases and the great majority of malaria deaths occur in ttopical Africa. Plasmodium falciparum is the main cause of severe clinical malaria and death. Distribution: 36 countries in sub-Saharan Africa *Distribution: Latin America f Distribution: Endemic in 88 countries on 4 continents. Two forms of the disease: cutaneous (caused by Leishmania major), and visceral (caused by L. donovani) Helminth parasites are contained in three phyla: Nematoda (roundworms), Platyhelminthes (flatworms) and Acantocephala (spiny-headed worms). Helminth infections are rarely fatal, but pose an enormous burden to human population in the tiopics Distribution: endemic in 74 developing countries with more than 80% of infected people living in sub-Saharan Africa Distribution: 35 countries in total. 28 in tropical Africa, where 99% of infected people live. Isolated foci in Latin America and Yemen. Distribution: Endemic in over 80 countries in Africa, Asia, South and Central America and the Pacific Islands. Three species are of significance, Wuchereria bancrofti, Brugia malayi and Brugia timori.
In Spite of the diversity of parasites, all face similar biological problems that relate to their parasitic lifestyle. Among them, the neutralization of the effector mechanisms deployed by the host immune system is of paramount importance. Resident macrophages and inflammatory-site phagocytic leukocytes (mostly neutrophils, but also monocytes and eosinophils, depending of the type of infection) are cells equipped to kill foreign organisms. They possess an oxidase system located in their plasma membrane, which becomes activated upon certain stimuli, for example, by interaction of cell receptors with antibodies bound to the foreign organism or with parasite molecular motifs (Figure la) [3]. Subsequently, 'respiratory burst' (increase in oxygen uptake not linked to respiration) takes place and produces superoxide anion and additional reactive oxygen species (ROS) [4]. Large amounts of nitric oxide (NO) are also produced by macrophages (and to a lesser extent by neutrophils) activated by a variety of immunological stimuli, such as y-interferon and tumor necrosis factor. NO reacts with superoxide to produce peroxynitrite and other reactive nitrogen species (RNS) (Figure lb) [5]. In addition, activated neutrophils and eosinophils release myeloperoxidase and eosinophil peroxidase, respectively, that catalyze the conversion of hydrogen peroxide and halides into hypohalous acids that are powerful oxidants and can form further damaging species [4]. Collectively, ROS and RNS are powerful oxidants and nitrating species: they can inactivate enzymes and initiate the process of lipid peroxidation and nitration, which leads to radical chain reactions that further damage membranes, nucleic acids and proteins (Figure Ic). These processes (and an additional arsenal of the host effector cells, such as hydrolytic enzymes) may
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Selenium: Its molecular biology and role in human health
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R 9 , RSS?, R3DK R 3 0 0 H nittotyrosine , inactivatlon of Fe/Sclusteis (metal OMdation) • Lipid peroxidation and nitration: LDOK LNO,, IDONO^, propagation of radical ctiain reactions • DMA strand breaks
(d) Bizymatic defenses SOD GR< •
FEH ONOOH-+NO, FSff?-* R3H R90OH^.R33H
Selenoproteins in parasites
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Figure 1. Reactive oxygen and nitrogen species generated by the host immune response and antioxidant defenses, (a) Recognition of parasites by host leukocytes (such as macrophages, neutrophils and eosinophils) occurs by pattern recognition receptors (PRR) that bind to pathogen-associated molecular patterns (PAMPs), or through antibodies (Ig), and leads to activation of host immune cells. Upon activation, these cells produce superoxide ("02") and nitric oxide ('NO) radicals. 'NO is produced in the cytosol (but can cross membranes) by inducible nitric oxide synthase (iNOS); 02" is produced by a multi-component, membraneassociated NADPH oxidase. Superoxide is released towards the extracellular space in the case of non-phagocytosable parasites {e.g., worms), or towards the phagosome (topologically equivalent to the extracellular space) in the case of intracellular parasites {e.g., protozoans), (b) 'NO and 'O2" react at diffusible controlled rate to produce peroxynitrite (ONOO"). Peroxynitrite can react in one-electron oxidations {e.g., with transition metal centers), two electrons oxidations (of a given target), or with CO2, redirecting its reactivity. It also decomposes spontaneously into other ROS and RNS such as 'OH and •NO2. In addition, activated neutrophils and eosinophils release myeloperoxidase and eosinophil peroxidases, respectively, which catalyze the conversion of hydrogen peroxide and halides into hj^sohalous acids, (c) Collectively, these products can inactivate enzymes, damage membranes and nucleic acids, and ultimately kill the parasitic organisms. (D) Parasites' defenses include antioxidant enzymes that directly scavenge superoxide, decreasing peroxynitrite formation (superoxide dismutases), and hydrogen and organic peroxide reductases (GPx and TPx). Some TPx have also been shown to reduce peroxynitrite catalytically. Repair mechanisms include methionine sulfoxide reductase, thioredoxin, and sulfiredoxin among others. *R'H denotes a hydrocarbon chain, or alcohol (R'H=ROH), or a thiol R'H=RSH)
ultimately lead to killing parasitic organisms. Yet, well-adapted parasites cope with the oxidative stress imposed by the host's immune response by a series of cellular chemicals and antioxidant enzymes that directly neutralize ROS and RNS (Figure Id), and constitute important model organisms to study antioxidant defense. Several antioxidant enzymes found in parasites belong to selenoprotein families. Glutathione peroxidase: tlie first selenoenzyme described in parasites Glutathione peroxidase was the first selenoenzyme to be characterized from a parasite. A cDNA from the platyhelminth Schistosoma mansoni encoding a GPx with a TGA in-frame at the active site was cloned in the early 1990s [6]. The protein encoded by this gene has biochemical properties similar to mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPx); its activity being higher with phosphatidyl choline hydroperoxide and other phospholipid hydroperoxides than with hydroperoxide substrates, such as cumene hydroperoxide and hydrogen peroxide [7]. GPx and superoxide dismutase, another antioxidant enzyme, co-localize in the tegument and gut epithelium of adult worms, which are the exposed interfaces of the parasite towards the host [8]. Additional evidence suggests that antioxidant enzymes, and GPx in particular, are vital for ROS neutralization and parasite survival within the host. Indeed, expression of GPx is developmentally regulated, with the highest levels present in the adult worm [8], the stage most resistant
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Selenium: Its molecular biology and role in human health
to oxidative stress and immune elimination [9]. In addition, GPx expression is upregulated by hydrogen peroxide and xanthine/xanthine oxidase generated ROS [10]. Recently, a search for GPx in Expressed Sequence Tag databases (dbEST) of platyhelminths identified a second GPx (GPx2) in S. mansoni and S. japonicum [11]. GPx2 also encodes a Sec residue at the active site and possesses an N-terminal signal peptide, which targets this isoform to the extracellular compartment, suggesting that this secreted variant would be important for extracellular hydroperoxide removal, helping to protect the parasite in its immediate environment. In this study, a GPxl ortholog whose 3 '-untranslated region revealed the presence of a SECIS element was also identified in Echinococcus granulosus (another flatworm) transcriptome using the SECISearch algorithm (Chapter 9 and http://genome.unl.edu/SECISearch.html) [12]. In contrast to platyhelminths, the corresponding Cys-containing enzymes appear to occur in nematodes [13], as reviewed in [14]. Nevertheless, recent datamining of nematode dbEST revealed some exceptions (see below) [15]. Free-living nematode Caenorhabditis elegans has no Sec-containing GPx encoded in its genome [15]. GSH- and Trx-reduction pathways in platyhelminth parasites are controlled by a single selenoenzyme In most living organisms, there are two analogous and mutually supporting enzymic systems that provide antioxidant defense to cells: the glutathione (GSH) and the thioredoxin (Trx) systems (Figure 2) [16,17]. These systems have overlapping yet distinct targets. GSH, due to its reactivity and intracellular concentration, is one of the most important cellular antioxidants, being efficient in rescuing small disulfide molecules and in reacting directly with ROS. The major function of Trx is to maintain cysteine residues in substrate proteins in the reduced form. In addition to their direct function as antioxidants, GSH and Trx provide electrons to GPx and Trx peroxidase (TPx), respectively, which reduce hydrogen peroxide and organic hydroperoxides, and to methionine sulfoxide reductase, which is also an important antioxidant repair enzyme. GSH and Trx are usually reduced by GSH and Trx reductases (GR and TR), respectively, at the expense of NADPH oxidation. Recent characterization of these systems in platyhelminth parasites has shown that 'conventional' GR and TR are absent; instead, the GSH and Trx systems are intermingled with the enzyme thioredoxin glutathione reductase (TGR), which provides reducing equivalents to both pathways (Figure 2).
