TPD52: Functional Elucidation of a Novel Lipid Droplet Binding Protein in Human Hepatocytes
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TPD52: functional elucidation of a novel lipid droplet binding protein in human hepatocytes. by Shi Bo Feng Biochemistry, McGill University, Montreal August 2015 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master © Shi Bo Feng (2015) Acknowledgements I wish to thank Transplant Quebec with donors and their families and patients consenting to liver resection. I thank Dr. Jarred Chicoine and Dr. Robert Sladeck for the plasmid design and construction, also for making tetracycline inducible TPD52 cell lines. I would also like to thank Dr. Fariba Kalantari and Dr. Ali Fazel for providing fractionated human primary liver samples and delipidated lipid droplet fractions. I also thank Dr. Min Fu for her help with confocal microcopy, Sarita Negi for the cryosection of human liver samples, and Eun Joo Lee for training me on various techniques. Mom and Dad, thank you so much for your support all these years. Finally, I would like to thank Dr. Robert S. Kiss and Dr. Tommy Nilsson for accepting me into their laboratories and for supporting me in so many ways so I could complete two fulfilling years in research. ii Abstract TPD52: functional elucidation of a novel lipid droplet binding protein in human hepatocytes. Shi Bo Feng McGill University, (2015) Supervisors: Robert S. Kiss & Tommy Nilsson Lipid droplets (LDs) are intracellular vesicles involved in the storage of triglycerides (TG) and cholesteryl ester (CE). Although LDs are used for storage, they also play a dynamic role in cell metabolism. LD overaccumulation in the liver causes non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), often resulting in liver dysfunction. We identified tumor protein D52 (TPD52) as a novel LD binding protein. By immunofluorescence and western blot, TPD52 was found on LDs in both HepG2 cells and human primary liver samples. Transient transfection of TPD52-eGFP fusion protein in HepG2 and inducible stable transfection in HEK293 showed that TPD52 stably localizes to LDs induced by oleic acid and by arachidonic acid. A fluorescence recovery after photobleaching (FRAP) experiment also showed the time-dependent recruitment of TPD52 to LDs. Moreover, an in situ biotinylation identification technique (Bio-ID) identified LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) as a potential TPD52 interacting protein. LAMTOR1 is a membrane protein found on late endosomes/lysosomes known to interact with components of the Ragulator complex regulating autophagy. LAMTOR1 was shown to localize to LDs in HepG2 and human primary liver samples by immunofluorescence. Implications of a functional link between TPD52 and LAMTOR1 are discussed. TPD52 may be linked to lipophagy, which has the potential of helping understanding NAFLD. TPD52 may be the key to develop potential treatment. iii Résumé TPD52: élucidation fonctionnelle d'une nouvelle protéine de la gouttelette lipidique de liaison dans les hépatocytes humains. Shi Bo Feng McGill University, (2015) Superviseurs: Robert S. Kiss & Tommy Nilsson Les gouttelettes lipidiques (LDS) sont des vésicules intracellulaires impliquées dans le dépôt des triglycérides (TG) et l'ester de cholestéryle (CE). Bien que LDs sont utilisées pour le dépôt, ils jouent également un rôle dynamique dans le métabolisme cellulaire. LD suraccumulation dans le foie provoque la maladie non alcoolique du foie gras (SNA) et la stéatohépatite non alcoolique (NASH), ce qui entraîne souvent un dysfonctionnement hépatique. Nous avons identifié la protéine tumorale D52 (TPD52) comme une nouvelle protéine de lié à la surface de LD. Par l’immunofluorescence et Western blot, TPD52 a été trouvé sur LD dans les deux cellules HepG2 et des échantillons de foie primaires humains. La transfection transitoire de TPD52-eGFP protéine de fusion dans des cellules HepG2 et transfection stable inductible dans les cellules HEK293 a montré que TPD52 localise de manière stable à LD induite par l'acide oléique et de l'acide arachidonique. Un recouvrement de fluorescence après photoblanchiment (FRAP) a également montré le recrutement en fonction du temps de TPD52 à LD. En outre, une technique in situ d'identification de biotinylation (Bio-ID) identifié LAMTOR1 (fin endosomal / lysosomial adaptateur, MAPK et mTOR activateur 1) en tant que protéine interagissant TPD52 de potentiel. LAMTOR1 est une protéine trouvé sur les endosomes tardifs / lysosomes connus pour interagir avec des composants du complexe autophagie Ragulator régulation. LAMTOR1 a été montré à localiser à LDS HepG2 et des échantillons de foie primaires humains par immunofluorescence. Conséquences d'un lien fonctionnel entre TPD52 et LAMTOR1 sont discutées. TPD52 peut être lié à lipophagy, qui a le potentiel d'aider la compréhension de NAFLD. TPD52 peut être la clé pour développer un nouveau traitement potentiel. iv Table of Contents Acknowledgements ..............................................................ii Abstract-Résumé ................................................................ iii List of Illustrations ............................................................. vii List of Figures .................................................................. viii INTRODUCTION................................................................ 1 Lipid and Lipid Droplet ......................................................... 1 LD structure ................................................................ 2 The PAT family proteins ................................................. 3 LD biogenesis .............................................................. 4 Disease related to LD accumulation: NAFLD & NASH................ 6 TPD52 family ..................................................................... 7 Structure..................................................................... 8 TPD52 association with cancer ....................................... 8 TPD52 expression in different cell lines ............................ 9 Potential function of TPD52 ............................................ 9 TPD52 and lipid metabolism ..........................................11 Autophagy/Lipophagy ........................................................ 11 Definitionof autophagy ................................................. 11 mTOR & Autophagy ..................................................... 12 LAMTOR1 & Autophagy ................................................ 13 Autophagy, lipid metabolism and NAFLD .........................13 Importance of the project and hypothesis ............................. 14 v RESULT........................................................................... 16 Part I: TPD52, an oncogene located on the LDs under a high oleate concentration..............................................................16 Part II: The heterogeneity of TPD52 expression on LDs surface 22 Part III: Exogenous TPD52 is expressed under various conditions65 Part IV: TPD52 family proteins (TPD52L1 & TPD52 L2) are also found on lipid droplet ................................................... 33 Part V: LAMTOR1- TPD52 interacting protein that is involved in autophagy .................................................................. 37 DISCUSSION & CONCLUSION.......................................... 42 MATERIALS & METHODS ................................................. 46 REFERENCE .................................................................... 50 vi List of Illustrations Illustration I: LD structure ................................................... 3 Illustration II: Schematic representation of LD biogenesis ........ 5 Illustration III: The spectrum range of NAFLD ........................ 7 Illustration IV: TPD52 gene map ........................................... 8 vii List of Figures Figure 1: Endogenous TPD52 co-localizes with PLIN2 on lipid droplet in hepG2 cell line ................................................ 18 Figure 2: Endogenous TPD52 associates with LDs in primary human liver tissue under physiological state ......................19 Figure 3: TPD52 expression level in human hepatic intracellular fractions shows TPD52’s localization on LDs ............ 21 Figure 4: TPD52 expression in human hepatic LD fractions is heterogeneous....................................................23 Figure 5: Exogenous TPD52 is found on LDs across different time points after oleate induction ................................. 27 Figure 6: Exogenous TPD52 is found on LDs generated using different lipid source ............................................ 29 Figure 7: FRAP analysis of TPD52 mobility on LD .................... 31 Figure 8: Both TPD52L1 and TPD52L2 are two known members of TPD52 family that localize on LD surface ................ 34 Figure 9: TPD52L1 expression in human hepatic LD fractions is heterogeneous and differs from TPD52 expression ... 36 Figure 10: LAMTOR1 co-localizes with PLIN2 on LDs ................ 39 Figure 11: Endogenous LAMTOR1 is located on LDs of NAFLD human liver .................................................................. 40 viii INTRODUCTION Lipid and Lipid Droplet Lipid droplets (LDs) are present in various eukaryotic cells as the storage organelles for excess free fatty acids and cholesterol (1). The mechanism and machinery to generate LDs is evolutionarily conserved from prokaryotic bacteria to humans (1,2). For storage purposes, free fatty acids are converted to triglycerides (TG), and cholesterol to cholesteryl ester (CE), and both are stored in LDs (1,2).