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Characterization of an NBEAL2-Interacting Partner

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Characterization of an NBEAL2-Interacting Partner Characterization of an NBEAL2-interacting partner By Kevin To A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Kevin To 2018 Characterization of an NBEAL2-interacting partner Kevin To Master of Science Department of Biochemistry University of Toronto 2018 Abstract Platelets are small, abundant blood cells involved in many processes. Loss of function of Neurobeachin-like 2 (NBEAL2) is causative for Gray Platelet Syndrome (GPS), where patient platelets lack the crucial secretory α-granules. Thus, NBEAL2 is required for α-granule formation in platelet precursor megakaryocytes, but the functions and interaction partners of this protein are largely unknown. Here, I verified VAMP-associated protein A (VAPA) as an NBEAL2-binding protein. Co-immunoprecipitation (Co-IP) experiments confirmed the NBEAL2-VAPA interaction, which was mapped to at least five atypical FFAT motifs located within the PH-BEACH domain of NBEAL2. Structural modeling and docking simulations implicated one motif, termed ncFFAT*, however, Co-IP experiments with individual acidic residues in ncFFAT* (E2218K and D2224K) suggested that these residues are not uniquely involved. Immunofluorescence microscopy of human megakaryocytes and megakaryocytic cells suggested a partial interaction of NBEAL2 and VAPA. These studies suggest that NBEAL2- VAPA interactions may facilitate platelet α-granule formation. ii Acknowledgements I would like to express my deepest appreciation to my supervisor, Dr. Walter Kahr, for his guidance, inspiration, and unending patience during my graduate experience. I am grateful to my supervisory committee Dr. Angus McQuibban and Dr. Aleixo Muise for their insight, guidance, and constructive criticisms. The work presented here would not have been possible without the contributions of my colleagues, former and current, from the Kahr and Trimble Labs. I am especially grateful to Dr. Ling Li for her boundless enthusiasm and positivity that allowed me to persevere through challenging times. I owe much of my technical proficiency to her wonderful teaching. I am also grateful to Dr. Fred Pluthero for insightful discussions, microscopy expertise, and boundless knowledge in random scientific facts. Thank you for unending support and enthusiasm. I thank other colleagues Stephanie Nguyen, Carrie Chen, Marko Drobac, and Richard Liu for their helpful advice and support, especially Richard Lo for providing the initial results for my project; Dr. William Trimble for insightful discussions; and to all members of the Trimble Lab for helpful advice and sharing of reagents. Last, but not least, I would like to thank my parents Hoa and Tieu for their faith and support throughout my graduate experience. iii Table of Contents Acknowledgements.................................................................................................................... iii Table of Contents ....................................................................................................................... iv List of Tables ........................................................................................................................... vii List of Figures .......................................................................................................................... viii List of Appendices ...................................................................................................................... x List of Abbreviations ................................................................................................................. xi Chapter 1: Introduction ............................................................................................................... 1 1.1 Platelets: effectors of haemostasis and other processes .............................................. 1 1.2 Platelet secretory granules ........................................................................................... 3 1.3 Platelet production by megakaryocytes....................................................................... 5 1.4 The endosome is a crucial structure in α-granule production ..................................... 7 1.4.1 Endosome maturation facilitates α-granule formation and release ................. 7 1.4.2 α-Granules are sorted at the endosome and trafficked into proplatelet shafts ........................................................................................................................ 8 1.5 NBEAL2 is implicated in α-granule formation as its absence causes Gray Platelet Syndrome ..................................................................................................... 10 1.6 NBEAL2 is predicted to have roles in vesicular and protein-scaffolding events ..... 13 1.7 Rationale and hypothesis ........................................................................................... 14 Chapter 2: Materials and methods ............................................................................................ 15 2.1 Lab reagents, antibodies, and plasmids ..................................................................... 15 2.2 Cell maintenance and growth .................................................................................... 15 2.3 Plasmid cloning ......................................................................................................... 17 2.3.1 Yeast plasmids .............................................................................................. 17 2.3.2 Cloning of mammalian overexpression constructs ....................................... 17 2.4 Yeast-2-Hybrid screen............................................................................................... 19 2.5 Mammalian transfections and preparing cell lysates ................................................ 22 2.6 Co-Immunoprecipitation preparation and procedures............................................... 23 2.6.1 Co-IP requiring pre-conjugated α-FLAG and control IgG beads ................. 23 2.6.2 Co-IP requiring self-conjugated α-HA, α-MYC, and control IgG beads ..... 24 2.7 Sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and western blotting .... 24 2.8 Culturing and differentiation of human megakaryocytes .......................................... 25 2.9 Immunofluorescent microscopy ................................................................................ 25 2.10 Structural prediction and protein-protein docking simulations ............................... 26 Chapter 3: Results ..................................................................................................................... 27 3.1 Yeast-two-hybrid screening ...................................................................................... 27 3.1.1 The majority of NBEAL2 truncations cloned into the Yeast-2-Hybrid pGBKT7 vector were unstable in DH5α E. coli ........................................... 27 3.1.2 NBEAL2 Fragment C was expressed in the yeast system and did not exhibit reporter autoactivation .................................................................................. 29 3.1.3 A small variety of binding partners were screened in Y2H with NBEAL2 iv Fragment C as bait ........................................................................................ 31 3.2 Verifying candidate binding partners to NBEAL2 ................................................... 33 3.2.1 Cross-referencing binding partners from Y2H and affinity purification-mass spectrometry screens showed no overlap ...................................................... 33 3.2.2 VAPA, among a list of AP-MS candidates, could be an NBEAL2-interacting protein ........................................................................................................... 33 3.3 Verifying the NBEAL2-VAPA interaction ............................................................... 35 3.3.1 Full length MYC-VAPA can co-immunoprecipitate with HA-VAPB ......... 35 3.3.2 Full length MYC-VAPA can co-immunoprecipitate with GFP-NBEAL2- FLAG in HEK293 and NBEAL2-stable Dami cells ..................................... 35 3.3.3 Endogenous VAPA can Co-IP with GFP-NBEAL2-FLAG in NBEAL2- stable Dami cells ........................................................................................... 39 3.4 Characterizing the interaction between NBEAL2 and VAPA .................................. 40 3.4.1 HA-VAPB does not Co-IP with GFP-NBEAL2-FLAG in HEK293 cells ... 40 3.4.2 The PH-BEACH domains of NBEAL2 may be responsible for the VAPA interaction ..................................................................................................... 40 3.4.3 HA-C5 is predicted to harbour several atypical FFAT motifs for VAPA- binding .......................................................................................................... 42 3.5 Searching for relevant ncFFAT motifs in the NBEAL2-VAPA interaction ............. 45 3.5.1 The structure of the PH-BEACH regions in NBEAL2 is predicted to be homologous to that of NBEA ...................................................................... 45 3.5.2 The PH-BEACH regions of NBEAL2 and NBEA share similar ncFFAT motifs ............................................................................................................ 45 3.5.3 Predicted ncFFAT motifs could be surface-exposed on NBEAL2 .............. 48 3.5.4 Protein docking simulations implicate the ncFFAT* motif of NBEAL2 in VAPA-binding .............................................................................................
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