Selenoproteins in parasites
361
(a) Comparison of the SSH, Tnc and llntod Tnc43SH systems
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362
Selenium: Its molecular biology and role in human health
Figure 2. Linked thioredoxin-glutathione systems, (a) Comparison of thioredoxin, glutathione and linked thioredoxin-glutathione systems. The glutathione system comprises (i) GR, GSH and Grx, whereas the thioredoxin system consists of (ii) TR and Trx. In linked Trx-GSH systems (iii), TGR functionally replaces TR, GR and Grx, providing reducing equivalents to targets of both systems. In all systems, NADPH is the upstream donor of reducing equivalents, (b) Components of the thioredoxin and glutathione systems. Redox centers of GR, TR, TGR, Grx and Trx are indicated, as well as the FAD prosthetic group and the ligands NADPH and GSH. TR and TGR possess a C-terminal extension missing in GR, which contains the Cterminal GCUG redox-active motif TGR possesses an N-terminal Grx domain that is absent in TR and GR. The Grx and Trx domains contain the CXXC redox center. Grx, unlike Trx, binds GSH. (c) Schematic representation of electron flow in TGR. TGR, like GR and TR, is a homodimer, with monomers oriented in a head-to-tail manner. Electrons flow from NADPH to FAD, to the CX4C redox center, to the C-terminal GCUG redox center of the second subunit, to the CX2C redox center of the Grx domain of the first subunit, and to targets, including GSSG (left scheme). Alternatively, electrons can flow, presumably directly, from the GCUG redox center to Trx (right scheme). The model proposes a flexible hinge, which connects the TR and Grx domains. This organization allows electrons to flow to the "in built' Grx domain or to Trx. Parts (a) and (b) in the figure reprinted with modifications from [11] with copyright with permission from Elsevier.
This protein is a second selenoenzyme family that has been characterized in platyhelminth parasites (reviewed in [11]). TGR is an oxidoreductase shown to possess TR, GR and Grx activities, achieving its broad substrate specificity by a fusion between Grx and TR domains (Figure 2b); this domain fusion was originally described in a mouse testis TGR [18]. Experimental and in silico data support the proposition that TGR is the single enzyme responsible for recycling both oxidized Trx and GSH in platyhelminth parasites. Treatment of S. mansoni adult worm extracts with auranofin, a known inhibitor of Sec-containing TRs, resulted in complete inhibition of TR and GR activities [19]. In addition, TGR was the single protein isolated from Taenia crassiceps (also a flatworm) extracts as a result of tracing GR and TR activities [20]. Examination of EST databases from Schistosoma species, which covers more than 90% of the gene content of this organism [21], revealed cDNAs encoding TGR, but not conventional TR or GR [11]. The biochemical characterization of E. granulosus and T. crassiceps TGR indicated that the native enzyme shuttles elecfrons from NADPH to oxidized Trx (TR activity), GSSG (GR activity) and glutathionemixed disulfides (Grx activity). The stoichiometric inhibitory effect of auranofin on both GR and TR activities of TGR indicates that the Seccontaining C-terminal redox center participates in elecfron fransfer to GSSG and oxidized Trx [20,22]. In addition, TR and Grrx domains can function either in coupled reactions or independently. Conventional TRs neither bind GSH nor possess GR activity; thus, the N-terminal Grx domain of TGR would reduce GSSG, accepting elecfrons from the Sec-containing C-terminal redox center. The idea that the C-terminal redox center donates elecfrons to the fused Grx domain implies that the Grx domain of TGR would be linked
Selenoproteins in parasites
363
to the TR domains by a flexible hinge to allow reduction of the oxidized Trx (Figure 2c). It is interesting to note that T. crassiceps TGR showed a hysteretic behavior in enzymatic assays with GSSG at high concentrations; this observation led the authors to propose a model in which TGR would possess high and low affinity sites for glutathione [20]. Clearly, further biochemical characterization and structural data on this multifunctional enzyme are needed that will shed light on the mechanism of catalysis, hi addition, molecular characterization of the corresponding gene could also provide clues regarding the mechanism of generation of isoforms. hideed, the analysis of TGR in E. granulosus revealed two trans-spliced cDNAs derived from a single gene [22]. These variants code for mitochondrial (mt) and cytosolic (c) TGRs, containing identical Grx and TrxR domains, but differing in their N-termini. These variants derive from alternative initiation of transcription, followed by trans-splicing. Similarly, mtTGR and cTGR variants also derived fi"om a single gene have been identified in S. mansoni [11]. Collectively, the results from platyhelminth studies strongly suggest that TGR is the main pyridine-nucleotide thiol-disulfide oxidoreductase in these organisms, in contrast to their hosts, where there is some redundancy of mechanisms for recycling oxidized Trx and GSH. Very little has been published about these pathways in the other phylum of helminth parasites (Nematoda), and to the best of our knowledge, nothing is known about Sec/Cys-containing TR or TGR in parasitic nematodes. However, no single genome has yet been completed from metazoan parasites. Selenoproteins of nematode parasites: old families, unusual SECIS An in silica analysis of Caenorhabditis elegans and Caenorhabditis briggsae (free-living nematodes) genomes revealed that these organisms encode a single a selenoprotein, TR [15], corroborating earlier experimental data [23]. However, no experimental studies have yet been performed with selenoproteins from parasitic nematodes. Nevertheless, in a recent study [15], the existing nematode ESTs were searched for selenoprotein genes using SECISearch and by screening for homologs of known selenoproteins. These analysis identified selenoprotein homologs of selK, selT, selW, Sepl5, selenophosphate synthetase and GPx. Two interesting points were noted from these analyses. First, various nematodes encode different selenoproteins, and the distribution of selenoprotein families within this phylum is mosaic. Second, it was found that all detected nematode selenoprotein genes contained an unusual form of SECIS element, with G rather than a canonical A at the conserved position preceding the quartet of non-Watson-Crick base pairs [15].
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Selenium: Its molecular biology and role in human health
Selenoproteins of protozoan parasites: waiting for surprises? Very little is known about selenoproteins from protozoan parasites. Recently, the presence of tRNA^^' was described in several species of the phylum Apicomplexa [24] (Lobanov et al., submitted). Plasmodium falciparum, which is the causative agent of malaria - the most overwhelming human parasitic infection, belongs to this phylum. The finding of tRNA^'" was consistent with the presence of putative EFsec and selenophosphate synthetase in P. falciparum and other Plasmodia. In addition, tRNA^*'' was observed in Toxoplasma, but not in Cryptosporidium parasites. Genomewide searches for SECIS elements in the six Plasmodium genomes revealed four selenoprotein genes. Interestingly, homology analyses of these proteins identified no hits outside Apicomplexa, suggesting that these selenoproteins do not exist in the apicomplexan hosts. These properties make the new selenoproteins attractive targets for anti-malaria drug development. The other reference in the literature to a parasite Sec-decoding protozoan is the description of a Cys-containing selenophosphate synthetase from Leishmania major [25]. Leishmania belongs to trypanosomatidae family, which also includes Trypanosoma brucei, and T. cruzi (Table 1), which are causative agents of disabling and fatal diseases in the poorest rural population of the third world [26]. Consistent with the finding of selenophosphate synthetase, recent bioinformatics analyses revealed three selenoprotein genes in several Trypanosoma genomes (Lobanov and Gladyshev, unpublished). Finally, no single reference could be found in the literature regarding a Sec-decoding amoebae, a traditional group of protozoa that include the parasitic amoebae of humans, Entamoebae histolytica. Parasite selenoproteins: drug or vaccine candidates? From a global perspective, the confrol of parasitic infections, which are a major cause of disability and mortality in many developing countries, remains as one of the most important challenges for medicine in the 21^' century [2]. Although there are safe and effective drugs to control some parasitic diseases, parasites can develop resistance to drugs rendering them ineffective, as it has been the case of certain antimalarial drugs [27]. Thus, effective vaccines and new drugs against parasitic organisms are needed. The task ahead is enormous considering that parasite and hosts are eukaryotic organisms; as yet, there is not a single vaccine for a human parasitic infection. Whether selenoproteins can be drug targets or generate immunity depends on premises that are not necessarily different from those for any other target protein: the validity of a drug target would rely on it being an essential protein, and sufficiently different from the host homolog(s) as to be selectively inhibited. Likewise, a good vaccine candidate should generate an
Selenoproteins in parasites
365
appropriate and selective immune response against the parasite, without inducing pathology to the host. In platyhelminths, TGR is an attractive pharmacological target because of the lack of redundant mechanisms (i.e., TR and GR) to provide reducing equivalents to essential enzymes. Inhibition of this enzyme could lead to impaired synthesis of DNA and antioxidant defenses, compromising parasite survival. TGR may also be a good vaccine candidate, since it is a large protein with a degree of identity to host enzymes below 60%. However, there are no studies regarding TGR as an immunogen. Contrary to TGR, there are promising studies on the use of GPx as a vaccine candidate. Vaccination of mice (not a natural host) against the platyhelminth S. mansoni with naked DNA constructs containing Sec-containing GPx showed significant levels of protection compared to a control group [28]. In this context, it is important to emphasize not only the fact that GPx appears to be important at the host parasite interface, but also that platyhelminth lack catalase and rely exclusively on GSH and Trx peroxidases for hydrogen peroxide removal. In the case of protozoan parasites, further studies are needed to identify and functionally characterize their selenoproteins. Nevertheless, it is highly significant that the four selenoproteins identified in Plasmodium sp have neither Sec nor Cys homologs in humans. Considering that Sec is usually located at the redox-active sites of enzymes, the selenol- and thiol-based redox systems may play vital an important role in the survival of protozoan parasites [29]. Finally, selenoproteins may be different to other proteins in one respect: electrophilic drugs, such as gold or platinum compounds, or alkylating agents that react preferentially with Sec over Cys may affect the parasite and the host to a different extent, depending on the relative importance of selenoproteins for the two organisms, and the presence/absence of Cyscontaining enzymatic back up systems. Acknowledgements This work has been supported by Fogarty International Research Collaboration Award TW006959 and Ministry of Education, Uruguay, PDT 29/171. References 1. 2. 3. 4. 5. 6.
