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Characterising the Role of Valosin Containing Protein (VCP) in Autophagy and Cell Differentiation

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Characterising the Role of Valosin Containing Protein (VCP) in Autophagy and Cell Differentiation Characterising the role of Valosin Containing Protein (VCP) in autophagy and cell differentiation. Autophagy vs. aberrant osteoclastogenesis in the IBMPFD mouse model Doctor of Philosophy Thesis, October 2015 Milka B. Budnik-Zawilska Project Supervisors: Dr Giles Watts, Prof Ian Clark Norwich Medical School, Health Policy and Practice University of East Anglia This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 1 Contents LIST OF TABLES ............................................................................................................................ 5 LIST OF FIGURES .......................................................................................................................... 6 ABSTRACT .................................................................................................................................... 9 ABBREVATIONS ......................................................................................................................... 10 ACKNOWLEDGMENTS ............................................................................................................... 15 CHAPTER 1: INTRODUCTION .................................................................................................... 17 1.1 Mutations in the VCP gene cause a degenerative disorder of muscle, bone and brain..... .............................................................................................................................. ...17 1.2 VCP is implicated in diseases other than IBMPFD .................................................... 21 1.3 Structure and function of Valosin Containing Protein (VCP) .................................... 23 1.4 VCP in the ubiquitin system ...................................................................................... 28 1.5 Protein quality control, stress granules and inclusion bodies .................................. 32 1.6 Autophagy in health and disease .............................................................................. 37 1.7 Paget disease of the bone and aberrant osteoclastogenesis ................................... 46 1.8 Summary ................................................................................................................... 53 CHAPTER 2: MATERIALS AND METHODS .................................................................................. 57 2.1 Cell culture ................................................................................................................ 57 2.2 Plasmids .................................................................................................................... 58 2.3 Cloning ...................................................................................................................... 61 2.3.1 Generating pQE9 plasmids encoding His-tagged wt p62 and wt LC3B. ............ 62 2.3.2 Generating VCP-deletion mutants. ................................................................... 66 2.4 Bacterial cell cultures ................................................................................................ 68 2.5 Reagents .................................................................................................................... 68 2.6 Transfection and Autophagy induction .................................................................... 69 2.7 Cycloheximide-chase degradation assay .................................................................. 70 2.8 Co-immunoprecipitation ........................................................................................... 70 2.9 Western blot analysis ................................................................................................ 71 2.9.1 Odyssey infrared imaging system (Licor) .......................................................... 72 2.9.2 WesternBreeze Chemiluminescent Immunodetection (Invitrogen #WB7104 and #WB7106) .................................................................................................................. 74 2.9.3 Quantification of Western Blots ....................................................................... 74 2.10 Immunostaining ........................................................................................................ 75 2.10 Light Microscopy and imaging .................................................................................. 76 2.11 Imaris analysis ........................................................................................................... 76 2 2.12 Filter-trap assay......................................................................................................... 77 2.13 VCP mouse Genotyping ............................................................................................ 78 2.14 Primary macrophages culture preparation and In vitro osteoclastogenesis ............ 80 2.14.1 Osteoclast (OCL) differentiation ‘monocyte separation protocol’ .................. 80 2.14.2 OCL differentiation ‘quick protocol’ .................................................................. 81 2.14.3 OCL differentiation ‘9-day protocol’ ................................................................. 81 2.15 TRAP staining of osteoclasts ..................................................................................... 82 2.16 Detection of Active NFκB p65 in the Bone Marrow Derived Macrophages ............. 83 2.17 Statistical Analysis. .................................................................................................... 84 CHAPTER 3: VCP IN AGGREGATE CLEARANCE .......................................................................... 87 3.1 Introduction .............................................................................................................. 87 3.2 VCP in autophagy positive cells co-localises not only with Ubiquitin but also with p62 and LC3. .......................................................................................................................... 89 3.3 VCP co-localises with ubiquitin, p62 and LC3 in cells expressing expanded polyglutamines ...................................................................................................................... 94 3.4 Cells expressing VCP mutants are more sensitive to protein aggregation ............... 96 3.5 Overexpression of wt VCP in the cell culture increases clearance of poly- ubiquitinated substrates. .................................................................................................... 102 3.6 Discussion ................................................................................................................ 104 CHAPTER 4: VCP FORMS A COMPLEX WITH p62 .................................................................... 110 4.1 Introduction ............................................................................................................ 110 4.2 VCP directly interacts with p62 in the mTOR/ autophagy dependant manner. ..... 115 4.3 Discussion ................................................................................................................ 123 CHAPTER 5: STABILITY AND DEGRADATION OF VCP ............................................................... 127 5.1 Introduction ............................................................................................................ 127 5.2 VCP stability is linked to p62 instability in cells. ..................................................... 128 5.3 Discussion ................................................................................................................ 136 CHAPTER 6: AUTOPHAGY AND OSTEOCLASTOGENESIS ......................................................... 140 6.1 Introduction ............................................................................................................ 140 6.2 Inhibition of the mTOR signalling suppresses both early and late stages of osteoclast differentiation .................................................................................................... 146 6.3 Osteoclast differentiation from the primary Bone marrow derived macrophages.150 6.4 Inducers of protein aggregation lead to increased NFκB activation and p65 nuclear translocation in the primary BMDM. .................................................................................. 153 6.5 Disccussion .............................................................................................................. 161 CHAPTER 7: DISCUSSION ......................................................................................................... 167 3 7.1 The VCP binds to p62 and is involved in the clearance of ubiquitinated protein aggregates by autophagy .................................................................................................... 167 7.2 VCP stability is linked to p62 instability .................................................................. 172 7.3 P62/VCP – dependant autophagy regulates osteoclastogenesis ........................... 175 7.4 Concluding remarks ................................................................................................ 182 REFERENCES ............................................................................................................................ 184 APPENDIX ...............................................................................................................................
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