A Cell Biological Approach to Lipid Absorption.

脂质吸收的细胞生物学方法。

• 批准号: 7079579
• 负责人: CHARLES Milton MANSBACH
• 金额: $ 32万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7079579
• 关键词: Golgi apparatus; apolipoprotein B; binding sites; chylomicrons; endoplasmic reticulum; fatty acid binding protein; gastrointestinal nutrient absorption; guanosinetriphosphatase activating protein; guanosinetriphosphatases; intracellular transport; laboratory rat; lipid metabolism; membrane fusion; membrane proteins; nutrition related tag; phosphorylation; protein protein interaction; transport proteins; vesicle /vacuole

DESCRIPTION (provided by applicant): The rate-limiting step in lipid absorption is the exit of pre-chylomicrons from the endoplasmic reticulum, which occurs by budding the pre-chylomicron transport vesicle from the surface of the endoplasmic reticulum membrane. This sealed vesicle transports chylomicrons anterograde to the cis Golgi where it fuses with the Golgi, delivers its chylomicron cargo to the Golgi lumen, and within the Golgi, the chylomicron acquires apolipoprotein Al on its surface. We propose to identify the site on apolipoprotein B48 to which Sar1 b and other cargo selective proteins bind as this will confirm if they all bind to the same apolipoprotein B48 peptide or if different binding sites are required for different proteins. We will also identify the proteins required for budding the pre-chylomicron transport vesicle, enable it to be vectorially transported to the Golgi, and to fuse with it, using both immunological and other approaches to determine protein-protein interactions. The GTP binding pocket in Sar1b is mutated in Chylomicron Retention Disease/Anderson's Disease leading to the question of promiscuous cargo selection for inclusion in the pre-chylomicron transport vesicle and production of a vesicle, which does not fuse, with the cis Golgi. We will determine why the COPII proteins, which are associated with the pre-chylomicron transport vesicle, are not uncoated prior to the docking of the vesicle with the cis Golgi unlike vesicles that transport proteins from the ER to the cis Golgi. This may be due to inhibition of the GTPase activating function of Sec23 by Sec23 Interactive Protein or by differential phosphorylation. Both possibilities will be tested for using recombinant Sec23 Interactive Protein and antibody directed towards it, and by using g32P-GTP loaded Sar1 b. In the absence of COPII proteins on the surface of the vesicles, no fusion with the Golgi occurs suggesting their functionality in SNARE pairing, a possibility that will be tested using specific antibodies. We will also mutate Sar1b to mimic Chylomicron Retention Disease/Anderson's Disease to test its function in a fusion assay of the vesicle with the cis Golgi. Experiments will be performed to test if Sar1 b activity is rate limiting for the generation of fusion competent pre-chylomicron transport vesicles and to test if one function of Sar1 b is to restrict access of budding competent proteins to the cargo selection protein, apolipoprotein B48. In the absence of the COPII proteins, vesicle budding increases 6 to 10 fold. We will compare the ability of Sar1a and Sar1b to bud with pre-chylomicron and protein vesicles to produce vesicles that are fusion competent with the cis Golgi to determine the specificity of each Sar1 for both vesicle types.

描述（由申请人提供）：脂质吸收的速率限制步骤是从内质网的出口，这是通过从内质网膜的表面萌芽的，这是通过萌芽的胶质体转运小囊而发生的。该密封的囊泡将乳糜微粒递送到顺式高尔基体中，在那里与高尔基体融合，将其乳糜微粒货物交付到高尔基体腔内，在高尔基体中，乳糜微粒在其表面上获得了载脂蛋白Al。 我们建议在载脂蛋白B48上识别SAR1 B和其他货物选择性蛋白结合的位点，因为这将确认它们是否与相同的载脂蛋白B48肽结合，或者是否需要不同蛋白质的不同结合位点。我们还将鉴定出萌芽的纤毛前转运囊泡所需的蛋白质，使其能够将其转运至高尔基体，并使用免疫学和其他方法来确定蛋白质蛋白质相互作用。 SAR1B中的GTP结合口袋在乳糜微粒保留疾病/安德森病中突变，导致选择了滥交货物的问题，以纳入胶囊前的转运囊泡和囊泡的产生，该囊泡不融合，而不会与顺式Golgi融合。我们将确定为什么在囊泡与顺式高尔基囊泡对接之前，与胶囊前转运囊泡相关的COPII蛋白与从ER转运到顺式高尔基的囊泡不同。这可能是由于Sec23互动蛋白或差异磷酸化对SEC23的GTPase激活功能的抑制。两种可能性都将用于使用针对其的重组SEC23相互作用蛋白和抗体，并使用G32P-GTP加载的SAR1 b。在囊泡表面上没有COPII蛋白的情况下，与高尔基体没有融合表明它们在圈套配对中的功能，这种可能性将使用特定的抗体进行测试。我们还将突变SAR1B以模仿乳糜微粒保留疾病/Anderson病，以测试其在与CIS Golgi的囊泡融合测定中的功能。将进行实验，以测试SAR1 B活性是否是融合融合效率的前胶囊前传输囊泡的速率限制，并测试SAR1 B的一种功能是否是限制萌芽的合理蛋白进入货物选择蛋白Apolipopropoprototin B48的访问。在没有COPII蛋白的情况下，囊泡萌芽增加了6至10倍。我们将比较SAR1A和SAR1B与胶囊前和蛋白囊泡产生与CIS Golgi融合的囊泡的能力，以确定两种囊泡类型的每个SAR1的特异性。