K Warren 1988 The Biology of Parasitism PT Englund, A Sher (Ed) Alan R. Riss Inc New York 3 WHO The world health report -changing history. 2004 (http://www.who.int/whr/2004/en/report04_en.pdf) DH McGuinness, PK Dehal, RJ Pleass 2003 Trends Parasitol 19:312 BG Halliwell, JMC Gutteridge 1999 Free Radicals in Biology and Medicine Oxford University Press Inc New York R Radi, G Peluffo, MN Alvarez, M Naviliat, A Cayota 2001 Free Radic Biol Med 30:463 DL Williams, RJ Pierce, E Cookson, A Capron 1992 Mol Biochem Parasitol 52:127
366 I. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26.
27. 28. 29.
Selenium: Its molecular biology and role in human health M Maiorino, C Roche, M Kiess, K Koenig, D Gawlik, M Matthes, E Naldini, R Pierce, L Flohe 1996 EurJBiochem 238:838 H Mei, PT LoVerde 1997 Exp Parasitol 86:69 GM Mkoji, JM Smith, RK Prichard 1988 Int J Parasitol 18:661 UE Zelck, B Von Janowsky 2004 Parasitology 128:493 G Salinas, ME Selkirk, C Chalar, RM Maizels, C Fernandez 2004 Trends Parasitol 20:340 GV Kryukov, VM Kryukov, VN Gladyshev \999 J Biol Chem 274:33888 L Tang, K Gounaris, C Griffiths, ME Selkirk 1995 J Biol Chem 270:18313 K Henkle-Duhrsen, A Kampkotter 2001 MolBiochem Parasitol 114:129 K Taskov, C Chappie, GV Kryukov, S Castellano, AV Lobanov, KV Korotkov, R Guigo, VN Gladyshev 2005 Nucleic Acids Res 2005 ZTi-.llll A Holmgren 2000 Antioxid Redox Signal 2:811 PG Winyard, CJ Moody, C Jacob 2005 Trends Biochem Sci 30:453 QA Sun, L Kimarsky, S Sherman, VN Gladyshev 2001 Proc Natl Acad Sci USA 2001 98:3673 HM Alger, AA Sayed, MJ Stadecker, DL Williams 2002 Int J Parasitol 32:1285 JL Rendon, IP del Arenal, A Guevara-Flores, A Uribe, A Plancarte, G MendozaHemandez 2004 Mol Biochem Parasitol 133:61 S Verjovski-Almeida, R DeMarco, EA Martins, PE Guimaraes, EP Ojopi, AC Paquola, JP Piazza, MY Nishiyama, Jr., JP Kitajima, RE Adamson, PD Ashton, MF Bonaldo, PS Coulson, GP Dillon, LP Farias, SP Gregorio, PL Ho, RA Leite, LC Malaquias, RC Marques, PA Miyasato, AL Nascimento, FP Ohlweiler, EM Reis, MA Ribeiro, RG Sa, GC Stukart, MB Soares, C Gargioni, T Kawano, V Rodrigues, AM Madeira, RA Wilson, CF Menck, JC Setubal, LC Leite, E Dias-Neto 2003 Nat Genet 2003 35:148 A Agorio, C Chalar, S Cardozo, G Salinas 2003 J Biol Chem 2003 Apr 1111%: 12920 VN Gladyshev, M Krause, XM Xu, KV Korotkov, GV Kryukov, QA Sun, BJ Lee, JC Wootton, DL Hatfield 1999 Biochem Biophys Res Commm 259:244 T Mourier, A Pain, B Barrell, S Griffiths-Jones 2005 RNA 11:119 PC Jayakumar, VV Musande, YS Shouche, MS Patole 2004 DNA Seq 15:66 CM Morel, T Acharya, D Broun, A Dangi, C Elias, NK Ganguly, CA Gardner, RK Gupta, J Haycock, AD Heher, PJ Hotez, HE Kettler, GT Keusch, AF Krattiger, FT Kreutz, S Lall, K Lee, R Mahoney, A Martinez-Palomo, RA Mashelkar, SA Matlin, M Mzimba, J Oehler, RG Ridley, P Senanayake, P Singer, M Yun 2005 Science 309:401 TE Mansour Chemotherapeutic Targets in Parasites: Contemporary strategies 2002 T Mansour (ed) Cambridge University Press Cambridge 4 KA Shalaby, L Yin, A Thakur, L Christen, EG Niles, PT Lo Verde 2003 Vaccine 22:130 S MuUer, E Liebau, RD Walter, RL Krauth-Siegel 2003 Trends Parasitol 19:320
Chapter 32. Incorporating 'omics' approaches to elucidate the role of selenium and selenoproteins in cancer prevention Cindy D. Davis and John A. Milner Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Summary: Epidemiologic and preclinical studies provide evidence that increasing dietary selenium (Se) may have cancer protective properties. However, variation in cancer incidence among and within populations with similar Se intake suggests that an individual's response may reflect interactions with genetic and/or environmental factors. The "omics" of nutrition (nutrigenomics, nutrigenetics, nutritional epigenomics, nutritional transcriptomics, proteomics and metabolomics) may assist in understanding the cellular and molecular events associated with the cancer protective effects of Se, as well as in identifying responders and non-responders. Approaches, that utilize transgenic and knockout mice with altered selenoprotein expression offer models to evaluate the importance of selenoproteins or small molecular selenocompounds in mediating the cancer protective effects of Se. While the challenges will be enormous, the potential rewards in terms of both cancer morbidity and mortality will be of equally great magnitude. Introduction Extensive evidence indicates that dietary selenium (Se) supplementation reduces the incidence of cancer in experimental animals. Adding Se to the diet or drinking water inhibits initiation and/or post-initiation stages of liver, esophageal, pancreatic, colon and mammary carcinogenesis and spontaneous liver and mammary tumorigenesis in several rodent models [reviewed in 1,2]. Similarly, ecological studies have usually found an inverse relationship between Se status and mortality from cancer of the large intestine, rectum, prostate, breast, ovary, lung, and leukemia [3]. Data from most case-control and cohort studies indicate its possible protective relationship with lung and prostate cancer, but data is not overly convincing for other cancer sites, including breast and colon/rectum [4]. A recent meta-analysis suggests that Se supplementation may afford some protection against lung cancer in populations where average Se levels are traditionally low [5]. Evidence
368
Selenium: Its molecular biology and role in human health
suggests that toenail Se may be a useful predictor of status [5]. Cohort studies also have identified low baseline serum or toenail Se concentrations as a risk factor for prostate cancer [6,7]. A recent intervention study provides the most compelling evidence for the protective effects of Se against cancer [8]. This randomized controlled trial was designed to test Se as a deterrent to the development of basal or squamous carcinomas. Secondary end-point analyses showed that the mineral resulted in a significant reduction in total cancer mortality (RR = 0.5), total cancer incidence (RR = 0.63), and incidence of lung (RR = 0.54), colorectal (RR = 0.42), and prostate (RR = 0.37) cancer [8]. Participants with baseline plasma Se concentration in the lowest two tertiles (^NMe,
^
^
^
20
Singh et al reported the GPx-like activity of a series of diaryl diselenides having intramolecular Se—N interactions [42]. It was found that the diselenides that have quite strong Se—N intramolecular interactions were less active, whereas the diselenides that contain a built-in basic amino group but no Se—N interactions showed excellent GPx activity [43]. The ferrocene-based diselenides (18-20 in Scheme 3) were reported to display much higher activities than those of the phenyl-based diselenides, and the enhancement in catalytic activity could be ascribed to the synergistic effect of redox-active ferrocenyl and internally chelating amino groups.
