A Cell Biological Approach to Hepatic Lipid Metabolism

描述（由申请人提供）：肝脏面临游离脂肪酸（FFA）流入的主要挑战。为了防止潜在的肝脏脂毒性，肝脏有效地将FFA转化为三酰甘油（TAG）用于储存，将其氧化为酮，或将其作为极低密度脂蛋白（VLDL）输出。在脊椎动物中，用于组装VLDL的TAG对于脂质向外周组织分配能量至关重要。然而，在VLDL的代谢中，产生LDL，其是胆固醇的主要载体并且与动脉粥样硬化的发展相关。VLDL从其合成位点（内质网（ER））到高尔基体的运动是其最终从肝细胞分泌所需的，并且代表了控制血浆中LDL浓度升高的潜在治疗靶点，LDL是动脉粥样硬化的决定因素。在分子水平上理解VLDL如何从ER中退出并将其递送到高尔基体是本申请的主题。我们的初步研究表明，VLDL和白蛋白（一种典型的肝脏分泌蛋白）分别在两个不同的囊泡从肝脏ER运输到高尔基体。尽管这两种货物在不同的囊泡中运输，但两种类型的囊泡都利用了ER囊泡出芽的相同启动子Sar 1。这表明，一个专门的囊泡是用于极低密度脂蛋白和不同的蛋白质参与选择货物的两种类型的运输囊泡。该资助的第一个目的是鉴定将VLDL包含在VLDL转运囊泡（VTV）中所需的货物选择蛋白。2D-DIGE分析显示，VTV含有5种蛋白质，这些蛋白质不存在于蛋白质囊泡中。在初步研究中，我们已经确定Sar 1b作为VTV出芽的启动子。此外，我们的数据显示载脂蛋白B（apo B）是ER-VLDL上的货物选择性蛋白。我们的第二个目标是确定从肝脏ER形成芽VTV的预芽复合物。初步研究表明，VTV在其大小、密度和蛋白质组成方面与含白蛋白的囊泡有很大不同。由于两种载体囊泡之间的主要差异，VTV表面可能具有不同的蛋白质，这是VTV与顺式高尔基体对接和融合所必需的。我们建议确定SNARE蛋白负责VTV的目标/对接到肝脏高尔基体和他们的同源SNARE蛋白的肝脏高尔基体。我们的初步数据表明，VTV与肝脏高尔基体的融合需要胞质因子。此外，当使用蛋白酶K处理的胞质溶胶或煮沸的胞质溶胶时，VTV不与肝高尔基体融合，这表明融合发生所需的胞质因子是蛋白质。该建议的另一个目的是确定控制VLDL向高尔基体递送的胞质蛋白。项目叙述：VLDL从其合成位点（内质网）转运至高尔基体是其最终从肝细胞分泌所必需的，并且代表了控制LDL浓度升高的潜在治疗靶点，LDL是胆固醇的主要载体和动脉粥样硬化的决定因素。参与VLDL选择进入VTV、VTV出芽及其与高尔基体融合过程的蛋白质的鉴定将为它们的抑制提供潜在的靶点，从而有可能控制VLDL从肝脏的分泌。

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.