Proteomics of Mouse Cortex Following Conditional Deletion of Psmc1 Proteasomal Subunit in Neurones
Total Page：16
File Type：pdf, Size：1020Kb
Proteomics of mouse cortex following conditional deletion of Psmc1 proteasomal subunit in neurones Jamal Ibrahim Elkharaz, B.Pharm, M.Sc. Thesis submitted to The University of Nottingham For the degree of Doctor of Philosophy October 2013 Abstract Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system and the formation of protein inclusions in surviving neurones. The mechanisms underlying neurodegeneration and neuroprotection in the nervous system remain elusive. Ubiquitin is one of the hallmarks of neuropathological inclusions in the majority of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, dysfunction of the ubiquitin proteasome system has been implicated in disease cause and/or progression. This thesis investigates a unique conditional genetic mouse model of neurodegeneration caused by conditional genetic 26S proteasomal depletion in mouse forebrain neurones. We have identified potential proteins targeted for ubiquitination in brain using bio-affinity chromatography of zinc finger protein ZNF216 coupled with mass spectrometry. This lead to the identification of several potential ubiquitinated proteins involved in gene expression and regulation. We have also investigated the global brain proteome in response to 26S proteasomal depletion in neurones using two-dimensional fluorescence difference in-gel electrophoresis coupled to mass spectrometry for protein identification. Several differentially expressed proteins were identified in the 26S proteasome-depleted cortex. Astrocytic intermediate filament proteins glial acid fibrillary protein and vimentin, as well as the antioxidant peroxiredxoin-6, were upregulated. Mitochondrial fumarate hydratase and stathmin-1, involved in the tricarboxylic acid cycle and cytoskeletal microtubule dynamics respectively, were downregulated. These proteins have been validated by biochemical and immunohistochemical approaches. Further analysis of oxidative stress revealed increased lipid and protein oxidation that may be involved in the neurodegeneration associated with 26S proteasomal depletion. However, we also show increased phospholipase A2 activity associated with peroxiredoxin-6 expression that may have additional roles in neurodegenerative and/or neuroprotective functions. Interestingly, the levels of reactive oxygen species were inversely correlated with the upregulation of peroxiredoxin-6. We suggest that peroxiredoxin-6 may play an important role in the brain in the protection against oxidative stress and our studies may improve our physiological and pathological understanding of neurodegenerative disease. I | P a g e Acknowledgments First and foremost, I would like acknowledge my gratitude to my supervisor Dr. Lynn Bedford, for her efforts, encouragement and shrewd advice throughout my time as her student. I was very lucky to have a supervisor who was so interested in my research work and who worked so keenly to provide me with the mouse model of dementia to work with for my PhD study. I must also express my appreciation to my second supervisor Dr Layfield Robert for his helpful suggestions, encouragement and kindness by providing me with some materials I needed for my research. I must also acknowledge Prof. John Mayer for welcoming me to his lab before he retired, and he even continued to visit us in the lab from time to time and encouraged me during my work, giving me suggestions and advice; for which I thank him. Finishing this work would have not been possible without the help of Dr Aslihan Ugun-Klusek, who gave me valuable advice and performed the mass spectrometry analysis, so my thanks also go out to her. Thanks to my colleagues in the past and in the present Dr. Simon Paine, Dr. Zahra Nooshin, Dr. Joanna Strachan, Dr James Cavy, Zakok Ahmed, for all their help and support. Many thanks to Maureen Mee and Karen Lawler. Also many thanks to Dr. Constantin Teodosiu for his advice and who allowed me to use the spectrophotometer in his lab and to Dr. David Tooth for mass spectrometry analysis. Finally I would also like to thank my wife Intissar for her support, encouragement and patience throughout my study, and to thank my parents who prayed for me. II | P a g e Publications Elkharaz J, Ugun-Klusek A, Constantin-Teodosiu D, Lawler K, Mayer RJ, Billett E, Lowe J, Bedford L (2013). Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones. Biochim Biophys Acta. 1832 (12): 1959-1968. Rezvani N, Elkharaz J, Lawler K, Mee M, Mayer RJ, Bedford L (2012). Heterozygosity for the proteasomal Psmc1 ATPase is insufficient to cause neuropathology in mouse brain, but causes cell cycle defects in mouse embryonic fibroblasts. Neurosci Lett. 521(2):130-135. III | P a g e Abbreviations 1DG one dimensional gel 2D-DIGE two dimensional fluorescence difference in-gel electrophoresis ACTN β-actin AD Alzheimer’s disease AMPS ammonium per-sulphate ANOVA analysis of variance ApoE apolipoprotein E Bcl-2 B-cell lymphoma 2 BSA bovine serum albumin CaMKIIα calcium calmodulin-dependent protein kinase IIα CNBr cyanogen bromide activated-Sepharose-4B CP 20S proteolytic core particle COX5A cytochrome c oxidase subunit 5A, mitochondrial Cre causes recombination DCFDA 2, 7- dichlororfluorescein diacetate DLB dementia with Lewy bodies DNPH 2,4- dinitrophenylhydrazine DUB deubiquitination ECL chemiluminescent substrate GAB gel application buffer GAPDH glyceraldehyde-3-phosphate dehydrogenase GFAP glial acidic fibrillary protein GST gluthathion-S-transferase HSP7C heat shock cognate 71 kDa protein IEF isoelectric focusing IF intermediate filaments IL-1β interleukins-1β IPG immobilized pH gradient IV | P a g e IPTG isopropyl-β-D-thiogalactopyrinosid Iso-T isopeptidase T LB Lewy bodies LC/MS-MS liquid chromatography/mass spectrometry MAP2 microtubule-associated protein-2 MDA malondialdehyde mFUMH mitochondrial fumarate hydratase MJ33 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol MPTP 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine MALDI-TOF matrix-associated laser desorption ionization-time of flight MT microtubule NEM N-ethylmaleimide NEUG neurogranin NF-қB nuclear factor kappa-B NO nitric oxide ONOO peroxynitrite P53 protein 53 or tumor protein 53 PB pale body PBST phosphate buffered saline-Triton PD Parkinson’s disease PDD Parkinson’s disease with dementia PLA2 phospholipase A2 PRDX-6 peroxiredoxin-6 PSMA proteasome subunit mouse-A PSMB proteasome subunit mouse-B PSMC proteasome subunit mouse-C PSMD proteasome subunit mouse-D RIPA radioimmunoprecipitation assay buffer ROS reactive oxygen species V | P a g e RNS reactive nitrogen species RP 19S regulatory particle Rpn regulatory particle non-ATPase subunits Rpt regulatory particle ATPase subunits RpS27A ribosomal protein S27a SEM standard error of the mean SEPT-7 septin-7 SNAB beta-soluble NSF attachment protein STMN-1 stathmin-1 TBHP tert-butyl hydrogen peroxide TBS Tris-buffered saline TBS-T Tris-buffered saline-Triton TCA trichloroacetic acid TCB thrombin cleavage buffer TEMED N, N, N, N- tetramethylene diamine TMOP 1, 1, 3, 3-tetramethoxypropane VIME vimentin Uba52 ubiquitin A-52 residue ribosomal gene Ubb polyubiquitin-B gene UBD ubiquitin binding domains Ubc ubiquitin conjugating enzyme UBP ubiquitin binding proteins UCHL1 ubiquitin carboxyl-terminal esterase L1 UCH ubiquitin carboxy-terminal hydrolases UPS ubiquitin proteasome system USP ubiquitin-specific proteases ZNF216 zinc finger protein-216 VI | P a g e Table of contents Chapter 1: The Introduction ……………………………………………………….1 1.1 Introduction .............................................................................................................. 2 1.2 The ubiquitin 26S proteasome system ..................................................................... 4 1.2.1 The 26S proteasome .......................................................................................... 4 1.2.2 The 20S core particle ........................................................................................ 4 1.2.3 The 19S regulatory particle ............................................................................... 5 1.2.4 Proteasome assembly ........................................................................................ 5 1.2.5 Ubiquitin ........................................................................................................... 9 1.2.6 Ubiquitination ................................................................................................... 9 1.2.6.1 The ubiquitin activation enzyme (E1): ................................................... 10 1.2.6.2 The ubiquitin conjugating enzyme (E2): ................................................ 10 1.2.6.3 The ubiquitin ligase enzyme (E3): .......................................................... 10 1.2.7 Unanchored polyubiquitin chains ................................................................... 12 1.2.8 Ubiquitin specific peptidases (deubiquitinating enzymes) ............................. 12 1.2.9 Ubiquitin binding proteins .............................................................................. 12 1.3 Protein homeostasis and neurodegenerative diseases ............................................ 13 1.3.1 Lewy body diseases ........................................................................................ 13 1.3.2 The Lewy body ............................................................................................... 14 1.3.3 Parkinson’s disease