Selenoprotein mimics
391
By analogy to diselenides, ditellurides and related compounds were proposed as GPx mimics. Engman et al [44] was first to report that some of these compounds (e.g. 21-22) show GPx-like activity. Modifying the basic structure of diphenyl ditelluride based on substituent effects and isosteric replacements further impacts the reactivity of diphenyl ditelluride. More recently, Mugesh et al [45] directly compared the thiol peroxidase activity of several ditellurides (e.g., 23-26 in Scheme 4) with that of their selenium analogues. All ditellurides were found to be much more efficient catalysts than the corresponding diselenides in reducing H2O2 with PhSH as cosubstrate. Scheme 4 ^R
"O'^'K!}"' "O'^'-^'O'' 0-^=-'^^ 22
21
oci
^ 24
23
22-1
NMej
Teh
25
26
Scheme 5 X-X
0
0)_ I ' f 27,28 X = Se, Te
32
0
29-31 X =Se,Te,Sec o HNCH2(CH2NHCH2)„CH2-N^
Se-S(
SeOgH
Q
j[
J
SeOgH
33-35 n = 1-3
Cyclodextrins (CDs) are cyclic oligosaccharides containing a hydrophobic cavity, in which many complexes can be formed via host-guest chemistry [46]. They have extensively been exploited as enzyme models and molecular receptors [47]. To elucidate the effect of substrate recognition on catalysis, a series of cyclodextrin-derived organoselenium and organotellurium compounds (e.g. 27-35) were developed as GPx models (Scheme 5) [48-51].
392
Selenium: Its molecular biology and role in human health
The first model compound 29 was prepared by attaching a diselenide group to the CD primary face [48]. Attachment of a ditelluride group onto cyclodextrin resuhed in GPx models 28 (2-TeCD) and 30 (6-TeCD) [50]. The catalytic efficiency of 2-TeCD-catalyzed reduction of hydroperoxides by GSH was found to be 350,000-fold higher than that involving diphenyl diselenide (PhSeSePh). Selenoenzyme transformation by chemical modification Transformation of natural enzymes into selenoenzymes Although many low molecular weight GPx mimics are known, they possess serious disadvantages: low activity, low solubility in water, and in some cases, toxicity. In this regard, natural proteins may have advantage as protein macromolecules carry molecular information for both substrate recognition and efficient catalysis. Engineering proteins by genetic and chemical methods is a valuable strategy for introducing new functions into protein scaffolds. So far, three corresponding protein design methods have been described: site-directed mutagenesis, chemical modification and the combination of both. By using these strategies, natural enzymes, proteins and antibodies have been used successfully to construct efficient selenoenzyme models [16]. Scheme 6 ICH2—Q_'^!^L_/2|l/4(H
"^°2
/||^^XxO;H
The first example in the field of selenoenzyme design is the chemical conversion of the active site serine residue of the bacterial serine protease subtilisin (EC 3.4.21.14) into selenocysteine [52]. The hydroxyl group of Ser221 could be selectively modified to introduce distinct functional groups into the active site of subtilisin. Inspired by the earlier work on the first semisynthetic enzyme, thiolsubtilisin [53], Wu and Hilvert prepared selenosubtilisin by using a similar method [54] (Scheme 6). The semisynthetic selenoenzyme exhibited significant GPx-like redox activity. It catalyzed the reduction of a variety of hydroperoxides by 3-carboxy-4nitrobenzenthiol (ArSH). The reduction of ter^butyl hydroperoxide (^ BuOOH) by ArSH was at least 70,000-fold faster than the reaction catalyzed by diphenyl diselenide, a well-studied antioxidant [54,55]. Since selenosubtilisin has the same substrate binding pocket as subtilisin, it was possible to rationalize and even predict its substrate selectivity. Thus, a series of different racemic hydroperoxides was chemically synthesized by the Schreier group and subjected to selenosubtlisin-catalyzed reactions [56,57].
Selenoprotein mimics
393
All alkyl aryl hydroperoxides showed an enrichment of enantiomers. In a similar fashion, Liu et al prepared a selenotrypsin by converting the active site serine into Sec [58]. The study revealed that GSH is not a particularly good substrate for selenotrypsin. Nevertheless, the data showed that it was possible to convert an active site serine into Sec in various serine proteases. Recent studies showed that tellurium is an excellent alternative element for the construction of GPx models [14-16]. However, introducing tellurium into proteins is currently a challenge. Following selenosubtilisin, Liu and coworkers developed a methodology to introduce tellurium into the binding pocket of subtilisin and yielded a first semisynthetic telluroen2yme tellurosubtilisin (Scheme 6) [59]. Like natural GPx, tellurosubtilisin can catalyze the reduction of ROOH by thiols efficiently and acts as an excellent GPx mimic.
Table 1. Catalytic activities of selenoprotein GPx mimics (Data from ref. 21). selenoenzyme mimic
GPx activity (U/nmol)
ebselen
1
printed protein
100-800
catalytic antibody
1100-24300
selenoGST
2000-6200
natural GPX
5780
Although seleno/tellurosubtilisin and selenotrypsin were generated via covalent modification of naturally occurring enzymes [54,58,59], it is a great challenge to prepare highly efficient semisynthetic enzymes that can rival natural selenoenzymes. Recently, Luo et al developed a method to mimic the action of GPx by chemically modifying a naturally occurring enzyme glutathione transferase (GST, EC.2.5.1.18) [60]. Taking advantage of the highly specific GSH binding site of GST, seleno-GST(Se-GST) was generated by chemical mutation using a method described for preparation of selenosubtilisin [54]. The selenium-containing Se-GST can efficiently catalyze the reduction of hydrogen peroxide with an activity that is greater than that for some natural counterparts (Table 1) [60].
394
Selenium: Its molecular biology and role in human health
Transformation of natural proteins into selenoenzymes To create an efficient artificial enzyme, the affinity for the substrate in the enzjmie-substrate complex must be reasonably high, and the catalytic groups should be adjacent to the reactive group of the substrate. An alternative approach to artificially creating such binding sites is the molecular imprinting technique [61]. Biopolymers can also be used as an alternative backbone for the imprinting procedure. This innovation has led to the development of the bioimprinting technique for the synthesis of proteinbased binding and catalytic sites. An imprinted enzyme model with GPx activity has been developed by a combination of bioimprinting and chemical mutation [62], A';,S-bis-2,4dinitrophenyl-glutathione (GSH-2DNP), a GSH derivative, was synthesized and acted as a template molecule (36 in Scheme 7). In the bioimprinting process, the imprinted molecule was allowed to interact with denatured proteins (e.g., egg albumin) to form a new conformation via hydrogen bonds, ion pairing and hydrophobic interactions. Scheme 7 N02 N02
--
H^
'>N02 '
O - < 0 > - "COOH - ^ ^ N J YO" ^ COOH 36
The new conformation was then fixed using the cross-linker glutaraldehyde. After removal of the imprinting molecule by dialysis, the serine residues located at the binding sites of the inprinted proteins were activated using phenylmethene sulfonyl fluoride and then converted into Sec in the presence of NaHSe. The imprinted protein exhibits GPx activity and is 100-800 fold more active than ebselen (Table 1). Transformation of antibodies into selenoenzymes One important way for designing binding sites is the use of antibodies. A recognition site for enzyme substrate is easy to generate by using a standard monoclonal antibody (McAb) preparation technique. This strategy was widely applied in the design of catalytic antibodies using transition state analogs as haptens. Recently, Luo et al employed this strategy in the design of GPx mimics [63-65]. The authors used substrate analogs instead of transition state analogs as haptens in order to generate monoclonal antibodies with the substrate binding site. In the design of catalytic antibodies, the polar groups of substrate GSH were modified by different hydrophobic
Selenoprotein mimics
395
groups and the modified substrates were used as a series of haptens (Scheme 8). Scheme 8 NH2
o
H
6
O
S
ISIH-,
"
o
o
HO
os-^^^o
6^°^
O H ,-^0 O
NO2
H3CO-^^'^V^N'^^V"OCH3 O H NH2 40
37-39 R = H, CHj, CHjCHjCHjCHj
The substrate binding sites were first made by monoclonal antibody preparation technique using hydrophobically modified GSH and GSSG as haptens (37-40 in Scheme 8). Thus, not only is the hydrophobic cavity of the antibody similar to that of the active site of native enzymes, but the affinity of the antibody active site for the substrate can also be adjusted to that of the native enzyme. The catalytic Sec was then incorporated into the McAb by chemical modification of the serine residue (Scheme 9) [63]. Surprisingly, these catalytic antibodies exhibited remarkably high catalytic efficiency which could rival the natural enzyme (e.g., rabbit liver GPx) (Table 1). In order to produce pharmaceutical proteins and elucidate the reason why this novel catalyst exhibited high catalytic efficiency, a selenium-containing single chain antibody was prepared (Se-ScFv) [65]. Scheme 9
MaAb production
y - y I 'S-CHjOH
PMSF
f - y " lS-CH20S02CH2-/>
NaHSe
(^ " ljS-CH2SeH
Similarly, Ding and coworkers [19] prepared a selenium-containing catalytic antibody (Se-4C5) by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T4) into Sec. Se-4C5 catalyzes the deiodination of T4 to 3,5,3'-triiodothyronine (T3) in the presence of dithiothreitol via a ping-pong mechanism, with a Vmax value of 270 pmol mg" ' min"'. Thus, Se-4C5 acted as a deiodinase mimic.
