A CHEMICAL APPROACH TO DISTINGUISH ATP-DEPENDENT PROTEASES By JENNIFER FISHOVITZ Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Irene Lee Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES Jennifer Fishovitz PhD Mary Barkley Irene Lee Thomas Gerken Anthony Berdis Michael Zagorski 11/19/10 TABLE OF CONTENTS Title page 1 Committee Sign-off sheet 2 Table of Contents 3 List of Tables 5 List of Schemes 6 List of Figures 7 Acknowledgments 10 List of Abbreviations 11 Abstract 15 CHAPTER 1: INTRODUCTION 17 CHAPTER 2: COMPARISON OF INHIBITION OF BACTERIAL AND HUMAN LON PROTEASE ACTIVITY 32 Introduction 33 Materials and Methods 35 Results and Discussion 42 Conclusions 56 CHAPTER 3: CHEMICAL TOOLS TO MONITOR ATP-DEPENDENT PEPTIDASE ACTIVITY IN ISOLATED MITOCHONDRIA 57 Introduction 58 Materials and Methods 61 3 Results and Discussion 66 Conclusion 78 CHAPTER 4: DESIGN AND CHARACTERIZATION OF ClpXP SPECIFIC N-TERMINAL BORONIC ACID INHIBITOR 79 Introduction 80 Materials and Methods 84 Results and Discussions 91 Conclusions 99 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS 100 Introduction 101 Materials and Methods 103 Results and Discussion 106 Current status and future directions 116 BIBLIOGRAPHY 119 4 LIST OF TABLES CHAPTER 2 2.1: Kinetic parameters for ADP inhibition of peptide cleavage by human Lon 43 2.2: Kinetic parameters of inhibition of human Lon peptidase activity by DBN93 as determined by global fitting of experimental data using DynaFit 52 CHAPTER 5 5.1: Enzyme-specific peptide substrates 117 5 LIST OF SCHEMES Scheme 2.1: Noncompetitive inhibition 44 Scheme 2.2: One-and two-step inhibition mechanisms 50 6 LIST OF FIGURES CHAPTER 1 1.1: Normal and Lon-deficient mitochondria 20 1.2: Cleavage of Insulin B chain by Lon and ClpXP 21 1.3: Domain layout of Lon protease monomer 23 1.4: Structures of Lon and ClpXP and simplified mechanism of proteolysis 24 1.5: Proposed mechanism for peptide hydrolysis by Lon 25 1.6: Continuous fluorescent peptidase assay used to monitor enzyme activity 27 CHAPTER 2 2.1: Structures of peptide-based substrates and inhibitors used in these experiments 34 2.2: Phosphate does not inhibit the ATPase activity of hLon 47 2.3: Pre-steady state ATP hydrolysis by human Lon 48 2.4: Time-dependent inhibition of human Lon peptidase activity by DBN93 50 2.5: Global fitting of peptide cleavage time courses in the presence of varying amounts of inhibitor 51 2.6: DBN93 inhibits protein degradation by human Lon 54 2.7: DBN93 does not significantly inhibit hLon ATPase activity in the presence of λN protein stimulation 56 7 CHAPTER 3 3.1: Peptide-based substrates are used to monitor activity of human Lon and human ClpXP 68 3.2: DBN93 does not inhibit human ClpXP peptidase activity 70 3.3: Identification of mitochondrial matrix proteins isolated from HeLa cells by Western Blot analysis 72 3.4: ATP-dependent peptide cleavage of FRETN 89-98 by isolated mitochondria is abolished by the addition of Lon specific inhibitor, DBN93 73 3.5: Immunodepletion of Lon from isolated mitochondria abolishes ATP-dependent peptide cleavage of FRETN 89-98 75 3.6: Chase experiments show CBN93 inhibition of StAR degradation in isolated mitochondria 77 CHAPTER 4 4.1: Complimentary peptide boronic acid strategies utilizing the P/S and P’/S’ sites 83 4.2: Purified samples of ClpX and ClpP 87 4.3: N-terminal peptidyl boronic acids synthesized by the Santos group and screened for ClpXP specific inhibitors 89 4.4: DBN93 inhibits casein degradation by human Lon but not ClpXP 93 4.5: Screening of N-terminal peptidic boronic acids using FRET 89-98 peptidase assay 95 8 4.6: WLS6a IC50 determination by fluorescent peptidase assay 96 4.7: Inhibition of ClpXP degradation of casein by WLS6a 98 4.8: WLS6a does not inhibit ClpXP ATPase activity 99 CHAPTER 5 5.1: Imidazole does not affect the ATPase activity of ClpX 108 5.2: Rate of ATPase activity by ClpX is not significantly affected by the amount of ClpP 109 5.3: Determination of kinetic parameters for ATP hydrolysis by ClpXP 110 5.4: Screening FRETN 89-98 alanine scan peptides for cleavage by ClpXP, human Lon, and E. coli Lon 113 5.5: Fluorescently labeled Cleptide is selectively cleaved by ClpXP in the presence of ATP 114 5.6: Cleavage of Cleptide by ClpXP can be monitored using the fluorescent peptidase assay 115 9 ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Irene Lee, for her patience, support and open door. She was a great teacher and always available when I needed help or a boost of confidence. I would be lost without the support of past and current lab members: Diana, Jessi and Hilary introduced me to “great days in the Lee lab” and convinced me that I was going to love Lon whether I wanted to or not. Jason Hudak worked as an undergraduate in our lab and spent a lot of time and energy cloning ClpXP for me to use. Many thanks go to Edward Motea for always making me laugh