Elevated levels of plasma very low-density lipoproteins (VLDLs) multiply the risk of atherosclerosis and coronary artery diseases, which currently poses an egregious impact to public health in the United States. VLDLs are synthesized in the liver and secreted into the blood. The rate-determining step in the secretion of VLDLs from the liver is their transport from their site of biogenesis, the endoplasmic reticulum (ER), to the Golgi. This step is physiologically regulatable and represents a potential therapeutic target in controlling elevated concentrations of plasma VLDLs. We propose to study how nascent VLDL exits from the ER and gets delivered to the Golgi at the molecular level. Our laboratory has shown that VLDL exits the ER in a specialized vesicle, the VLDL transport vesicle (VTV) which is different from other ER-derived vesicles e.g. protein transport vesicle (PTV) and pre-chylomicron transport vesicle (PCTV) in its size, buoyant density and protein composition. We reported earlier the VTV proteome, which revealed that reticulon-3A2 (RTN3A2) and small Valosine Containing Peptide (VCP)-interacting protein (SVIP) are uniquely present in VTV. Our preliminary studies show that apolipoproteinB100 (apoB100), a VLDL structural protein, plays a key role in VLDL- packaging into the VTV. Interestingly, we found that RTN3A2 selectively interacts with VLDL-apoB100 and is required for VLDL-exit but not for albumin-exit from the same hepatic ER. The first specific aim of this proposal is to identify cargo-selecting binding sites on RTN3A2 and cargo-selective binding sites on apoB100 for VLDL inclusion in VTV. In preliminary studies, we have identified another small Mr protein, SVIP, which is concentrated in VTVs as compared with their parent hepatic ER membranes. Our results show that SVIP interacts specifically with apoB100, the primary VTV-cargo protein and Sar1 but not with albumin, a PTV-cargo protein suggesting a role for SVIP in VTV-mediated transport of the VLDL. Our second specific aim will be to establish the role of SVIP in VTV-biogenesis and the ER-exit of apoB100 in primary hepatocytes. We found in our preliminary studies that the formation of VTV-Golgi fusion-competent SNARE-complex requires cytosolic proteins. In preliminary studies, we identified a functionally active cytosolic fraction that suppots SNARE- complex formation. SDS-PAGE analysis revealed that the active fraction contains only 3 proteins. We have identified two of these proteins.
In specific aim three of this grant, we propose to identify the third protein and test the functional roles of these proteins in VTV-Golgi fusion-complex assembly.

Public Health Relevance

Hypersecretion of VLDLs from the liver accelerates the development of atherosclerosis. The rate-determining step in the secretion of VLDLs is their transport from hepatic endoplasmic reticulum to the Golgi. This proposal aims to find out the proteins regulating the intracellular VLDL transport and secretion. Identification of proteins regulating this process would offer potential targets to control VLDL secretion from the liver.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Hepatobiliary Pathophysiology Study Section (HBPP)
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Burgess-Beusse, Bonnie L
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University of Central Florida
Other Basic Sciences
Schools of Medicine
United States
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Thibeaux, Simeon; Siddiqi, Shaila; Zhelyabovska, Olga et al. (2018) Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid-binding protein. J Biol Chem 293:1910-1923
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Tiwari, Samata; Siddiqi, Shaila; Zhelyabovska, Olga et al. (2016) Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking. J Biol Chem 291:12514-26
Siddiqi, Shadab A (2015) In Vitro Analysis of the Very-Low Density Lipoprotein Export from the Trans-Golgi Network. Curr Protoc Cell Biol 67:11.21.1-17
Hossain, Tanvir; Riad, Aladdin; Siddiqi, Shaila et al. (2014) Mature VLDL triggers the biogenesis of a distinct vesicle from the trans-Golgi network for its export to the plasma membrane. Biochem J 459:47-58
Tiwari, Samata; Siddiqi, Shaila; Siddiqi, Shadab A (2013) CideB protein is required for the biogenesis of very low density lipoprotein (VLDL) transport vesicle. J Biol Chem 288:5157-65
Tiwari, Samata; Siddiqi, Shadab A (2012) Intracellular trafficking and secretion of VLDL. Arterioscler Thromb Vasc Biol 32:1079-86
Rahim, Abdul; Nafi-valencia, Erika; Siddiqi, Shaila et al. (2012) Proteomic analysis of the very low density lipoprotein (VLDL) transport vesicles. J Proteomics 75:2225-35
Sengupta, B; Siddiqi, S A (2012) Hepatocellular carcinoma: important biomarkers and their significance in molecular diagnostics and therapy. Curr Med Chem 19:3722-9
Shukoor, Mohammed I; Tiwari, Samata; Sankpal, Umesh T et al. (2012) Tolfenamic acid suppresses cytochrome P450 2E1 expression in mouse liver. Integr Biol (Camb) 4:1122-9

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