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（由申请人提供）：脂质吸收的限速步骤是前乳糜微粒从内质网退出，这是通过前乳糜微粒转运囊泡从内质网膜表面出芽而发生的。这种密封的囊泡将乳糜微粒顺行运输到顺式高尔基体，在那里它与高尔基体融合，将其乳糜微粒货物输送到高尔基体腔，并且在高尔基体内部，乳糜微粒在其表面获得载脂蛋白Al。 我们建议鉴定 Sar1 b 和其他货物选择性蛋白结合的载脂蛋白 B48 上的位点，因为这将确认它们是否都结合相同的载脂蛋白 B48 肽，或者不同的蛋白是否需要不同的结合位点。我们还将鉴定出芽前乳糜微粒运输囊泡所需的蛋白质，使其能够矢量运输至高尔基体，并与其融合，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滞留病/安德森病中发生突变，导致了混杂的货物选择以包含在乳糜微粒前运输囊泡中以及产生不与顺式高尔基体融合的囊泡的问题。我们将确定为什么与前乳糜微粒转运囊泡相关的 COPII 蛋白在囊泡与顺式高尔基体对接之前没有被脱壳，这与将蛋白质从内质网转运到顺式高尔基体的囊泡不同。这可能是由于 Sec23 相互作用蛋白或差异磷酸化抑制了 Sec23 的 GTP 酶激活功能。这两种可能性都将使用重组 Sec23 相互作用蛋白和针对它的抗体以及使用加载 g32P-GTP 的 Sar1 b 进行测试。在囊泡表面不存在 COPII 蛋白的情况下，不会发生与高尔基体的融合，这表明它们在 SNARE 配对中具有功能，这种可能性将使用特定抗体进行测试。我们还将突变 Sar1b 以模拟乳糜微粒滞留病/安德森病，以测试其在囊泡与顺式高尔基体融合测定中的功能。将进行实验以测试 Sar1b 活性是否限制融合能力前乳糜微粒运输囊泡的生成，并测试 Sar1b 的一项功能是否是限制出芽能力蛋白接触货物选择蛋白载脂蛋白 B48。在缺乏 COPII 蛋白的情况下，囊泡出芽增加 6 至 10 倍。我们将比较 Sar1a 和 Sar1b 与前乳糜微粒和蛋白囊泡出芽以产生与顺式高尔基体融合的囊泡的能力，以确定每个 Sar1 对于两种囊泡类型的特异性。