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）：该项目的目标是了解含有新生 apoB 的脂蛋白颗粒的生物发生机制，并确定该过程所需的 apoB 因素和结构特征。拟议的研究由四个假设驱动：1) 人类 apoB-100 的 N 端 beta-alpha1 结构域，即成熟蛋白的前 1000 个残基，通过形成折叠成三角形卵黄蛋白样“脂质袋”发夹桥提供了一种启动脂蛋白组装的机制，而无需微粒体甘油三酯转移蛋白（MTP）的结构要求。 2) 最初的脂质转移到脂质袋是由磷脂转移蛋白（PLTP）介导的。 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) beta-alpha1 结构域的 betaC 区域与 MTP 一起提供了将脂质递送到脂质袋中的机制。这些假设将通过四个具体目标进行检验：1）确定发夹桥和四个盐桥：Arg997-Glu720、Glu998-His719、Asp999-Lys718 和 Arg1000-Asp717 在粒子组装启动中的作用。这一目标将通过定点诱变来破坏假定的盐桥和分泌颗粒的表征来实现。 2) 研究 PLTP 在新生 apoB 颗粒初始脂化中的假定作用。这将通过以下方式实现： (a) 在缺乏 PLTP 的哺乳动物细胞中，在有或没有 PLTP 共表达的情况下，apoB 的羧基末端截短形式的表达等于或大于 apoB:1000； (b)使用siRNA使表达截短形式apoB的McA-RH7777细胞中的PLTP失活； (c) PLTP 和截短形式的 apoB 在 PLTP 缺陷的小鼠肝细胞中共表达。 3) 鉴定 apoB 中对 MTP 和向新生颗粒添加大量脂质有高要求的结构域。这将按照特定目标 2 的描述，使用缺乏 MTP 的细胞和 MTP 缺陷的小鼠肝细胞来实现。我们建议绘制对连续包含 apoB-100 N 端区域的颗粒脂质成分化学计量的影响，并鉴定 beta1 结构域中赋予 apoB:1000 以外颗粒稳定性的序列。 4）。测试 beta-alpha1 结构域的 betaC 区域（β 桶）作为脂质转移至脂质袋的通道的假设。这将通过对β桶结构能力和与MTP结合至关重要的残基进行定点诱变来实现。