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IDENTIFICATION of NATIVE 20S PROTEASOMAL SUBSTRATES in the HALOARCHAEON Haloferax Volcanii THROUGH DEGRADOMIC and PHOSPHOPROTEOMIC ANALYSIS

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IDENTIFICATION of NATIVE 20S PROTEASOMAL SUBSTRATES in the HALOARCHAEON Haloferax Volcanii THROUGH DEGRADOMIC and PHOSPHOPROTEOMIC ANALYSIS IDENTIFICATION OF NATIVE 20S PROTEASOMAL SUBSTRATES IN THE HALOARCHAEON Haloferax volcanii THROUGH DEGRADOMIC AND PHOSPHOPROTEOMIC ANALYSIS By PHILLIP A. KIRKLAND A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Phillip Kirkland 2 To my mother, Kelly Kirkland; my grandmother Judy Jansen; and to the loving memory of Mr. and Mrs. L.V. Stevens. 3 ACKNOWLEDGMENTS I would like to express my deepest gratitude to Dr. Julie Maupin for her patience and guidance. Many thanks go to the Maupin lab staff (Matt Humbard, Wei Li, and Guanguin Zhou) and to former graduates Dr. Chris Reuter and Gosia Gil for all of their help and support. My sincerest thanks go to my supervisory committee (Dr. James Preston, Dr. Madeline Rasche, Dr. Richard Lamont, Dr. Peter Kima and Dr. Eric Triplett) for their insight. I owe a debt of gratitude to John Moore, Phi Do, Dr. Franz St. John, Youngnyun Kim, John Rice and the entire Microbiology and Cell Science Department. I am also extremely grateful to all of those who have offered help and support with instrumentation, bioinformatics and general proteomic endeavors including Dr. Stan Stevens, Scott McClung, Dr. Marjorie Chow and Dr. Alfred Chung and the entire ICBR staff as well as Dr. Jennifer Busby and Valerie Cavett at Scripps Florida. Finally, I am eternally grateful for the support and encouragement of my family and friends: my mom, Kelly Kirkland, my grandmother Judy Jansen, my fiancé Shelley Cooper, Jeremy and Matt Jones, William Knapp, and the Smith, Bright, Blair, Stevens and Jansen families and my great grandparents, the late Lavert V. and Una B. Stevens. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT...................................................................................................................................15 CHAPTER 1 LITERATURE REVIEW .........................................................................................................17 Introduction.............................................................................................................................17 Proteasome Structure and Function........................................................................................17 Core Particle Structure and Arrangement........................................................................18 Associated ATPase Regulatory Components..................................................................20 The 19S regulatory particle......................................................................................20 Proteasome-activating nucleotidases........................................................................21 Bacterial ARC ATPase.............................................................................................22 Other proteasome-associated complexes .................................................................22 Protein Degradation................................................................................................................23 Protein Degradation Signals............................................................................................24 Ubiquitination and other PTM’s ..............................................................................25 The N-end rule .........................................................................................................28 PEST sequences .......................................................................................................30 Oxidative damage and hydrophobic patch exposure................................................32 Known Substrates and Cellular Influences of the Proteasome........................................34 Cell cycle..................................................................................................................34 Transcription and translation....................................................................................36 Cellular metabolism and stress response..................................................................38 Proteomics and Biological Mass Spectrometry......................................................................41 The Current State of Bottom-Up Proteomics ..................................................................42 Protein/peptide separation ........................................................................................43 Post-translational modification analysis...................................................................46 Shotgun proteomics and quantitative, high-throughput strategies ...........................49 Mass spectrometry and bioinformatics ....................................................................53 Special Considerations for Extreme Proteomes ..............................................................60 Proteomes with extreme pI value bias......................................................................60 Hydrophobic proteomes and membrane proteomics................................................62 Project Rationale and Design..................................................................................................64 5 2 METHODS AND MATERIALS..............................................................................................67 Chemicals and Reagents.........................................................................................................67 Strains, Plasmids and Culture Conditions ..............................................................................67 Construction of panA Knockout Mutant GG102....................................................................69 Protein Preparation and Quantification ..................................................................................70 Standard Protein Extraction.............................................................................................71 Trizol-Mediated Protein Extraction.................................................................................71 Protein Modification...............................................................................................................73 Protein Reduction, Alkylation and Tryptic Digestion.....................................................73 Peptide Methyl Esterification..........................................................................................73 Phosphoprotein Enrichment and Purification.........................................................................74 Immobilized Metal Affinity Chromatography ................................................................74 Titanium Dioxide Phosphopeptide Enrichment ..............................................................74 Nickel Purification of Polyhistadine-Tagged Proteins....................................................74 2D PAGE Analysis and Imaging............................................................................................75 Liquid Chromatography and Mass Spectrometry...................................................................76 Reversed Phase HPLC Coupled with Nano-ESI-QTOF (QSTAR) MS/MS...................76 Three-Dimensional LCQ Deca Ion Trap MS ..................................................................77 MS Data and Protein Identity Analyses..................................................................................77 Radioactive 35S Pulse-Chase Labeling of H. volcanii Proteins ..............................................78 Immunoprecipitation of Radiolabeled H. volcanii Proteins ...................................................79 3 OPTIMIZING ISOELECTRIC FOCUSING OF HALOPHILIC PROTEINS THROUGH A TRIZOL-BASED SAMPLE PREPARATION METHOD ................................................86 Introduction.............................................................................................................................86 Results and Discussion ...........................................................................................................87 4 EFFECT OF CLASTO-LACTACYSTIN BETA LACTONE ON THE PROTEOME OF Haloferax volcanii ..................................................................................................................95 Introduction.............................................................................................................................95 Results.....................................................................................................................................96 Discussion.............................................................................................................................101 Ribosomal Proteins........................................................................................................101 Elongation Factor 1A ....................................................................................................102 DJ-1/ThiJ/PfpI Superfamily ..........................................................................................103
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