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The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line

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The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line Brett Hawley Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Brett Hawley, Ottawa, Canada, 2012 ABSTRACT The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha- synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research. ii ACKNOWLEDGEMENTS I would like to take this opportunity to thank my supervisor, Dr. Daniel Figeys, for his support and guidance throughout my studies in his lab. It was a great experience to work in his lab and I am very thankful I was given the chance to learn and work under him. I would also like to thank the members of my lab for all their assistance in learning new techniques and equipment in the lab. In particular, I would like to thank Hu Zhou, Rui Chen, and Zhibin Ning. They provided me with a lot of assistance in working with and operating the HPLC and mass spectrometers and discussed results and troubleshooting numerous times. iii TABLE OF CONTENTS Abstract ......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii List of Figures .............................................................................................................................. vi List of Tables ............................................................................................................................. viii List of Abreviations ..................................................................................................................... ix CHAPTER 1. INTRODUCTION I. Interactome Proteomics Through Affinity Purification-Mass Spectrometry ............ 1 II. The Interactome of Several Alzheimer’s disease Related Genes .............................. 9 III. The Purification and Analysis of the Platelet Activating Factor Receptor ............. 23 IV. Purification and Comparison of Eh Domain Protein 1-4 in Association with Low Density Lipoprotein Receptor and Proprotein convertase subtilisin/kexin type 9 ............................................................................................. 35 V. Summary ................................................................................................................. 42 CHAPTER 2. METHODS .......................................................................................................... 46 CHAPTER 3. RESULTS I. The Interactome of Several Alzheimer’s disease Related Genes ............................ 61 II. The Purification and Analysis of the Platelet Activating Factor Receptor ........... 110 III. Purification and Comparison of Eh Domain Protein 1-4 in Association with Low Density Lipoprotein Receptor and Proprotein convertase iv subtilisin/kexin type 9 ........................................................................................... 140 CHAPTER 4. DISCUSSION I. The Interactome of Several Alzheimer’s disease Related Genes .......................... 156 II. The Purification and Analysis of the Platelet Activating Factor Receptor ........... 165 III. Purification and Comparison of Eh Domain Protein 1-4 in Association with Low Density Lipoprotein Receptor and Proprotein convertase subtilisin/kexin type 9 ........................................................................................... 170 CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS I. Conclusions ........................................................................................................... 174 II. Future Directions ................................................................................................... 175 III. Bibliography .......................................................................................................... 178 v LIST OF FIGURES Figure 1-1: LC-MS/MS Configuration. Figure 1-2: The Alternate Pathways in the Processing of Amyloid Precursor Protein. Figure 1-3: A Three Step Process to the Appearance of Clinical Symptoms of Alzheimer’s Disease. Figure 1-4: The Three Pathways in PAF Synthesis. Figure 1-5: Effects of platelet-activating factor (PAF) on the cell is dependent on the presence of the PAF receptor and whether PC (O-16:0/2:0) or PC (O-18:0/2:0) is present. Figure 1-6: The Signaling Pathways of the Activated PAFR. Figure 1-7: The Role of EHD Protein Isoforms in Endocytic Transport. Figure 1-8:Experimental protocol for the transfection, immunopurification, digestion and analysis of interacting proteins bound to a bait protein. Figure 3-1: Restriction digest of AD-related clones in pCMV6 expression vector. Figure 3-2: Western blot (WB) validation of expression of FLAG-tagged recombinant proteins. Figure 3-3: Example of silver-stained gels obtained from the optimized immunopurification of the FLAG-tagged bait proteins and their interaction partners from HEK 293T cells. Figure 3-4: Illustration of the Transition from In-gel digestion to MS/MS Sequencing Results. Figure 3-5: PICALM recruits clathrin, adaptor proteins and cargo proteins to the site of endocyctosis during the early stages of the clathrin-coated vesicle cycle. Figure 3-6: The predicted interactors of APOE based on a model where APOE is a factor in mitochondrial dysfunction and subsequent cell stress. Figure 3-7: Co-immunopurification validation of interactors identified by mass spectrometry analysis. Figure 3-8: Western blot (WB) testing for actin following immunopurificationof FLAG-tagged recombinant proteins. Figure 3-9: Loading control used to ensure equal quantity of protein used in each immunopurification experiment. vi Figure 3-10: Restriction digest of PAFR in gateway expression vectors. Figure 3-11: Western blot (WB) validation of expression of FLAG-tagged PAFR in 3X FLAG expression vector. Figure 3-12: Restriction digest of PAFR in pCMV6 entry vector. Figure 3-13: Western blot (WB) validation of expression of FLAG-tagged PAFR in pCMV6 entry. Figure 3-14: Comparison of different lysis bufferes in the immunopurification of FLAG-tagged PAFR in HEK 293T cells. Figure 3-15: Silver-stained gel obtained from the immunopurification of FLAG-tagged PAFR, the empty vector control and their interaction partners from HEK 293T cells. Figure 3-16: Experimental protocol for immunopurification of EHD proteins, LDLR, PCSK9 and bound interactors. Figure 3-17: Example of silver-stained gels obtained from the optimized immunopurification of the FLAG-tagged bait proteins and their interaction partners from HEK 293T cells. Figure 3-18: Example of silver-stained gels obtained from the optimized immunopurification of the FLAG-tagged bait proteins and their interaction partners from HEK 293T cells. Figure 3-19: Example of silver-stained gels obtained from the optimized immunopurification of FLAG-tagged bait proteins or V5-tagged bait proteins and their interaction partners from HEK 293T cells. Figure 4-1: Overview of the early stages of clathrin-mediated endocytosis and PICALM’s role in the process. vii LIST OF TABLES Table 3-1: The results of the in-gel digestion of cells transfected with the 6 tagged AD genes studied (APOE, CLU1, CLU2, LDLR, PICALM and SNCA) and cells transfected with the negative control, which is the empty vector (pCMV-Entry). The top 50 results for each bait are presented. Table 3-2: SAINT probability scores for the results of the MS analysis of in-gel digested HEK 293T cells transfected with the 6 tagged AD genes studied (APOE, CLU1, CLU2, LDLR, PICALM and SNCA) and cells transfected with the negative control, which is the empty vector (pCMV-Entry). Only interactors with a score >0.200 are shown. Table 3-3: The results of the in-gel digestion of cells transfected with the either tagged PAFR or the negative control, which is the empty vector (pCMV-Entry). All results are presented. Table 3-4: The results of the in-solution digestion of cells transfected with the either tagged PAFR or the negative control, which is the empty vector (pCMV-Entry). All results are presented. Table 3-5: The results of the in-solution digestion of cells transfected with the either tagged PAFR or the negative control, which is the empty vector (pCMV-Entry) following a membrane isolation protocol. All results are presented. Table 3-6: The results of the in-solution digestion of cells singly transfected with the DDK tagged EHD1-4 or LDLR, or cells doubly transfected with LDLR-V5 and DDK tagged
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