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肪毒性，肝脏有效地将FFA转化为三酰基甘油（TAG）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的能量分布至关重要。而在VLDL的代谢过程中产生LDL，它是胆固醇的主要载体，与动脉粥样硬化的发生有关。VLDL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，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。了解VLDL如何从内质网退出并在分子水平上传递到高尔基体是当前应用的主题。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）分别通过两个不同的囊泡从肝脏内质网转运到高尔基体。尽管两种货物在不同的囊泡中运输，但两种类型的囊泡都使用相同的ER囊泡出芽引发剂Sar1。这表明一个专门的囊泡被用于VLDL，并且不同的蛋白质参与了两种类型的运输囊泡的货物选择。该资助的第一个目的是确定在VLDL-运输囊泡（VTV）中包含VLDL所需的货物选择蛋白。2D-DIGE分析显示，VTV含有5种在蛋白质囊泡中不存在的蛋白质。在初步研究中，我们已经确定Sar1b是VTV出芽的启动剂。此外，我们的数据显示载脂蛋白B （apoB）是ER-VLDL上的货物选择性蛋白。我们的第二个目标将是确定从肝脏内质网形成芽VTV的芽前复合体。初步研究表明，VTVs与含白蛋白的囊泡在大小、密度和蛋白质组成上有很大的不同。由于两个载体囊泡之间的主要差异，VTV表面可能有不同的蛋白质，这是VTV与顺式高尔基体对接和融合所必需的。我们建议鉴定负责将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，VTVs与肝脏高尔基体的融合需要细胞质因子(s)。此外，当使用蛋白酶K处理的细胞质或煮沸的细胞质时，vtv不能与肝脏高尔基体融合，这表明发生融合所需的细胞质因子是一种蛋白质。本研究的另一个目的是确定控制VLDL向高尔基体传递的细胞质蛋白。项目描述：VLDL从其合成部位内质网运输到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。鉴定参与VLDL选择进入VTV、VTV出芽及其与高尔基体融合过程的蛋白质，将为它们的抑制提供潜在的靶点，从而有可能控制肝脏的VLDL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.