396
Selenium: Its molecular biology and role in human health
Transformation of proteins into selenoenzymes by genetic engineering Since Sec is encoded by a stop codon UGA, it is difficult to prepare selenoproteins by traditional recombinant DNA technology. The most suitable approach to bioincorporating selenium is the auxotrophic expression technique. In 1975, the first use of selenium in sulfur pathways in E.coli was reported by Cowie & Cohen [66]. Following this early work, there was considerable interest in the insertion of selenium analogs of sulfur-containing amino acids into proteins. Moroder and Budisa further developed this strategy and incorporated selenomethionine, telluromethionine and their isosteric analogs into proteins in order to solve the phase problem in protein X-ray crystallography [67,68]. Furthermore, Bock et al used a similar auxotrophic expression system and incorporated Sec into thioredoxin [69]. The biosynthetic substitution of the catalytically essential cysteine (Cysl49) of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by Sec led to selenoGAPDH that displayed GPx-like properties [70]. This selenoenzyme catalyzed the reduction of hydroperoxides with aryl thiols instead of GSH as this enzyme lacked a GSH-binding site. Similarly to the studies on seleno-GAPDH, Liu et al converted glutathione transferase (Lucilia cuprina LuGSTl-1) into selenoenzyme (seleno-LuGSTl-1) by means of auxotrophic expression [71]. The Ser9 in the GSH-binding site was mutated to cysteine and then biosynthetically substituted to selenocysteine in an auxotrophic expression system. This novel selenium-dependent enzyme exhibited high catalytic activity toward H2O2 in the presence of GSH, which was similar to that of the native GPx. For the first time, a seleniumcontaining enzyme with such remarkable GPx activity was generated by genetic engineering. It is now clear that the design of selenoprotein mimics plays important roles in understanding biochemical functions and reaction mechanisms. It is becoming apparent that selenoprotein mimics possess therapeutic potential against various diseases and that their fiinctions range from antioxidants to anticancer and antiviral agents. It can be anticipated that as our understanding of the basic biology and biochemistry of selenoproteins increases, future efforts will result in even more sophisticated approaches to the rational development of new selenoprotein mimics. References 1. 2. 3. 4. 5. 6. 7. 8.
JR Andreesen, L Ljungdahl 1973 J Bacterial 116:867 DC Turner, TC Stadtman 1973 Arch Biochem Biophys 154:366 L Flohe, EA GOnzler, HH Schock 1973 FEBSLett 32:132 JT Rotruck, AL Pope, HE Ganther, et al 1973 Science 179:588 GV Kryukov, S Castellano, SV Novoselov et al 2003 Science 300:1439 VN Gladyshev, GV Kryukov, DE Fomenko, DL Hatfield 2004 Annu Rev Nutr 24:579 T Tamura, TC Stadtman 1996 Proc Natl Acad Sci U.S.A. 93:1006 JR Arthur, F Nicol, GJBeckett 1990 Biochem J 111:52,1
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398 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.
Selenium: Its molecular biology and role in human health JQ Liu, MS Jiang, GM Luo, GL Yan, JC Shen 1998 Biotechnol Lett 20:693 SZ Mao, ZY Dong, JQ Liu et al 2005 J Am Chem Soc 127:11588 XJ Ren, P Jemth, PG Board et al 2002 Chem Biol 97:89 G Wulff 1995 Angew Chem Int Ed Engl 34:1812 J Liu, G Luo, S Gao, K Zhang, X Chen, J Shen 1999 Chem Commun 199 GM Luo, ZQ Zhu, L Ding et al 1994 Biochem Biophys Res Commun 198:1240 L Ding, Z Liu, ZQ Zhu, GM Luo, DQ Zhao, JZ Ni 1998 Biochem J 2,2,2:251 XJ Ren, SJ Gao, DL You et al 2001 Biochem J 359:369 DB Cowie, GN Cohen, 1957 Biochim Biophys Acta 26:252 N Budisa, B Steipe, P Demange et al 1995 Eur J Biochem 230-JS& N Budisa, C Minks, FJ Medrano et al 1998 Proc Natl Acad Sci USA 95:455 S MuUer, H Senn, B Gsell, W Vetter, C Baron, A Bock 1994 Biochemistry 33:3404 S Boschi-Muller, S Muller, AV Dorsselaer et al 1998 FEBS Letters 439:241 HJ Yu, JQ Liu, A Bock, J Li, GM Luo, JC Shen 2005 J Biol Chem 280:11930
Chapter 35. Update of human dietary standards for selenium Orville A. Levander Beltsville Human Nutrition Research Center, U. S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA
Raymond F. Burk Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Summary: An update of the human dietary standards for selenium is presented, including the year 2000 Dietary Reference Intakes (U.S.A.), the 1996 standards of the World Health Organization (WHO), and a recent relevant intervention trial carried out in China. Two criteria have been used by official bodies to set recommendations. One is the prevention of Keshan disease, the only proven selenium-responsive disease. The other is full expression of all selenoproteins as indicated by optimization of a plasma biomarker, either glutathione peroxidase activity or selenoprotein P concentration. An average per capita intake of 20 |xg selenium/day will prevent Keshan disease in a population but will not allow optimization (full expression) of selenoproteins. Using plasma glutathione peroxidase activity as the selenium biomarker to be optimized, the RDA for adults in the U.S.A. was set at 55 )ig in 2000. The recent trial in China utilized selenoprotein P as a biomarker and its results suggest that an upward revision of the current RDA will be needed. Even higher intakes of selenium have been postulated to prevent cancer. Intervention trials now underway in the U.S.A. are evaluating that possibility and the safety of large selenium supplements. Introduction In South Dakota during the 1930s, selenium was identified as the toxic agent in animal feeds and forages that caused the livestock poisoning known as "alkali disease" [1]. Plants that grew in certain areas of the Great Plains of the United States took up so much selenium from the selenium-rich soils that they became toxic to poultry and livestock. In cattle the disease is characterized by hair and hoof loss and a generalized emaciated appearance. For an extensive description of selenosis in farm animals, consult the monograph by Rosenfeld and Beath [2].
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There were suggestions in the literature that selenium might have a beneficial effect under certain conditions (e.g., see the work of Pinsent with bacteria [3]), but the prevailing opinion was that selenium was a toxin that played no positive role in metabolism. Moreover, selenium compounds were thought to be carcinogenic. Then in 1957 Schwarz and Foltz [4] announced their discovery that traces of dietary selenium could protect vitamin Edeficient rats from developing liver necrosis. Very soon thereafter, seleniumresponsive diseases were found in a variety of economically important farm animals including turkeys, chickens, sheep, swine, and cattle [5]. The need for selenium in human nutrition was shown by Chinese scientists who demonstrated in 1979 that this essential trace mineral protected against Keshan disease, a cardiomyopathy affecting young children and women of child bearing age residing in low-selenium areas in China. This finding increased greatly the interest in the human selenium requirement [6]. The current and previous dietary standard for selenium, the year 2000 Dietary Reference hitake (DRI) [7] and the 1989 RDA [8], respectively, were both based on maximization (or optimization) of plasma glutathione peroxidase activity so their values were rather similar. A proposal to base the next selenium standard on the full expression of selenoprotein P would lead to a higher value for the standard and is evaluated below. This review concludes with a discussion about the possible use of selenium as a cancer chemoprevention agent. Such a practice, if justified by studies that are in progress and planned, might result in substantially elevated recommendations of selenium intake. RDAs - tenth edition (1989) hiterest in the possible beneficial effects of selenium in human health continued to grow well into the 1980s and this was reflected in the Tenth Edition of the RDAs [8]. Literature citations increased six-fold. This was due not only to the increasing number of papers dealing with selenium but also to the expressed desire of the RDA Committee to make the RDA book more "scientific" and one in which every step of the derivation of the RDA was "transparent" so that the logic and reasoning behind the derivation of the dietary standard was clear and open for everybody to see. Fortunately for selenium researchers, several studies from China allowed the RDA Committee to pinpoint human selenium requirements with increased precision such that it was possible to advance selenium to full RDA status for the first time. One group of studies examined the dietary selenium intake needed to prevent Keshan disease in regions of China where it was endemic [9]. The disease did not occur in those areas in which the selenium intake by adults was 17 jxg/day or more. Thus, 17 ^g/day was suggested as a minimum daily requirement based on disease prevention.
