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: 7222722
• 负责人: NASSRIN DASHTI
• 金额: $ 40.54万
• 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/7222722
• 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）：该项目的目的是了解含有脂蛋白颗粒的新生apob的生物发生的机制，并确定此过程所需的APOB的因素和结构特征。拟议的研究由四个假设驱动：1）人类APOB-100的N端β-Alpha1结构域，即成熟蛋白的第一个1000个残基，将成熟蛋白的第一个残基折叠成一个三角形脂维素蛋白的“脂质”，不用a hairpin-bridge的构建机械构成的机械化，以供ahairpin-broke构成。甘油三酸酯转移蛋白（MTP）。 2）初始脂质转移到脂质袋中是由磷脂转移蛋白（PLTP）介导的。 3）MTP介导的甘油三酸酯（TG）募集需要两亲性Beta1结构域中特定基序的翻译。 4）Beta-Alpha1结构域的BETAC区域与MTP一起提供了一种将脂质输送到脂质口袋中的机制。这些假设将通过四个特定目的来检验：1）确定发夹桥和四个盐桥的作用：ARG997-GLU720，GLU998-HIS719，ASP999-LYS718和ARG1000-ASP717在粒子组件的开始中。该目标将通过定位的诱变来实现，以破坏推定的盐桥和分泌颗粒的特征。 2）研究PLTP在新生APOB颗粒初始脂质中的推定作用。这将通过：（a）羧基末端截断的APOB的表达等于或超过ApoB：1000在缺乏PLTP的哺乳动物细胞中，具有PLTP的共表达； （b）使用siRNA表达APOB的截短形式的MCA-RH7777细胞中PLTP的失活； （c）PLTP缺乏小鼠肝细胞中PLTP和APOB的截短形式的共表达。 3）确定APOB中赋予MTP和大量脂质添加到新生粒子的域中的域。使用缺乏MTP和MTP缺陷型小鼠肝细胞的细胞，将根据特定目标2的描述来实现这一点。我们建议绘制对APOB-100的N末端区域的顺序包含颗粒的脂质成分的化学计量法的影响，并识别Beta1结构域中赋予APOB以外粒子中稳定性的序列：1000。 4）。为了检验β-Alpha1结构域的BetAC区域（β桶）的假设，是脂质转移到脂质口袋中的通道。这将通过位于位置的诱变的残基的诱变来完成，对β枪管的结构能力至关重要并与MTP结合。