A Cell Biological Approach to Lipid Absorption.

脂质吸收的细胞生物学方法。

• 批准号: 7226007
• 负责人: CHARLES Milton MANSBACH
• 金额: $ 29.06万
• 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/7226007
• 关键词: Address; Anderson syndrome; Antibodies; Apolipoproteins; Back; Binding; Binding Sites; Biological; Biological Assay; Body Weight decreased; Carbohydrates; Carrier Proteins; Cells; Chylomicrons; Clinical; Coatomer Protein; Complex; Condition; Cytosol; Data; Diet; Dietary intake; Disease; Docking; Eating; Endoplasmic Reticulum; Essential Fatty Acids; Fatty acid glycerol esters; GTP Binding; GTPase-Activating Proteins; Generations; Golgi Apparatus; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Immunoprecipitation; In Vitro; Intake; Intestines; Laboratories; Lipids; Maintenance; Mediating; Membrane; Molecular; Mutate; Names; Pathway interactions; Peptides; Phosphorylation; Play; Process; Production; Protein Binding; Proteins; Range; Rate; Recombinants; Relative (related person); Role; SNAP receptor; Site; Specificity; Surface; Testing; Transport Vesicles; Treatment Protocols; Vesicle; absorption; apolipoprotein B-48; crosslink; lipid transport; liver fatty acid-binding protein; mouse Gdi2 protein; protein protein interaction; research study; seal; secretory protein

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(申请人提供)：脂类吸收的限速步骤是前乳糜粒从内质网上退出，这是通过将前乳糜粒运输囊泡从内质网膜表面萌发而发生的。这个封闭的小泡将乳糜粒顺行运输到顺式高尔基体，在那里它与高尔基体融合，将乳糜体运送到高尔基体腔，在高尔基体腔内，乳糜体在其表面获得载脂蛋白A1。 我们建议确定载脂蛋白B48上Sar1b和其他货物选择蛋白结合的位置，因为这将确认它们是否都与相同的载脂蛋白B48肽结合，或者不同的蛋白质是否需要不同的结合部位。我们还将利用免疫学和其他方法确定前乳糜粒运输囊泡萌发所需的蛋白质，使其能够向外运输到高尔基体，并与其融合，以确定蛋白质-蛋白质相互作用。在乳糜粒滞留症/安德森病中，Sar1b中的GTP结合口袋发生突变，导致混杂的货物选择包括在乳糜粒前运输小泡中，并产生一个不与顺式高尔基体融合的小泡。我们将确定为什么与前乳糜粒转运囊泡相关的COPII蛋白在囊泡与顺式高尔基体对接之前没有被覆盖，这与从内质网向顺式高尔基体运输蛋白质的囊泡不同。这可能是由于Sec23相互作用蛋白或通过差异磷酸化抑制了Sec23的GTP酶激活功能。这两种可能性都将通过使用针对它的重组Sec23相互作用蛋白和抗体以及通过使用g32P-GTP负载的Sar1b进行测试。在小泡表面没有COPII蛋白的情况下，不会与高尔基体融合，这表明它们在圈套配对中具有功能，这种可能性将使用特定的抗体进行测试。我们还将突变Sar1b以模拟乳糜粒滞留症/安德森病，以在囊泡与顺式高尔基体的融合实验中测试其功能。将进行实验，以测试Sar1b活性是否对融合能力前乳糜粒运输囊泡的产生具有速率限制，并测试Sar1b的一个功能是否是限制萌芽中的能力蛋白对货物选择蛋白载脂蛋白B48的访问。在没有COPII蛋白的情况下，囊泡发芽增加6到10倍。我们将比较Sar1a和Sar1b与前乳糜粒和蛋白质小泡发芽的能力，以产生与顺式高尔基体融合的小泡，以确定每种Sar1对这两种小泡类型的特异性。