A Cell Biological Approach to Lipid Absorption.

脂质吸收的细胞生物学方法。

• 批准号: 7906344
• 负责人: CHARLES Milton MANSBACH
• 金额: $ 4.99万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7906344
• 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; Cytosol; Data; Diet; Dietary intake; Disease; Docking; Eating; Endoplasmic Reticulum; Essential Fatty Acids; Fatty Acid-Binding Protein 1; Fatty acid glycerol esters; GTP Binding; GTPase-Activating Proteins; Generations; Golgi Apparatus; 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; Recombinants; Relative (related person); Role; SNAP receptor; Site; Specificity; Surface; Testing; Transport Vesicles; Treatment Protocols; Vesicle; absorption; apolipoprotein B-48; crosslink; lipid transport; 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乳糜微粒从内质网出口，这是通过从内质网膜表面出芽乳糜微粒运输囊泡发生的。这个封闭的囊泡将乳糜微粒顺行运输到顺性高尔基体，在那里它与高尔基体融合，将乳糜微粒货物运送到高尔基体腔，在高尔基体内，乳糜微粒在其表面获得载脂蛋白Al。