Lipids are essential for cellular functions, and the liver plays a crucial role in regulating lipid balance through processes like fatty acid metabolism and very low-density lipoprotein (VLDL) biosynthesis. Disruptions in these mechanisms contribute to diseases such as atherosclerotic cardiovascular diseases, diabetes, obesity, and metabolic syndromes. VLDL transport vesicles (VTVs) are specialized vesicles responsible for transporting nascent VLDL from the endoplasmic reticulum (ER) to the Golgi apparatus. VTVs, like the canonical protein transport vesicles (PTVs), rely on coatomer complex II (COPII) proteins for their formation from the hepatic ER membrane. Proteomic analysis and western blot identified proteins unique to the VTV proteome, namely cell death-inducing DFF45-like effector (CideB), small valosin-containing protein-interacting protein (SVIP), and reticulon 3 (RTN3). While CideB and SVIP have been identified previously as VLDL-sorting protein and intracellular trafficking protein respectively, the specific function of RTN3 and the factors affecting its expression remain unclear. This study aimed to investigate the effect of dietary fat on RTN3 expression, validate its association with COPII proteins, and identify the endogenous multiprotein complex involved in VTV biogenesis. Western blot and confocal analysis revealed that RTN3 protein expression was significantly elevated in hepatocytes (HepG2 cells) in response to dose and time-dependent oleic acid treatment. In contrast, SVIP and CideB levels remained stable under similar nutrient conditions, highlighting the unique role of RTN3. Coimmunoprecipitation experiments confirmed an interaction between RTN3 and COPII complex marker Sar1B, providing evidence that RTN3 regulates VTV formation through its interaction with the COPII component of VTV. Further analysis revealed the presence of an endogenous VTV complex in HepG2 cells composed of CideB, SVIP, RTN3, and the COPII complex marker Sar1B. These findings provide valuable insights into the assembly process of VTVs and underscore the critical role of RTN3 in VTV formation and lipid trafficking. Comprehending these processes can contribute to a better understanding of lipid-related diseases and potentially guide the development of therapeutic interventions.
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Master of Science (M.S.)
College of Medicine
Burnett School of Biomedical Sciences
Length of Campus-only Access
Masters Thesis (Campus-only Access)
College of Medicine
Mishra, Rutuparnna, "Identification of an Endogenous Multiprotein Complex Required to Generate the VLDL Transport Vesicle from the Hepatic ER" (2023). Electronic Theses and Dissertations, 2020-. 1799.
Restricted to the UCF community until August 2028; it will then be open access.