Molecular Mechanisms of Biogenesis of Apolipoprotein B-Containing Lipoproteins

载脂蛋白 B 脂蛋白生物合成的分子机制

• 批准号: 7088085
• 负责人: NASSRIN DASHTI
• 金额: $ 41.77万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088085
• 关键词: apolipoprotein B; cell line; gene expression; genetically modified animals; laboratory mouse; lipid biosynthesis; lipid metabolism; lipid structure; lipid transport; molecular assembly /self assembly; mutant; phospholipids; protein binding; protein localization; protein structure function; site directed mutagenesis; small interfering RNA; stoichiometry; transport proteins; triglycerides

DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms involved in the biogenesis of nascent apoB- containing lipoprotein particles and to identify the factors and structural features of apoB that are necessary for this process. The proposed research is driven by four hypotheses: 1) The N-terminal beta-alpha1 domain of human apoB-100, i.e., the first 1000 residues of the mature protein, folds into a triangular lipovitellin-like "lipid pocket" via the formation of a hairpin-bridge that provides a mechanism for the initiation of lipoprotein assembly without the structural requirement for microsomal triglyceride transfer protein (MTP). 2) The initial lipid transfer to the lipid pocket is mediated by phospholipid transfer protein (PLTP). 3) Translation of specific motifs in the amphipathic beta1 domain is required for MTP-mediated triglyceride (TG) recruitment. 4) The betaC region of the beta-alpha1 domain, together with MTP, provides a mechanism for delivering lipids into the lipid pocket. These hypotheses will be tested by four specific aims: 1) To establish the role of the hairpin-bridge and the four salt bridges: Arg997-Glu720, Glu998-His719, Asp999-Lys718, and Arg1000-Asp717 in the initiation of particle assembly. This aim will be accomplished by site-directed mutagenesis to disrupt the putative salt bridges and characterization of the secreted particles. 2) To investigate the putative role of PLTP in the initial lipidation of nascent apoB particles. This will be accomplished by: (a) expression of carboxyl-terminally truncated forms of apoB is equal to or more than apoB:1000 in mammalian cells that lack PLTP, with and without coexpression of PLTP; (b) inactivation of PLTP in McA-RH7777 cells expressing truncated forms of apoB using siRNA; (c) coexpression of PLTP and truncated forms of apoB in PLTP-deficient mouse hepatocytes. 3) To identify the domains in apoB that confer a high requirement for MTP and bulk lipid addition to the nascent particle. This will be accomplished as described for Specific Aim 2 using cells that lack MTP and MTP-deficient mouse hepatocytes. We propose to map the effects on the stoichiometry of lipid component of the particles of sequential inclusion of the N-terminal region of apoB-100 and to identify the sequences in the beta1 domain that confer stability in the particle beyond apoB:1000. 4). To test the hypothesis that the betaC region (beta barrel) of beta-alpha1 domain serves as a channel for lipid transfer into the lipid pocket. This will be accomplished by site- directed mutagenesis of residues critical for the structural competence of the beta barrel and binding to MTP.

描述(由申请人提供)：本项目的目标是了解新生载脂蛋白颗粒的生物发生机制，并确定这一过程所必需的载脂蛋白B的因子和结构特征。这项研究由四个假设驱动：1)人apoB-100的N-末端β-α1结构域，即成熟蛋白的前1000个残基，通过形成发夹桥折叠到一个三角形的脂卵黄蛋白样“脂袋”中，该桥为启动脂蛋白组装提供了一种机制，而不需要微粒体甘油三酯转移蛋白(MTP)的结构要求。2)磷脂转运蛋白(PLTP)是由磷脂转运蛋白(PLTP)介导的向脂囊的初始转移。3)MTP介导的甘油三酯(TG)募集需要两亲性β1结构域中特定基序的翻译。4)β-α1结构域的Betac区域与MTP一起，提供了一种将脂类输送到脂袋中的机制。1)建立发夹桥和四个盐桥：Arg997-Glu720、Glu998-His719、Asp999-Lys718和Arg1000-Asp717在启动颗粒组装中的作用。这一目标将通过定点突变来实现，以破坏可能的盐桥和分泌颗粒的特征。2)探讨PLTP在新生载脂蛋白B颗粒初始脂化中的作用。这将通过以下途径实现：(A)在缺乏PLTP的哺乳动物细胞中，羧基末端截短形式的apoB的表达等于或大于apoB：1000，但同时存在和不表达PLTP；(B)在使用siRNA表达截短形式的apoB的MCA-RH7777细胞中，PLTP处于失活状态；(C)在PLTP缺失的小鼠肝细胞中，PLTP和截短形式的apoB共表达。3)确定apoB中对MTP和向新生颗粒添加大量脂类的高要求结构域。这将按照特定目的2的描述使用缺乏MTP的细胞和MTP缺陷的小鼠肝细胞来完成。我们建议绘制apoB-100 N-末端区域的顺序包涵体对颗粒脂质成分化学计量比的影响，并鉴定在apoB：1000以外的颗粒中提供稳定性的Beta1结构域序列。4)。为了验证这一假设，即β-α1结构域的Betac区域(β桶)作为脂质转移到脂袋中的通道。这将通过对对β桶的结构能力和与MTP结合至关重要的残基进行定点突变来实现。