A Cell Biological Approach to Hepatic Lipid Metabolism

肝脏脂质代谢的细胞生物学方法

• 批准号: 7908829
• 负责人: Shadab A Siddiqi
• 金额: $ 28.71万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908829
• 关键词: Albumins; Antibodies; Apolipoproteins B; Atherosclerosis; Binding; Biological; Blocking Antibodies; Cells; Cholesterol; Chylomicrons; Coat Protein Complex I; Complex; Coupled; Cytosol; Data; Development; Docking; Endopeptidase K; Endoplasmic Reticulum; Event; Face; Far-Western Blotting; Furuncles; Gel; Generations; Golgi Apparatus; Grant; Guanosine Triphosphate; Hepatic; Hepatocyte; Immunoblotting; Immunoprecipitation; Intestines; Ketones; Lipids; Liver; Low-Density Lipoproteins; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Metabolism; Molecular; Movement; N-terminal; Nonesterified Fatty Acids; Peripheral; Plasma; Precipitation; Process; Proteins; Recombinants; Reporting; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Site; Surface; Testing; Tissues; Transport Vesicles; Triglycerides; Vertebrates; Very low density lipoprotein; Vesicle; density; gel electrophoresis; lipid metabolism; low density lipoprotein inhibitor; prevent; protein complex; protein transport; research study; secretory protein; target SNARE proteins; therapeutic target; vesicular SNARE proteins

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.

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毒性，肝脏有效地将 FFA 转化为三酰甘油 (TAG) 进行储存，将其氧化为酮，或将其作为极低密度脂蛋白 (VLDL) 输出。用于组装 VLDL 的 TAG 对于脊椎动物将脂质分配到外周组织以获取能量至关重要。然而，在极低密度脂蛋白（VLDL）代谢过程中，会产生低密度脂蛋白（LDL），它是胆固醇的主要载体，与动脉粥样硬化的发展有关。 VLDL 从其合成位点内质网 (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（动脉粥样硬化的决定因素）的潜在治疗靶点。了解 VLDL 如何从 ER 退出并在分子水平上递送至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）分别在两个不同的囊泡中从肝脏 ER 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利用相同的 ER 囊泡出芽引发剂 Sar1。这表明 VLDL 使用了专门的囊泡，并且不同的蛋白质参与了两种类型运输囊泡中每种运输囊泡的货物选择。该资助的首要目的是确定将 VLDL 纳入 VTV 所需的货物选择蛋白。 2D-DIGE 分析表明，VTV 含有 5 种蛋白质囊泡中不存在的蛋白质。在初步研究中，我们已确定 Sar1b 是 VTV 出芽的启动子。此外，我们的数据显示载脂蛋白 B (apoB) 是 ER-VLDL 上的货物选择性蛋白。我们的第二个目标是确定从肝脏 ER 中形成 VTV 芽的芽前复合体。初步研究表明，VTV 在大小、密度和蛋白质组成方面与含有白蛋白的囊泡有很大不同。由于两个载体囊泡之间存在重大差异，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需的。我们建议鉴定负责将 VTV 靶向/对接至肝脏高尔基体的 SNARE 蛋白及其在肝脏高尔基体上的同源 SNARE 蛋白。我们的初步数据表明，VTV 与肝脏高尔基体的融合需要胞质因子。此外，当使用用蛋白酶 K 处理的胞质或煮沸的胞质时，VTV 不与肝脏高尔基体融合，这表明发生融合所需的胞质因子是蛋白质。该提案的另一个目的是确定控制 VLDL 向高尔基体输送的胞质蛋白。项目叙述：VLDL 从其合成位点（即内质网）转运至高尔基体是其最终从肝细胞分泌出来所必需的，并且是控制 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抑制这些蛋白质提供潜在的靶点，从而有可能控制肝脏的 VLDL 分泌。