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In another study, a "physiological" selenium requirement was determined by following increases in glutathione peroxidase activity in the plasma of men living in a Keshan disease area who were given graded doses of selenomethionine as a supplement over a period of several months [10]. At a total intake of 41 jig/day or more (11 p.g from diet), the plasma glutathione peroxidase activity became optimized. Therefore, it was concluded that Chinese men had a physiological requirement of 41 fig/day. In order to convert this figure to an RDA for North American males, it was necessary to apply a correction factor for differences in body weight (79/60) and to apply a safety factor (1.3) to allow for individual differences in requirement. Thus, the calculation for adult males became: 41 X 79/60 X 1.3 = 70^g/day For adult North American females, the calculation was: 41 X 63/60 X 1.3 = 55ng/day A more detailed explanation of the RDA calculations for adults was presented elsewhere [11]. Because of the lack of data, the RDAs for young adults also served as the basis of RDAs for the elderly. Likewise, because of the lack of data, RDAs for infants and children were based on adult values with extrapolations downward on the basis of body weight plus a factor arbitrarily allowed for growth. The RDA during pregnancy was calculated using a factorial technique based on the fetal accretion of selenium. The RDA during lactation provided sufficient selenium to avoid depletion of the mother and permit a satisfactory selenium content in the breast milk. The Tenth Edition discussed selenium toxicity only in general terms [8]. An episode of human selenosis in China was described in which hair loss and fingernail changes were observed on intakes approximating 5000 jig selenium/day. It was pointed out that sensitive and specific biochemical indices of selenium overexposure were not available and no attempt was made to establish a safe upper limit of selenium intake. World Health Organization (1996) In 1996, the World Health Organization (WHO) published its dietary standards for several trace elements, including selenium [12]. WHO has the responsibiUty for setting recommendations that apply to many different countries around the globe (United Nations member states) that have highly varied national diets. For that reason, the Organization tends to suggest nutrient intakes that are often somewhat lower than those set in the U.S.A. This also turned out to be true for selenium since large parts of the U. S. Great Plains, a major wheat production area, have soils that are rich in
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selenium and relatively generous amounts of the trace element are incorporated into the food chain. Intakes of selenium exceeding 100 i^g/day are not uncommon in the U.S. and so meeting an RDA of 55 to 70 }ig/day is not difficult. On the other hand, meeting the 1989 RDA for selenium could be quite a challenge for some other coimtries. Many parts of China, for example, routinely consume much lower amounts of selenium in their diet [9] and New Zealanders rarely ingest such RDA levels [13]. Likewise, Finland had a low-selenium food supply before deciding in 1985 to add selenium to its fertilizers [14]. In fact, dietary surveys indicate that several European countries would have problems achieving intakes as high as the 1989 RDA, including Belgium, Denmark, France, Germany, United Kingdom, Slovakia, and Sweden [reviewed in [15]]. The selenium intake in Switzerland was somewhat higher because of the common use of North American wheat rich in selenium. So it is not surprising that WHO was reluctant to set a dietary standard that so many of its member states could not attain, especially in the absence of any evidence of signs of human selenium deficiency outside of China. The reader will recall that the rationale used by the 1989 RDA Committee for its selenium recommendation was full expression of plasma glutathione peroxidase activity. The WHO Committee decided that such full activity was probably not necessary for human health and that only two-thirds full activity of plasma glutathione peroxidase still afforded sufficient protection against oxidative stress. This conclusion was based on observations that blood cells metabolized hydrogen peroxide satisfactorily until their glutathione peroxidase activity fell to one-quarter or less of normal. Of course, if one selects a lower target glutathione peroxidase activity for the biochemical criterion of adequate nutriture, this allows a lower dietary standard to be proposed also. In this case, the WHO Committee (formal designation: Joint FAO/IAEAAVHO Expert Consultation on Trace Elements in Human Nutrition) came up with 40 and 30 |J.g/day for the lower limit of the safe range of population mean dietary selenium intake that would meet the normative requirement of most adult males and females, respectively. As defined by WHO, the normative requirement referred to the "level of intake that serves to maintain a level of tissue storage or other reserve that is judged by the Expert Consultation to be desirable" [12]. WHO also defined a basal requirement that referred to the "intake needed to prevent pathologically relevant and clinically detectable signs of impaired function attributable to inadequacy of the nutrient." For selenium, the basal requirement was taken from the quantity needed to protect against Keshan disease. The lower limit of the safe range of population mean dietary selenium intake that would meet the basal requirement of most adult males
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and females was calculated to be 21 and 16 p-g/day, respectively, after adjusting for body weight.
Table 1. The 1996 WHO Lower Limits of the Safe Ranges (Basal and Normative) of Population Mean Intakes of Dietary Selenium (ng/day)
Life Stage
Aee fvears)
Basal
Normative
Infants
0-0.25 0.25-0.5 0.5-1.0
3 5 6
6 9 12
1-3 3-6 6-10
10 12 14
20 24 25
Males
10-12 12-15 15-18 18+
16 19 21 21
30 36 40 40
Females
10-12 12-15 15-18 18+
16 16 16 16
30 30 30 30
Pregnancy
18
39
Lactation 0-3 months 3-6 months 6-12 months
21 25 26
42 46 52
Children
Adapted from [12].
The WHO Committee also attempted to deal with the question of tolerances of high dietary selenium intakes. On the basis of considerable fieldwork with human selenosis in China, Yang and associates proposed 750850 ^g as a marginal level of daily safe dietary selenium intake [16], defined as "the level of selenium intake at which few individuals have functional signs of excessive intake and above which the tendency to exhibit functional signs is apparent and symptoms may first appear among ... susceptible individuals [whose] selenium intake [is] further increased". The Committee
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Selenium: Its molecular biology and role in human health
took one-half of the average of this range because of the uncertainty surrounding the harmful dose of selenium for people to suggest a maximal daily safe dietary selenium intake of 400 |ig. Dietary reference intakes (2000) The new millennium saw a host of changes in the way that dietary standards for selenium (and many other nutrients) were handled in the U. S. A. [7]. First of all, selenium was grouped with a variety of so-called "dietary antioxidants" (vitamins C and E and the carotenoids) instead of with the trace elements where it had traditionally been put. This change made sense because selenium, due to its multitude of roles protecting against oxidative stress, really had more in common with the nutritional antioxidants than it did with a collection of various microminerals. Another substantial change was in the dietary standards themselves [7]. The general term "Dietary Reference Intakes" was used to describe not only the RDA, but also Adequate Intake (AI), Tolerable Upper Intake Level (UL), and Estimated Average Requirement (EAR). Each of these terms has a particular role in describing the dietary standards of a nutrient and it might be worthwhile to repeat here their meanings as presented by the Panel on Dietary Antioxidants and Related Compounds: Recommended Dietary Allowance {RDA): the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 to 98 percent) healthy individuals in a particular life stage (which considers age, and when applicable, pregnancy and lactation) and gender group. Adequate Intake (AI): a value based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of healthy people that are assumed to be adequate—used when an RDA cannot be determined. Tolerable Upper Intake Level (UL): the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the risk of adverse effects increases. Estimated Average Requirement (EAR): a daily average nutrient intake value that is estimated to meet the requirement of half the healthy individuals in a life stage and gender group." Thus, the redefinition of the RDA echoes the 1989 version [8], which states that they are "... the levels of intake of essential nutrients that... are judged ... to be adequate to meet the known nutrient needs of practically all healthy persons." The AI is reminiscent of the "Estimated Safe and Adequate Daily Dietary Intake" which was a dietary standard to be used when insufficient data were available to posit an RDA. The UL represents the first formal attempt by an "RDA Committee" to establish a ceiling of intake for the nutrients being considered by the group. In the "Dietary Reference Intakes"
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(what the handbook on dietary standards in North America is now called— the title is no longer "Recommended Dietary Allowances") an entire chapter is devoted to describing a model for the development of ULs for nutrients. The EAR occupies a critical place in the new dietary standards, for without it, there can be no RDA. These two entities are related by the equation: RDA = EAR + 2 SD where SD is the standard deviation of the EAR. If the SD was unknown, the 2000 Committee generally assumed a coefficient of variation of 10% for the EAR so that RDA = 1.2 X EAR The 2000 Committee based its EAR on 2 intervention trials designed to estimate selenium requirements by determining the intake needed to optimize plasma glutathione peroxidase activity. The first trial was carried out in China [10] and in fact was the same study that served as the basis for the 1989 RDA [8]. The selenium intake needed to optimize plasma glutathione peroxidase in that work was 41 (ig/day, which came to 52 j^g/day after adjustment for Western body weight. The second intervention trial was from New Zealand [13] and the 2000 Committee interpreted that research as suggesting an EAR of 38 ^ig/day. Although other interpretations of the New Zealand trial may be possible [15], the average of both the New Zealand and Chinese trials, 45 |ig/day, was selected as the EAR. The RDA for adult males then was calculated as 45 X 1.2 to yield 55 ng/day. Thus, by using a lower base requirement figure than the 1989 Committee (45 vs. 52 (ig/day after adjustment for body weight) and a smaller correction factor for individual variation (1.2 vs. 1.3), the 2000 Committee arrived at a lower RDA figure for adult males than the 1989 Committee (55 vs. 70 |ig/day). Given the reported greater susceptibility of women to develop Keshan disease, their RDA was also set at 55 |ig/day despite their smaller body weight. The 2000 Committee could find no data available to calculate an EAR for children or adolescents, so the RDAs for them were extrapolated from young adult values. Similarly, there were no data that specifically addressed the selenium requirement for elderly persons and the 2000 Committee found no information that suggested that the aging process impaired selenium absorption or utilization, so their RDA was the same as young adults. A major philosophical shift occurred in the way that requirements were presented for infants up to one year if age. Because "No functional criteria of selenium status have been demonstrated that reflect response to dietary intake in infants", the 2000 Committee rescinded the 1989 RDA, so to speak.
