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Identification of the Role of Protein Kinases in VLDL TraffickingTasin, Fahim Rejanur 01 January 2024 (has links) (PDF)
The liver maintains lipid homeostasis in the body by uptake, synthesis and delivery of lipid molecules. An important component is very low-density lipoprotein (VLDL) which is by hepatocytes. The significance of this molecule lies in the fact that abnormal VLDL metabolism may lead to NAFLD, fibrotic liver, or atherosclerotic heart diseases in humans. Intracellular VLDL transport tightly regulates its secretion from the liver. Our prior research studies have demonstrated that this transport process relies on ATP, GTP, and the presence of cytosolic proteins, suggesting the involvement of protein phosphorylation. To delve deeper into the mechanisms governing VLDL secretion, our laboratory has initiated a study aimed at identifying the role of specific protein kinases responsible for VLDL phosphorylation and subsequent secretion in human hepatoma cells. Utilizing various small molecule inhibitors, such as H89 dihydrochloride, Akti-1/2 and Calphostin C targeting Protein Kinase A, B, and C, respectively, we employed a pulse-chase assay supplemented with radioactive 3H-oleic acid-based Scintillation Counting and Western Blot analyses to identify the VLDL secretion. Findings from Western Blot analysis of VLDL protein-ApoB100 levels in the cell culture media, revealing a significant reduction in ApoB100 secretion from hepatocytes upon inhibition of the protein kinase C family. The distribution of intracellular VLDL molecules have also been shown to be higher in protein kinase C inhibition indicating lower secretion with the treatment. Furthermore, the pulse chase assay followed by immunocytochemistry-based imaging led to the finding that both ER to Golgi and post-Golgi trafficking is disturbed with the inhibition of protein kinase C. This study provides valuable insights into the intricate mechanisms underlying VLDL secretion from the liver, shedding light on the pivotal role of specific protein kinases in this process.
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Molecular Regulators Of Post-golgi Vldl Transport Vesicle (pg-vtv) BiogenesisRiad, Aladdin 01 January 2013 (has links)
Amongst its numerous functions, the liver is responsible for the synthesis and secretion of very low-density lipoprotein (VLDL). VLDL particles play the important role of facilitating the transport of lipids within the aqueous environment of the plasma; yet high plasma concentrations of these particles result in the pathogenesis of atherosclerosis, while low VLDL secretion from the liver results in hepatic steatosis. VLDL synthesis in the hepatocyte is completed in the Golgi apparatus, which serves as the final site of VLDL maturation prior to its secretion to the bloodstream. The mechanism by which VLDL’s targeted transport to the plasma membrane is facilitated has yet to be identified. Our lab has identified this entity. Our findings suggest that upon maturation, VLDL is directed to the plasma membrane through a novel trafficking vesicle, the Post-Golgi VLDL Transport Vesicle (PG-VTV). PG-VTVs containing [3H] radiolabeled VLDL were generated in a cell-free in vitro budding assay for study. First, the fusogenic capabilities of PG-VTVs were established. Vesicles were capable of fusing with the plasma membrane and delivering the VLDL cargo for secretion in a vectorial manner. The next goal of our study is to characterize key regulatory molecular entities necessary for PG-VTV biosynthesis. A detailed analysis was undertaken to determine the PG-VTV proteome via western blot and two-dimensional difference in gel electrophoresis. The identification of key molecular regulators will potentially offer therapeutic targets to control VLDL secretion to the bloodstream.
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