Molecular Mechanisms of Biogenesis of Apolipoprotein B-Containing Lipoproteins

描述（由申请人提供）：本项目的目标是了解新生含apoB脂蛋白颗粒生物发生的机制，并鉴定该过程所需的apoB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-α 1结构域，即，成熟蛋白的前1000个残基通过发夹桥的形成折叠成三角形的卵黄脂蛋白样“脂质袋”，发夹桥提供了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)最初的脂质转移到脂质袋是由磷脂转移蛋白（PLTP）介导的。3)两亲性β 1结构域中特定基序的翻译是MTP介导的甘油三酯（TG）募集所必需的。4)β-α 1结构域的β C区与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：Arg 997-Glu 720、Glu 998-His 719、Asp 999-Lys 718和Arg 1000-Asp 717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，以破坏推定的盐桥和分泌颗粒的表征。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，在有和没有PLTP共表达的情况下，羧基末端截短形式的apo B的表达等于或大于apo B：1000;（B）在表达截短形式的apo B的McA-RH 7777细胞中使用siRNA灭活PLTP;（c）在PLTP缺陷的小鼠肝细胞中共表达PLTP和截短形式的apo B。3)确定apoB中对MTP和大量脂质添加到新生颗粒的高要求的结构域。这将按照特定目标2所述，使用缺乏MTP和MTP缺陷型小鼠肝细胞的细胞来完成。我们建议映射的顺序包含的N-末端区域的载脂蛋白B-100的颗粒的脂质组分的化学计量的影响，并确定在β 1结构域中的序列，赋予稳定性的颗粒超过载脂蛋白B：1000。4）。检验β-α 1结构域的β C区（β桶）作为脂质转移到脂质袋中的通道的假设。这将通过对β桶结构能力和与MTP结合关键的残基进行定点诱变来实现。

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.