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Selenium: Its molecular biology and role in human health
and replaced it with an AI based on the "mean selenium intake of infants fed principally with human milk." This fundamental change in viewing infant requirements was not limited to selenium. In fact, other nutrients have also been accorded only AI status for infants, including calcium, magnesium, vitamin D, thiamin, and riboflavin. The 2000 Committee selenium AI for infants for the first and second 6 months of life are 15 and 20 ng/day, respectively, up 50% and 33% from their 1989 counterparts, respectively. Using somewhat different assumptions, the 2000 Committee came up with RDAs for pregnancy and lactation that were slightly less than those set by the 1989 Committee (60 vs. 65 fxg/day and 70 vs. 75 |ig/day, respectively). Table 2. The 2000 Dietary Reference Intakes (DRI) for Selenium (^g/day)
Life Stage
Age
DRI (ng/day)
Infants
0-6 mo 7-12 mo
15* 20*
Children
1-3 y 4-8 y
20 30
Males
9-13 y 14-70 y >70y
40 55 55
Females
9-13 y 14-70 y >70y
40 55 55
Pregnancy
60
Lactation
70
Adapted from [7]; values with asterisk are AI, others are RDA.
Another innovation in the 2000 DRI was the establishment of an upper limit of intake. The UL for selenium was based on the criteria of h^ir and nail brittleness and loss due to dietary overexposure in a high-selenium region in China. Intakes of selenium from food sources were inferred from blood levels. A No-Observed-Adverse-Effect-Level (NOAEL) was calculated to be 800 |ig/day. An imcertainty factor of 2 was chosen to protect sensitive individuals, thereby leading to a UL of 400 fig/day for adults 19 years of age
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407
and older, a figure in agreement with upper limits set by others [12]. Finding no evidence of teratogenicity or selenosis in infants of mothers consuming high but not toxic amounts of selenium, the 2000 Committee kept to 400 Jig/day UL for pregnant and lactating women. The UL for infants 0 to 6 months old consuming human breast milk exclusively was set at 45 |ig/day based on the lack of any adverse effects (NOAEL) reported in such infants consuming breast milk containing 60 |ig selenium/L. The ULs for older infants, children, and adolescents were extrapolated on the basis of body weights. Recent trial using Selenoprotein P and glutathione peroxidase as biomarkers A report appeared in 2005 that described a selenium intervention trial carried out in 2001 in a low-selenium area of China [17]. The trial was designed to determine the selenium intake needed to 'optimize' the plasma selenium biomarkers, glutathione peroxidase and selenoprotein P. These 2 selenoproteins are accessible representatives (biomarkers) of the entire family of selenoproteins [18]. Optimization of them is used as an indicator of optimization (full expression) of all the selenoproteins in the body. A given selenoprotein is 'optimized' when providing additional selenium does not result in an increase in its concentration. There is a 'hierarchy' of selenoproteins with respect to their claim on available selenium. This means that when the selenium available will not allow optimization (full expression) of all selenoproteins, the selenoproteins most essential to the organism receive selenium and selenoproteins that are less essential do not. Thus, the "least essential" selenoprotein will be the last to be optimized when selenium availability is increased from inadequate to adequate, according to this concept. Animal experiments have suggested that liver glutathione peroxidase is the lowest selenoprotein in the hierarchy [19]. Because human tissues cannot be sampled for routine studies and blood can, plasma selenoproteins have been used as the best available representatives of the selenoproteins in the body. The subjects studied were farmers in a low-selenium region of Sichuan Province. Their average dietary selenium intake was 10 |j,g per day. hiitial plasma glutathione peroxidase activity was 40% of tj^ical U.S. values and selenoprotein P concentration was 23% of typical U.S. values. Subjects were supplemented for 20 weeks with placebo or several dose levels of selenite or L-selenomethionine (henceforth selenomethionine). Glutathione peroxidase became optimized with a supplement of 37 i^g of selenium per day as selenomethionine. The same level of glutathione peroxidase was achieved with 66 |j,g per day as selenite. The selenomethionine results are close to those of the 1983 study in China [10] and the 1995 study in New Zealand [13] and essentially confirm their results.
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Selenium: Its molecular biology and role in human health
Selenite has not been evaluated previously in this type of study and its lower bioavailability than that of selenomethionine is noteworthy. Selenoprotein P did not become optimized in this trial, even when 61 ng selenium per day was administered as selenomethionine. Thus, selenoprotein P is lower in the hierarchy of human selenoproteins than plasma glutathione peroxidase and is therefore the better biomarker for optimization of the selenoproteins in the body. It seems likely that use of selenoprotein P as a selenium biomarker will lead to an increase in the selenium dietary intake recommendations. However, the results in this study do not allow an estimation of the amount of selenium needed because optimization of selenoprotein P was not achieved. Moreover, it is possible that supplementation at the dose levels used in this study for more than 20 weeks will optimize selenoprotein P. Thus, a study that includes higher supplemental dose levels and a longer supplementation period is needed. This study has pointed out that different biomarkers are optimized at different selenium intakes and that therefore the choice of biomarker is important. Presently, selenoprotein P appears to be a better biomarker than plasma glutathione peroxidase activity. The study has confirmed the results of the 2 earlier studies that used plasma glutathione peroxidase. It indicates, however, that the current selenium recommendations, based on glutathione peroxidase, are likely to be too low. Another important finding of this study is the greater bioavailability of selenium in the form of L-selenomethionine than in the form of selenite. This has implications for formulation of selenium supplements and addition of selenium to foods. Selenium as a possible cancer chemoprevention agent In total, almost 150,000 individuals have participated in phase III nutritional intervention studies to prevent cancer [20]. Besides selenium, nutritional intervention agents have included beta-carotene, alpha-tocopherol, retinal, and various vitamin and mineral combinations. Several lines of evidence have suggested that selenium might be a suitable candidate for testing as a chemoprevention agent. First, experiments with laboratory animal models indicated that various selenium compounds protect against tumorigenesis under a variety of conditions [21]. Second, about half of the 36 epidemiological studies evaluated by the FDA implied some value of selenium against cancer [22]. Finally, the selenium intervention trial of Clark and colleagues [23] found that subjects given 200 jxg selenium/day in yeast form to prevent skin cancer had lower incidences of several cancers than did the placebo group. However, no effect on skin cancer was observed. Because of the positive results in these studies, the U.S. National Cancer Institute is sponsoring a large trial (32,400 men) called SELECT (the
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Selenium and Vitamin E Cancer Prevention Trial) [24]. SELECT is a phase III randomized, placebo-controlled test of selenium (200 (j.g/day as Lselenomethionine) and/or vitamin E (400 lU/day) to prevent prostate cancer in U.S. men. The subjects in SELECT are presumed to be selenium replete, with full expression of their selenoproteins. Thus, if SELECT demonstrates a preventive effect of selenium and/or vitamin E on prostate cancer, it will indicate that there is a health-related function of selenium independent of selenoproteins. Studies to characterize the dose of selenium needed to achieve the chemopreventive effect would then be desirable to inform strategies of supplementation. SELECT will also evaluate safety. Further consideration of the use of selenium to prevent cancer will depend on its being safe. The Food and Nutrition Board set an Upper Limit of selenium intake in their 2000 DRJ's at 400 Jig/day [7]. Intakes of subjects in the SELECT trial will be in the 300+ Hg/day range and determination of the safety of that intake over years will be important. Orderly progression of selenium chemoprevention studies is important. Only when data are produced that show selenium to be safe and effective in cancer prevention can recommendations to the public be made. References 1. 2. 3. 4. 5.
6. 7. 8.
9. 10.
11. 12. 13. 14. 15. 16. 17.
