A Cell Biological Approach to Hepatic Lipid Metabolism

描述(申请人提供)：血浆极低密度脂蛋白(VLDL)水平升高会使动脉粥样硬化和冠状动脉疾病的风险成倍增加，这目前对美国的公众健康构成了严重影响。极低密度脂蛋白在肝脏中合成，并分泌到血液中。从肝脏分泌极低密度脂蛋白的速率决定步骤是它们从其生物发生部位--内质网--转运 (呃)，到高尔基。这一步骤在生理上是可调节的，并代表了一种潜在的治疗方法 控制血浆极低密度脂蛋白浓度升高的目标。我们建议在分子水平上研究新生的极低密度脂蛋白是如何从内质网退出并被运送到高尔基体的。我们的实验室已经证明，VLDL以一种特殊的囊泡形式存在于内质网，即VLDL运输囊泡(VTV)，它在大小、浮力密度和蛋白质组成上不同于其他ER来源的囊泡，如蛋白质运输小泡(PTV)和前乳糜粒运输小泡(PCTV)。我们早些时候报道了VTV蛋白质组，发现VTV中存在网状蛋白-3A2(RTN3A2)和含小分子Valosine多肽(VCP)相互作用蛋白(SVIP)。我们的初步研究表明，载脂蛋白B100(ApoB100)是一种极低密度脂蛋白结构蛋白，在极低密度脂蛋白包装成VTV的过程中起着关键作用。有趣的是，我们发现RTN3A2选择性地与VLDL-apoB100相互作用，并且是VLDL退出所必需的，而不是白蛋白退出同一肝脏ER所必需的。这项建议的第一个具体目标是确定RTN3A2上的货物选择性结合位点和apoB100上的货物选择性结合位点，以便将极低密度脂蛋白纳入VTV。在初步研究中，我们已经鉴定出另一种小的MR蛋白SVIP，与其母细胞的肝脏ER膜相比，它集中在VTV中。我们的结果表明，SVIP与主要的VTV-Cargo蛋白apoB100和Sar1特异地相互作用，但不与白蛋白相互作用，白蛋白是一种PTV-Cargo蛋白，提示SVIP在VTV介导的VLDL运输中发挥作用。我们的第二个特定目标是确定SVIP在VTV生物发生中的作用以及apoB100在原代肝细胞中的ER退出。我们在初步研究中发现，VTV-高尔基体融合活性SNARE-复合体的形成需要胞浆蛋白。在初步研究中，我们确定了一种功能活跃的胞浆组分，它支持圈套复合体的形成。经SDS-PAGE分析，活性部分仅含有3种蛋白质。我们已经鉴定了其中的两种蛋白质。在这项资助的具体目标三中，我们建议鉴定第三种蛋白质，并测试这些蛋白质在VTV-高尔基融合复合体组装中的功能作用。

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.