A Cell Biological Approach to Hepatic Lipid Metabolism

肝脂质代谢的细胞生物学方法

• 批准号: 9097680
• 负责人: Shadab A Siddiqi
• 金额: $ 31.59万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9097680
• 关键词: Albumins; Amino Acids; Apolipoproteins B; Atherosclerosis; Binding Sites; Biogenesis; Biological; Biological Assay; Blood; Cells; Chylomicrons; Co-Immunoprecipitations; Complex; Coronary Arteriosclerosis; Coupled; Cytosol; Data; Development; Endopeptidase K; Endoplasmic Reticulum; Figs - dietary; Furuncles; Goals; Golgi Apparatus; Grant; Health; Hepatic; Hepatocyte; Hyperlipidemia; Immunoblotting; In Vitro; Laboratories; Liquid Chromatography; Liver; Mediating; Membrane; Methodology; Molecular; Mutation; N-terminal; Parents; Pathogenesis; Peptides; Performance; Plasma; Play; Precipitation; Process; Propionates; Proteins; Proteome; Proteomics; Public Health; Reporting; Risk; Role; SNAP receptor; Series; Site; Small Interfering RNA; Sorting - Cell Movement; Structural Genes; Surface Plasmon Resonance; Techniques; Testing; Transport Vesicles; United States; Very low density lipoprotein; Vesicle; crosslink; density; insight; knock-down; lipid metabolism; polypeptide; protein transport; research study; tandem mass spectrometry; therapeutic target

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：血浆极低密度脂蛋白（VLDL）水平升高会增加动脉粥样硬化和冠状动脉疾病的风险，目前这对美国的公共卫生造成了严重影响。VLDL在肝脏中合成并分泌到血液中。从肝脏分泌VLDL的速率决定步骤是它们从它们的生物合成位点--内质网--转运 (ER)到高尔基体。这一步是生理上可调节的，代表了一种潜在的治疗方法。 控制血浆VLDL浓度升高的目标。我们建议在分子水平上研究新生的VLDL如何从ER中退出并传递到高尔基体。我们的实验室已经表明，极低密度脂蛋白在一个专门的囊泡，极低密度脂蛋白运输囊泡（VTV），它不同于其他ER衍生的囊泡，如蛋白运输囊泡（PTV）和前乳糜微粒运输囊泡（PCTV）的大小，浮力密度和蛋白质组成。我们先前报道了VTV蛋白质组，揭示了Reticulon-3A 2（RTN 3A 2）和小的Valosine Containing Peptide（VCP）相互作用蛋白（SVIP）在VTV中是唯一存在的。我们的初步研究表明，载脂蛋白B100（apoB 100），一种VLDL结构蛋白，在VLDL包装入VTV中起关键作用。有趣的是，我们发现RTN 3A 2选择性地与VLDL-apoB 100相互作用，并且是VLDL离开所需的，但不是白蛋白从相同的肝ER离开所需的。该提议的第一个具体目的是鉴定RTN 3A 2上的货物选择性结合位点和apoB 100上的货物选择性结合位点，用于VTV中的VLDL包含。在初步研究中，我们已经确定了另一个小的先生蛋白，SVIP，这是集中在VTV相比，他们的父母肝ER膜。我们的研究结果表明，SVIP与载脂蛋白B100，主要的VTV货物蛋白和Sar 1，但不与白蛋白，PTV货物蛋白表明SVIP在VTV介导的VLDL运输的作用。我们的第二个具体目标是建立SVIP在VTV生物发生和原代肝细胞中apoB 100的ER退出中的作用。在我们的初步研究中，我们发现VTV-高尔基体融合感受态SNARE复合物的形成需要胞浆蛋白。在初步研究中，我们鉴定了一种功能活性的胞质组分，其支持SNARE复合物的形成。SDS-PAGE分析表明，活性组分仅含有3种蛋白。我们已经发现了其中的两种蛋白质。在本基金的具体目标三中，我们建议鉴定第三种蛋白质，并测试这些蛋白质在VTV-高尔基体融合复合物组装中的功能作用。