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Role of Ancient Ubiquitous Protein 1 in Hepatic Apob Degradation and VLDL Production

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Role of Ancient Ubiquitous Protein 1 in Hepatic Apob Degradation and VLDL Production Role of Ancient Ubiquitous Protein 1 in Hepatic ApoB Degradation and VLDL production by Mostafa Zamani A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Mostafa Zamani 2016 Role of Ancient Ubiquitous Protein 1 in Hepatic ApoB Degradation and VLDL production Mostafa Zamani Master of Science Department of Biochemistry University of Toronto 2016 Abstract Apolipoprotein B-100 (apoB100) is the main structural protein of atherogenic lipoproteins and is a key risk factor for development of coronary heart disease. The molecular mechanisms that regulate apoB100 degradation or secretion are not clearly understood but evidence supports intracellular lipid supply as a major decisive factor. Ancient Ubiquitous Protein 1 (AUP1) has been found to be present on the surface of Lipid Droplets (LDs), and has been implicated in ER-Associated Degradation. AUP1 knockdown dramatically increased levels of cellular apoB100 and facilitated secretion of apoB100 containing VLDL-sized particles from HepG2 cells. Knocking down AUP1 also increased Triglyceride (TG) levels in apoB100 containing VLDL particles. AUP1 was found to interact with apoB100 intracellularly based on co-immunoprecipitation experiments as well as in situ proximity ligation assay. Finally, modulating AUP-1 altered LD size in HepG2 cells and its knockdown increased the average size of LDs. ii Acknowledgments I would like to thank my family, especially my beautiful mom. I thank Dr. Khosrow Adeli for his generous support and guidance and the members of my thesis advisory committee. I also thank Rianna Zhang, our lab technician, for helping and teaching me how to do different experiments. And I appreciate my lab mates for making everything more enjoyable, especially Mark Naples, Sarah Farr and Jennifer Taher. iii Table of Contents Acknowledgments ............................................................................................................................... iii Table of Contents ................................................................................................................................. iv List of Tables ......................................................................................................................................... vi List of Abbreviations .......................................................................................................................... ix Introduction ........................................................................................................................................... 1 1. Overview of lipid metabolism ......................................................................................................................... 1 1.1. Exogenous lipid pathway ............................................................................................................................. 1 1.2. Endogenous lipid pathway .......................................................................................................................... 2 1.3. Reverse Cholesterol Transport ................................................................................................................. 2 1.4. Lipoproteins ...................................................................................................................................................... 2 1.5. Apolipoprotein B-100 ................................................................................................................................... 4 1.6. Lipid Droplets (LDs) ...................................................................................................................................... 5 1.7. Regulation of ApoB100 and VLDL assembly ....................................................................................... 7 1.7.1. Regulation of ApoB100 expression and translation ................................................................... 7 1.7.2. Co-translation regulation of apoB100 .............................................................................................. 7 1.7.6. Crosstalk between the ERAD and post-ER quality control pathways .............................. 15 1.8. Ancient ubiquitous protein 1 .................................................................................................................. 17 1.9. Rationale of the study ................................................................................................................................ 18 1.10. Objectives of the study ............................................................................................................................... 19 Material and Methods ...................................................................................................................... 20 2.1. Cell Culture ...................................................................................................................................................... 20 2.2. Treatment ........................................................................................................................................................ 20 2.3. siRNA Transfection ...................................................................................................................................... 20 2.4. In situ Proximity Ligation Assay (PLA) ............................................................................................... 21 2.5. RNA Extraction and Reverse transcription polymerase chain reaction (RT-PCR) .......... 21 2.6. Immunoblotting ............................................................................................................................................ 22 2.7. Crosslinking and Co-immunoprecipitation (Co-IP) ...................................................................... 23 2.8. Metabolic Labeling, Ultracentrifugation and Fractionation ...................................................... 23 2.9. Triglyceride Labeling, Lipid Extraction and Thin Layer Chromatography (TLC) ............ 24 2.10. Immunofluorescent Staining and Confocal Microscopy .............................................................. 25 iv 2.11. Lipid Droplet Fluorescent Staining and Confocal Microscopy ................................................. 25 Results ................................................................................................................................................... 27 3.1. Endogenous AUP1 physically interacts with apoB100 in HepG2 cells and controls its degradation ..................................................................................................................................................................... 27 3.1.1. Inhibition of proteasome in HepG2 cells decreases apoB100 degradation and promotes cellular apoB100 accumulation ........................................................................................................ 27 3.1.2. AUP1 interaction with apoB100 in HepG2 cells ........................................................................ 29 3.2. Co-Immunoprecipitation experiments ............................................................................................... 30 3.3. Knockdown of AUP1 decreased ubiquitination level of apoB in HepG2 cells. .................. 31 3.3.1. AUP1 knockdown in HepG2 cells ..................................................................................................... 31 3.3.2. AUP1 and ubiquitination ..................................................................................................................... 32 3.4. Knockdown of AUP1 increased both accumulated cellular apoB and secreted apoB in HepG2 cells. .................................................................................................................................................................... 33 3.5. Knockdown of AUP1 increased secretion of VLDL-sized apoB100 containing particles…………………………………………………………………………………………………………………………....34 3.6. Knockdown of AUP1 increased metabolically labeled TG in VLDL-sized particles secreted from HepG2 cells with or without MER-ERK inhibition ........................................................... 38 3.7. AUP1 knockdown increased the average size of LDs in HepG2 cells .................................... 38 Discussion ............................................................................................................................................ 42 4.1 AUP1 as an important regulator of apoB degradation in HepG2 cells .................................. 43 4.2 AUP1 as an important regulator of VLDL Secretion in HepG2 cells ....................................... 44 4.3 AUP1 as an important regulator of LD size in HepG2 cells ........................................................ 45 4.4 Future directions and concluding marks ........................................................................................... 47 References ................................................................................................................................... 45 v List of Tables Table 1) Lipoprotein Subclasses ...................................................................................... 4 Table 2) List of primers ................................................................................................... 22 vi List of Figures Figure 1) Lipoprotein composition ................................................................................................
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