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: 7421086
• 负责人: NASSRIN DASHTI
• 金额: $ 40.85万
• 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/7421086
• 关键词: Address; Amino Acids; Apolipoproteins B; Binding; Biogenesis; Cells; Competence; Goals; Hepatocyte; Human; Lampreys; Lipids; Lipoprotein (a); Lipoprotein (a-); Lipoproteins; Mammalian Cell; Maps; Mediating; Mediator of activation protein; Modeling; Molecular; Molecular Conformation; Mus; N-terminal; Phospholipid Transfer Proteins; Phospholipids; Primary carcinoma of the liver cells; Process; Protein Secretion; Publishing; Rattus; Research; Research Personnel; Role; Sequence Homology; Site-Directed Mutagenesis; Small Interfering RNA; Sodium Chloride; Testing; Translations; Triglycerides; apolipoprotein B-100; base; beta barrel; lipovitellin; microsomal triglyceride transfer protein; mutant; particle; phospholipid exchange proteins; programs; protein folding; stoichiometry

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新生含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颗粒的因素和结构特征。提出的研究基于四个假设：1)人类apoB-100的n端β - α 1结构域，即成熟蛋白的前1000个残基，通过形成发夹桥折叠成一个三角形的脂磷脂样“脂袋”，这提供了一种启动脂蛋白组装的机制，而不需要微粒体甘油三酯转移蛋白（MTP）的结构要求。2)脂质向脂袋的初始转移是由磷脂转移蛋白（PLTP）介导的。3) mtp介导的甘油三酯（TG）募集需要翻译两亲性beta1结构域的特定基序。α - α - 1结构域的β - ac区域与MTP一起，提供了一种将脂质输送到脂质袋的机制。这些假设将通过四个具体目标进行验证：1)建立发针桥和四个盐桥：Arg997-Glu720， Glu998-His719， Asp999-Lys718和Arg1000-Asp717在粒子组装起始中的作用。这一目标将通过定点诱变来实现，以破坏假定的盐桥并表征分泌颗粒。2)探讨PLTP在新生载脂蛋白颗粒初始脂化中的作用。这将通过以下方式实现：(a)在缺乏PLTP的哺乳动物细胞中，无论是否有PLTP共表达，羧基末端截断形式的apoB表达等于或大于apoB:1000；(b)用siRNA灭活表达截短的载脂蛋白ob的McA-RH7777细胞中的PLTP；(c) PLTP缺失小鼠肝细胞中PLTP和截短型载脂蛋白ob的共表达。3)确定载脂蛋白ob中对新生颗粒的MTP和大量脂质添加有高要求的结构域。这将在Specific Aim 2中使用缺乏MTP和MTP缺陷的小鼠肝细胞来完成。我们建议绘制序列包含apoB-100 n端区域的颗粒对脂质成分化学计量学的影响，并确定β 1结构域中赋予apoB:1000以上颗粒稳定性的序列。4）. 为了验证β - α - 1结构域的β - ac区域（β桶）作为脂质转移到脂质口袋的通道的假设。这将通过对β桶结构能力至关重要的残基的定点诱变和与MTP的结合来实现。