NOTE TO USERS This reproduction is the best copy available PEROXISOME SENESCENCE AND THE ROLE OF CATALASE IMPORT IN CELLULAR AGING by JAY I KOEPKE DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2006 MAJOR: PHARMACOLOGY Approved by: Advisor Date UMI Number: 3221036 UMI Microform 3221036 Copyright 2006 by ProQuest Information and Learning Company All rights reserved This microform edition is protected against unauthorized copying under Title 17, United States Code ProQuest Information and Learning Company 300 North Zeeb Road P.O Box 1346 Ann Arbor, MI 48106-1346 DEDICATION I dedicate this thesis to my wife Amy, who has been a constant source of encouragement and support Without her devotion and tolerance I not believe I would have been able to accomplish the work herein She is an inspiration and I am truly grateful for having her by my side And to my unborn child, may you benefit from your mother’s everlasting affection as I have ii ACKNOWLEDGMENTS I would first like to offer my uppermost gratitude to my advisor, Dr Stanley R Terlecky, whose enthusiastic approach to science as well as life’s circumstances has left a lasting impression and will bolster my future endeavors His encouragement, guidance, and friendship helped facilitate my graduate career I would like to sincerely thank my committee members – Drs Nicholas G Davis, Ladislau C Kovari, Roy B McCauley, and Raymond R Mattingly for their helpful suggestions and supervision throughout this process I also acknowledge collaborators such as Dr Paul A Walton and Dr Marc Fransen for their expertise and contributions to my research project I genuinely thank past and present members of the Terlecky laboratory – in particular Julie Legakis, Ferdous Barlaskar, Chris Wood, and Laura Terlecky They provided invaluable support for many of the studies herein I am grateful to my parents Walt and Helen Koepke – who have been a permanent fixture in my overall development I also owe appreciation to my siblings, Jeff and Jill, and my in-laws Thomas and Wiesia Petroske iii TABLE OF CONTENTS Chapter Page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF TABLES v LIST OF FIGURES vi CHAPTERS CHAPTER – Introduction CHAPTER – Materials and Methods .29 CHAPTER – Peroxisome Senescence in Human Fibroblasts 48 CHAPTER – Catalase Inactivation Drives Cells Towards a Senescence-like Phenotype 75 CHAPTER – Restoration of Peroxisomal Catalase Import 100 REFERENCES 127 ABSTRACT 155 AUTOBIOGRAPHICAL STATEMENT 157 iv LIST OF TABLES TABLE PAGE Table Human peroxisomal hydrogen peroxide-producing and -degrading enzymes and their relative PTS1 strength 28 v LIST OF FIGURES FIGURE PAGE Figure Quantitative in vitro assay for peroxisomal protein import 21 Figure Senescence-associated β-galactosidase activity in human diploid fibroblasts (HDFs) 50 Figure Peroxisomes in early, middle, and late passage HDFs 52 Figure Ultrastructure of early and late passage HDFs 54 Figure Peroxisomes in co-cultured early and late passage HDFs .56 Figure PTS1(-SKL)-protein import in early, middle, and late passage HDFs 59 Figure Analysis of Pex5p binding 62 Figure Quantitative analysis of peroxisomal protein import 64 Figure Catalase import in early and late passage cells 65 Figure 10 Pex5p’s association with organelle membranes 67 Figure 11 Hydrogen peroxide accumulates in aging HDFs 70 Figure 12 Effect of hydrogen peroxide on PTS1(-SKL)-protein import and PTS1 receptor localization 72 Figure 13 Inhibition of catalase by treatment of cells with 3-AT 77 Figure 14 Accumulation of ROS upon acute inhibition of catalase with 3-AT 80 Figure 15 Chronic catalase inhibition increases peroxisomal numbers 82 Figure 16 The effect of catalase inhibition on proliferation of HDFs .86 vi Figure 17 Protein carbonylation in normal and 3-AT treated cells 87 Figure 18 DNA damage in human fibroblasts 90 Figure 19 PTS1(-SKL)-protein import in untreated and 3-AT treated HDFs .91 Figure 20 Mitochondrial membrane potential and ROS production 94 Figure 21 Gelatin zymograpy of conditioned media from HDFs 98 Figure 22 Binding of Pex5p to catalase derivatives 104 Figure 23 Peroxisomal import of catalase derivatives 106 Figure 24 Peroxisomal import of catalase derivatives in early and late passage cells 108 Figure 25 Localization of catalase-SKL and Pmp70p in late passage cells 109 Figure 26 Association of catalase-SKL with endogenous catalase in cells 111 Figure 27 CPP-mediated catalase-SKL transduction into cultured human fibroblasts 114 Figure 28 Distribution of transduced catalase-SKL in mouse skin 116 Figure 29 Effects of transducible catalase-SKL administration on hydrogen peroxide levels in the skin of aged mice 117 Figure 30 Amplification plots of real-time PCR analysis of mouse transgenic DNA 119 Figure 31 Peroxisome deterioration spiral 122 vii 143 Miura, S., Kasuya-Arai, I., Mori, H., Miyazawa, S., Osumi, T., 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Traffic 7, 97-107 Yildirim O, Ates NA, Tamer L, Musler N, Ercan B, Atik U, Kanik A (2004) Changes in antioxidant enzyme activity and malondialdehyde