and providing the lab with great (paraphrased) music. Sandra Craig took me under her wing and taught me the intricacies of cell culture, thank you for your help and for assuring me the cells were tougher than I thought they were, at least until they weren’t. James Becker and Kristin synthesized a number of the peptides I used in my studies and Kristin synthesized boronic acid peptide. To the “newbies”, Iteen and Natalie, thanks for making sure I stopped at some point during the day to eat lunch. Now it’s your turn to love Lon like the rest of us. Special thanks to Dr. Anthony Berdis for numerous instances of help over the years. Collaborations with Dr. Carolyn Suzuki at UMDNJ, Dr. Webster Santos and Ken Knott at Virginia Tech, and Anton Simeonov and Tim Foley at NIH have been invaluable and are much appreciated. Last, but not least, I want to thank my family and friends, for their unwavering support and encouragement. Now when you ask me if my thesis is done, I can finally answer, “Yes.” 10 LIST OF ABBREVIATIONS λN Lambda N protein: a λ phage protein that allows E. coli RNA polymerase to transcribe through termination signals in the early operons of the phage AAA+ ATPases Associated with a variety of cellular Activities Abu Aminobutyric acid Abz Anthranilamide ACN Acetonitrile ADP Adenosine diphosphate Aloc Allyloxycarbonyl ATP Adenosine triphosphate BME Beta-mercaptoethanol Boc tert-Butyloxycarbonyl BSA Bovine Serum Albumin Bz Benzoic acid amide Cam Chloramphenicol Cbz Carboxybenzyl CBN93 Non-fluorescent C-terminal boronic acid, Cbz-YRGIT-Abu-B(OH)2 Cleptide Y(NO2)-FAPHMALVPV-K(Abz) dansyl 5-(dimethylamino)naphthalene-1-sulfonyl chloride DBN93 fluorescent C-terminal boronic acid, dansyl-YRGIT-Abu-B(OH)2 DE52 diethylaminoethyl cellulose anion exchange resin used in purification of Lon 11 DMEM Dulbecco's Modified Eagle Medium DMSO dimethyl sulfoxide DTT dithiothreitol E. coli Escherichia coli, a gram negative bacteria EDTA Ethylenediaminetetraacetic acid eLon E. coli Lon protease FBS Fetal Bovine Serum Fmoc Fluorenylmethyloxycarbonyl FRET Fluorescence Resonance Energy Transfer FRETN 89-98 Y(NO2)-YRGITCSGRQ-K(Abz) FRETN 89-98Abu Y(NO2)-YRGIT-Abu-SGRQ-K(Abz) HATU (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) hClpXP Human ClpXP protease HeLa Immortal cervical cancer cells widely used in scientific research HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) hLon Human Lon protease IPTG Isopropyl-β-D-thio-galactoside Kburst Burst rate constant for ATP hydrolysis kcat Vmax/[E] kcat/Km Substrate specificity constant Ki Inhibition constant Km Michaelis constant equal to [substrate] required to reach ½ Vmax kobs rate/[E] Kan kanamycin 12 KPi Potassium phosphate LB Lauria-Bertani medium Mg(OAc)2 Magnesium actetate Mito Mitochondria isolated from HeLa cells MOPS 3-(N-morpholino)propanesulfonic acid N Hill coefficient NaCl Sodium chloride NaPi Sodium phosphate Ni-NTA agarose Nickel-nitrilotriacetic acid agarose: resin used to purify 6xHis- tagged proteins NO2 Nitro P11 Phosphocellulose cation exchange resin used to purify Lon PCR Polymerase chain reaction PEI-cellulose Polyethyleneimine-cellulose pen/strep Penicillin/Streptomycin Pi Inorganic phosphate S. Typhimurium Salmonella enterica serovar Typhimurium SB Super Broth SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis SSD Sensor and Substrate Discrimination StAR Steroidogenic Acute Regulatory protein TBST Tris-buffered saline containing 0.05% Tween 20 TFA trifluoroacetic acid Tris tris(hydroxymethyl)aminomethane 13 UV ultraviolet v rate 14 A Chemical Approach to Distinguish ATP-dependent Proteases Abstract By JENNIFER FISHOVITZ Mammalian Lon and ClpXP are two ATP-dependent proteases that are found in the mitochondrial matrix and have been implicated in protecting the mitochondria against damage from oxidative stress. Our lab is interested in understanding the role of Lon and ClpXP in the regulation of levels of oxidatively damaged proteins. There has been shown to be a transient increase in ATP-dependent proteolysis activity following oxidative stress, but there has been no functional assay to distinguish between the activities of these two enzymes. Previously, genetic knock-down studies have been employed with similar proteases, but can lead to changes in cellular metabolism that obscure the accurate detection of substrates. Because Lon and ClpXP have been shown to have different peptide cleavage specificities in degrading protein substrates, I have developed a series of chemical tools which allow for the profiling these enzymes individually at a post-translational level. I now have enzyme-specific peptide substrates and enzyme-specific inhibitors that can be used to inactivate one protease while analyzing the activity of the other in order to identify their respective role in maintenance of mitochondrial function in healthy and oxidatively damaged tissue.