AL Moxon, M Rhian 1943 Physiol Rev 23:305 I Rosenfeld, O Beath 1964 Selenium. Geobotany, biochemistry, toxicity, and nutrition Academic Press New York J Pinsent 1954 Biochem J 57:10 K Schwarz, CM Foltz 1957 JAmer Chem Soc 79:3292 Subcommittee on Selenium, Committee on Animal Nutrition, Board on Agriculture, National Research Council 1983 Selenium in Nutrition Revised Edition National Academy Press Washington p 174 Keshan Disease Research Group 1979 Chinese Medical Journal 92:471 Institute of Medicine 2000 Selenium. In: Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids National Academy Press Washington pp 284-324 Subcommittee on the Tenth Edition of the RDAs, Food and Nutrition Board, National Research Council 1989 Recommended Dietary Allowances lO"" Edition National Academy Press Washington GQ Yang, KY Ge, JS Chen, XS Chen 1988 World Rev Nutr Diet 55:98 G-Q Yang, L-Z Zhu, S-J Liu, L-Z Gu, P-C Qian, J-H Huang, M-D Lu 1987 In: Selenium in biology and medicine.. Part B (eds. GF Combs Jr, JE Spallholz, OA Levander, JE Oldfield) AVI New York pp 589-607 OA Levander 1991 J Am Diet Assoc 91:1572 Trace Elements in Human Nutrition and Health. Report of a Joint FAO/IAEA/WHO Expert Consultation 1996 World Health Organization, Geneva pp 343 AJ Duffield, CD Thomson, KE Hill, S Williams 1999 Am J Clin Nutr 70:896 A Aro, G Alfthan, P Varo 1995 Analyst 120:841 MP Rayman 2000 Lancet 356:233 G Yang, S Yin, R Zhou, L Gu, B Yan, Y Liu, Y Liu 1989 J. Trace Elem. Electrolyes Health Dis y.\22 Y Xia, KE Hill, DW Byrne, J Xu, RF Burk 2005 Am J Clin Nutr 81:829
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Selenium: Its molecular biology and role in human health
18. GV Kryukov, S Castellano, SV Novoselov, AV Lobanov, O Zehtab, R Guigo, VN Gladyshev 2003 Science 300:1439 19. J-G Yang, KE Hill, RF Burk \9S9 JNutr 119:1010 20. PR Taylor, P Greenwald 2005 J Clin Oncol 23:333 21. CIp 1998JiV«/r 128:1845 22. PR Trumbo 2005 JNutr 135:354 23. LC Clark, GF Combs Jr, BW Tumbull, EH Slate, DK Chalker, J Chow, LS Davis, RA Glover, GF Graham, EG Gross, A Krongrad, JL Lesher, HK Park, BB Sanders, CL Smith, JR Taylor 1996 JAMA 276:1957 24. SM Lippman, PJ Goodman, EA Klein, HL Pames, IM Thompson Jr., AR Kristal, RM Santella, JL Probstfield, CM Moinpour, D Albanes, PR Taylor, LM Minasian, A Hoque, SM Thomas, JJ Crowley, JM Gaziano, JL Stanford, ED Cook, NE Fleshner, MM Lieber, PJ Walther, FR Khuri, DD Karp, GG Schwartz, LG Ford, CA Coltman Jr 2005 J Natl Cancer Inst 97-M
Index
Acquired immunodeficiency syndrome, 299-310 immunity, 300-302 metabolic syndrome, 302-304 micronutrient deficiencies, 304-305 selenium deficiency, 305-306 selenium supplementation, 306-308 wasting, 302-304 Adhesion molecules, cytokines, 314-315 Aging methionine sulfoxide reduction, 126-127 selenium in, 318-320 AIDS. See Acquired immunodeficiency syndrome Aminoacyl-tRNA recycling, 83-95 Antibodies, mimics of selenoprotein, selenoenzyme transformation, 394-395 Apoptosis, selenium-induced, 379-385 mitochondrial dysfunction, selenium-induced, 382-384 oxidative stress, selenium-induced, 381-382 safe levels of exposure, 380-381 selenium toxicity, 380 thiol modification, selenium-induced, molecules targeted for, 382 Atom detection, selenium, 224-225 Bacteria biosynthesis of selenocysteine, 14-15 decoding UGA with selenocysteine, 24-25 E. coli SECIS element, in gene expression, 16-11 incorporation of selenocysteine by, 12 SECIS element interaction, 18-21 SelB, domain structure, 18-21 termination vs. readthrough, 11-14 translation factor, SelB, 15-18 tRNAS'^'', 12-13 Bioinformatics tools for, selenoprotein identification, 99-102 Bios)mthesis of selenocysteine, 14—15 Brain function
effects of selenoprotein P deletion, 115 epilepsy, 238-239 neurodegenerative disorders, 239-241 Parkinson's disease, 236-237 selenium selenoproteins, 233-248 stroke, 234-236 transgenic selenoprotein-deficient mouse models, 241-245 Cancer prevention, 249-264, 367-368 animal models, 250 clinical trials, 250-252 epidemiological evidence, 250 15-kDa selenoprotein, 141-148 in cancer prevention, 142-143 dietary selenium, 146 glycoprotein glucosyltransferase, 143-142 thiol-disulfide oxidoreductase function, 145-146 mechanisms, 252-254 metabolic bases, 254-260 metabolomics, 373-374 nutrigenetics, 369-370 nutritional epigenetics, 370-371 nutritional transcriptomics, 371-372 proteomics, 373-374 Se-metabolities, 255-260 selenium dietary standards, 408^09 selenoenzymes, 254—255 selenoprotein gene variation, 277-286 GPx-1 in cancer etiology, 279-282 polymorphisms, 283-284 selenoproteins, 279 Sepl5, 282-283 thiol proteomics, 265-276 BiP/GRP78 over-expression, 272-273 methylseleninic acid, 266 monomethylated selenium, as protein redox modulator, 266-269 redox-modified proteins, 269 unfolded protein response, ER stress and, 269-271
412
Selenium: Its molecular biology and role in human health
UPR signaling, 271-272 Catalytic mechanisms, methionine sulfoxide reduction, 128 Cell-mediated immunity, 315-316 Conditional knockout mouse models, 339-340 Conformation-specific SEClS-binding activities, SBP2, eukaryotic selenoprotein synthesis, 69-70 Coxsackievirus, 287-290 Cys-containing counterparts, methionine sulfoxide reduction. Sec-containing proteins compared, 131 Cytoplasmic supramolecular complex, supramolecular complexes, selenocysteine biosynthesis, isolation, 89-91 Cytosolic, mitochondrial thioredoxin reductase knockout mice, 195-206 phenotype Txnrdl knockout embryos, 199-200 Txnrd2 knockout embryos, 201-202 Txnrdl/Txnrd2 embryonic expression profile, 198-199 embryonic lethality, 198 heart-specific inactivation of, 202-204 mouse models with conditional alleles for, 197-198 Decoding selenocysteine, 39-50 genetic code, 4 8 ^ 9 phenotype, dynamic process of evolution, 45^6 Sec decoding common origin, 40-41 lost trait, 41-42 Sec-tRNA^'^'' synthesis, non-canonical mechanism, 46-48 selenophosphate synthetase, 42-45 Decoding selenophosphate synthetase trait, 41^2 Decoding UGA with selenocysteine, 24-25 Deiodinases, endocrine function, 207-219 adaptive thermogenesis, 212-214 Dl overexpression, h5fperthyroidism, 217 D3 overexpression in hemangiomas, 216 deiodinases conservation, 3D structure, 208 fasting, changes in iodothyronine deiodination, 214-215 illness, changes in iodothyronine deiodination, 214-215 thyroid hormone homeostasis, 211
tissue-specific control of thyroid hormone action, 211-212 ubiquitination pathway, D2 inactivation, 209-211 Detection of selenium atom, 224-225 Diabetes, 173-182 early research, 175-176 glutathione peroxidase-1 in, 173-182 early research, 175-176 insulin function, 180 metabolic impact, 175 selenoprotein expression, 174-175 insulin function, 180 metabolic impact, 175 Dietary standards, selenium, 399^10 cancer chemoprevention, 408^09 dietary reference intakes, 404-407 glutathione peroxidase, as biomarker, 407-408 RDAs, 400-401 selenoprotein P, as biomarker, 407^08 World Health Organization, 401-404 Domain structure, SelB, 18-21 Drosophila, selenoproteins, 343-353 SelG/SelK/G-rich,351 SelM/BthD/SelH, 350-351 synthesis machinery, 344-348 intake, 344 selenocysteyl-tRNA, 344-345 selenophosphate synthetase, 345-348 translational machinery, 348-349 Drug development, parasite selenoproteins, 364-365 E. coll SECIS element, in gene expression, 26-27 EEFSec, SBP2 interactions, eukaryotic selenoprotein synthesis, 66-67 Eicosanoid metabolism, 313-314 Endocrine function, deiodinases and, 207-219 adaptive thermogenesis, 212-214 Dl overexpression, hyperthyroidism, 217 D3 overexpression, hemangiomas, 216 deiodinases conservation, 3D structure, 208 fasting, changes, iodothyronine deiodination, 214—215 illness, changes, iodothyronine deiodination, 214-215 thyroid hormone homeostasis, 211 tissue-specific control of thyroid hormone action, 211-212
Index ubiquitination pathway, D2 inactivation, 209-211 Endogenous Sec factors, supramolecular complexes, selenocysteine biosynthesis, supramolecular complexes composed of, 92 En2ymes, natural, mimics of selenoprotein, selenoenzyme transformation, 392-393 Epilepsy, selenium selenoproteins, 238-239 Eukaryotic Sec bios}Tithesis, supramolecular complexes, selenocysteine biosynthesis, protein factors, 86-87 Eukaryotic selenocysteine tRNAs, 29-37 biosynthesis, 32-34 evolution, insertion machinery, 34—36 insertion machinery, 34-36 mammalian Sec tRNA[s«]s