level in patients with age-related macular degeneration Ophthalmologica 218:202-206 Youssef J A and Badr M Z (2005) Aging and enhanced hepatocarcinogenicity by peroxisome proliferator-activated receptor alpha agonists Ageing Research Reviews 4, 103-118 Zoeller RA, Lake AC, Nagan N, Gaposchkin DP, Legner MA, Lieberthal W (1999) Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether Biochem J 338:769-776 154 Zoeller RA, Morand OH, Raetz CRH (1988) A possible role for plasmalogens in protecting animal cells against photosensitized killing J Biol Chem 263:11590-11596 155 ABSTRACT PEROXISOME SENESCENCE AND THE ROLE OF CATALSE IMPORT IN CELLULAR AGING by JAY I KOEPKE August 2006 Advisor: Dr Stanley R Terlecky Major: Pharmacology Degree: Doctor of Philosophy Peroxisomes are ubiquitous subcellular organelles required for proper functioning of eukaryotic cells They efficiently compartmentalize enzymes responsible for a number of essential cellular processes, including the metabolism of certain specific fatty acid chains via β-oxidation These and other oxidative reactions produce hydrogen peroxide, which is, in most instances, immediately processed in situ to water and oxygen The responsible peroxidase is the heme-containing tetrameric enzyme, catalase What has emerged during our investigation is that there are circumstances in which the tightly regulated balance of hydrogen peroxide producing and degrading activities in peroxisomes is upset - leading to the net production and accumulation of hydrogen peroxide and downstream reactive oxygen species The factor most essentially involved is catalase, which is missorted in aging, 156 missing or present at reduced levels in certain disease states, and inactivated in response to exposure to specific xenobiotics Our results indicate that the import of peroxisomal proteins containing a type targeting signal (PTS1) decline with age in human diploid fibroblasts Compromised to an even greater extent is the import of catalase - an enzyme with a poorly recognized PTS1 Taken together, these data suggest that as cells age, the ability to maintain a balance of hydrogen peroxide-generating and degrading activities within peroxisomes is disrupted This leads to peroxisomal dysfunction, which increases the oxidative load experienced by cells and presumably contributes to the aging process The overall goal of this thesis is to describe the molecular events associated with the development and advancement of peroxisomal senescence and to describe its effects on cells In addition, results of these efforts to increase levels of peroxisomal catalase and restore oxidative balance in cells 157 AUTOBIOGRAPHICAL STATEMENT Jay I Koepke EDUCATION/EMPLOYMENT: 1993 – 1997 1997 – 2000 2000 – 2003 2003 – 2006 Alma College, Alma, MI B.S Biology Wayne State University Department of Biological Sciences, Detroit, MI M.S Molecular Biotechnology Wayne State University School of Medicine Department of Pharmacology, Detroit, MI Research Assistant Wayne State University School of Medicine Department of Pharmacology, Detroit, MI Ph.D Pharmacology (Dr Stanley R Terlecky, advisor) PUBLICATIONS: Robinson, K.A., Koepke, J.I., Kharodawala, M., and Lopes, J.M (2000) A network of yeast basic helix-loop-helix interactions Nucleic Acids Research, 28: 4460-4466 Legakis, J.E., Koepke, J.I., Jedeszco, C., Barlaskar, F., Terlecky, L.J., Walton, P.A., and Terlecky, S.R (2002) Peroxisome senescence in human fibroblasts Molecular Biology of the Cell, 13: 4243-4255 Logsdon, B.C., Vickrey, J.F., Martin, P., Proteasa, G., Koepke, J.I., Terlecky, S.R., Wawrzak, Z., Winters, M.A., Merigan, T.C., and Kovari, L.C (2003) Crystal structure of a multidrug-resistant HIV-1 protease clinical isolate reveals an expanded active site cavity Journal of Virology, 78: 3123-3132 Wood, C.S., Koepke, J.I., Teng, H., Boucher, K.K., Katz, S., Chang, P., Terlecky, L.J., Papanayotou, I., Walton, P.A., and Terlecky, S.R (2006) Hypocatalasemic fibroblasts accumulate hydrogen peroxide and display age-associated pathologies Traffic, 7: 97-107 Koepke, J.I., Walton, P.A., Boucher, K.K, Terlecky, L.J., Nakrieko, K.A., Wood, C.S., and Terlecky, S.R (2006) Restoration of peroxisomal catalase import in aging cells Manuscript submitted Terlecky, S.R., Koepke, J.I., and Walton, P.A (2006) Peroxisomes and Aging Manuscript submitted Koepke, J.I and Terlecky, S.R (2006) Catalase Inactivation drives cells towards a senescent-like Phenotype In preparation ... an increase in the amount of peroxisome- associated Pex5p (Dodt and Gould, 1996) and compromised protein import An intraperoxisomal organizer of the import machinery, Pex8p, links the docking and. .. dolichol, and other isoprenoids; the committed step in ether phospholipids (plasmalogen) synthesis; and the catabolism of certain purines, polyamines, and amino acids (Wanders, 2004) Many of these... with either the basic domain of HIV-1 Tat or arginine residues at the aminoterminus A double-stranded oligonucleotide encoding amino acids from either the basic domain of HIV-1 Tat (amino acids