A Cell Biological Approach to Hepatic Lipid Metabolism

描述(由申请人提供)：肝脏面临着游离脂肪酸(FFA)流入的重大挑战。为了防止潜在的肝脏脂肪毒性，肝脏有效地将FFA转化为三酰甘油(TAG)进行存储，将它们氧化为酮，或将它们作为极低密度脂蛋白(VLDL)输出。在脊椎动物中，用来组装极低密度脂蛋白的标签对于将脂质分配到周围组织以获取能量至关重要。然而，在极低密度脂蛋白的代谢过程中，会产生低密度脂蛋白，低密度脂蛋白是胆固醇的主要载体，与动脉粥样硬化的发展有关。极低密度脂蛋白从其合成部位内质网(ER)移动到高尔基体是其最终从肝细胞分泌所必需的，是控制血浆低密度脂蛋白浓度升高的潜在治疗靶点，低密度脂蛋白是动脉粥样硬化的决定因素。在分子水平上了解极低密度脂蛋白如何从内质网退出并传递到高尔基体是目前应用的主题。我们的初步研究表明，极低密度脂蛋白和白蛋白(一种典型的肝脏分泌蛋白)分别在两个不同的小泡中从肝脏内质网运输到高尔基体。尽管这两种货物是在不同的囊泡中运输的，但两种类型的囊泡都利用相同的ER囊泡萌发的启动剂Sar1。这表明，极低密度脂蛋白利用了一个专门的小泡，不同的蛋白质参与了两种运输小泡中每种类型的货物的选择。这笔赠款的第一个目的是确定将极低密度脂蛋白包括在极低密度脂蛋白运输囊泡中所需的货物选择蛋白(S)。2D-DGE分析表明，VTV含有5种蛋白，这些蛋白不存在于蛋白泡中。在初步研究中，我们已经确定Sar1b是VTV发芽的启动子。此外，我们的数据显示载脂蛋白B(ApoB)是ER-VLDL上的货物选择蛋白。我们的第二个目标是确定从肝脏ER形成BUD VTV的预芽复合体。初步研究表明，VTV在大小、密度和蛋白质组成上与含白蛋白的囊泡有很大不同。由于两个载体囊泡之间的主要差异，VTV的表面可能有不同的蛋白质，这些蛋白质是VTV与顺式高尔基体对接和融合所必需的。我们建议确定负责VTV靶向/对接到肝脏高尔基体的SNARE蛋白及其在肝脏高尔基体上的同源SNARE蛋白。我们的初步数据显示，VTV与肝脏高尔基体融合需要胞浆因子(S)。此外，当使用蛋白酶K处理的胞浆或煮熟的胞液时，VTV不与肝脏高尔基体融合，这表明发生融合所需的胞浆因子(S)是一种蛋白质(S)。这项提议的另一个目的是确定控制极低密度脂蛋白向高尔基体输送的胞浆蛋白。项目简介：极低密度脂蛋白从合成部位、内质网到高尔基体的运输是肝细胞最终分泌所必需的，是控制低密度脂蛋白浓度升高的潜在治疗目标，低密度脂蛋白是胆固醇的主要载体和动脉粥样硬化的决定因素。VLDL选择进入VTV、VTV萌发及其与高尔基体融合的过程中涉及的蛋白质的鉴定将为它们的抑制提供潜在的靶点，从而潜在地控制肝脏VLDL的分泌。

DESCRIPTION (provided by applicant): The liver faces a major challenge of free fatty acid (FFA) influx. To prevent the potential hepatic lipotoxicity, the liver efficiently converts FFA to triacylglycerol (TAG) for storage, oxidizes them to ketones, or exports them as very-low density lipoproteins (VLDLs). TAG utilized to assemble VLDL is crucial for the distribution of lipids to peripheral tissues for energy in vertebrates. On metabolism of VLDL, however, LDL is generated which is the major carrier of cholesterol and is associated with the development of atherosclerosis. The movement of VLDL from its site of synthesis, the endoplasmic reticulum (ER) to the Golgi is required for its eventual secretion from the hepatocyte and represents the potential therapeutic target in controlling elevated concentrations of LDL in the plasma, the determinant of atherosclerosis. To understand how VLDL exits from the ER and its delivery to the Golgi at the molecular level is the subject of the current application. Our preliminary studies show that VLDL and albumin (a typical liver secretory protein) are transported separately in two different vesicles from the liver ER to the Golgi. Despite the two cargoes being transported in different vesicles, the same initiator of ER vesicle budding, Sar1, is utilized by both types of vesicles. This suggests that a specialized vesicle is utilized for VLDL and that different proteins are involved with the selection of cargo for each of the two types of transport vesicles. The first aim of this grant is to identify the cargo selecting protein(s) required for the inclusion of VLDL in the VLDL-transport-vesicle (VTV). 2D-DIGE analyses revealed that VTV contains 5 proteins that are not present in protein vesicles. In preliminary studies we have identified Sar1b as the initiator of VTV budding. Further, our data show apolipoprotein B (apoB) to be the cargo selective protein on ER-VLDL. Our second aim will be to identify the pre-budding complex that forms to bud VTV from the liver ER. Preliminary studies show that VTVs greatly differ from albumin containing vesicles in their size, density and protein composition. Because of the major differences between the two carrier vesicles, the VTV may have different proteins on its surface, which are required for docking and fusion of the VTV with the cis Golgi. We propose to identify the SNARE proteins responsible for the targeting/docking of VTVs to the liver Golgi and their cognate SNARE proteins on the liver Golgi. Our preliminary data show that fusion of VTVs with the liver Golgi requires cytosolic factor(s). Further, VTVs do not fuse with the liver Golgi when cytosol treated with proteinase K or boiled cytosol was used, suggesting that the cytosolic factor(s) required for fusion to occur is a protein(s). Another aim of this proposal is to determine cytosolic proteins that control the delivery of VLDL to the Golgi. Project Narrative: Transport of VLDL from its site of synthesis, the endoplasmic reticulum to the Golgi is required in its eventual secretion from the hepatocyte and represents a potential therapeutic target in controlling elevated concentrations of LDL, the major carrier of cholesterol and determinant of atherosclerosis. The identification of proteins involved in the process of VLDL selection into VTVs, VTV budding and its fusion with Golgi would offer potential targets for their inhibition and thus potentially to control VLDL secretion from the liver.