Assembly Pathway of VLDL-A Three Step Process

描述(申请人提供)：拟议研究的长期目标是开发一种肝脏组装极低密度脂蛋白(VLDL)的模型。目前流行的极低密度脂蛋白组装模型是两步模型，在该模型中，在粗面内质网(ER)中形成一个小的、致密的、富含磷脂的、含apoB的颗粒(步骤1)，然后在ER内添加大量的甘油三酯(步骤2)。我们实验室的研究表明，极低密度脂蛋白在内质网内的组装并不完整，并导致了我们的工作假设：极低密度脂蛋白在肝脏中组装的过程分三步进行，前两步发生在内质网中，最后一步是甘油三酯和磷脂的添加，发生在通往高尔基体或在高尔基体内的路线上。为了验证这一工作假说，我们提出了以下具体目标：具体目标1.确定高尔基体在极低密度脂蛋白组装的第三步中的作用。这些研究将利用体内和体外方法进一步确定高尔基体内新生极低密度脂蛋白的脂化机制，并评估微粒体甘油三酯转移蛋白(MTP)在这一过程中的作用。具体目的#2.评估囊泡从内质网到高尔基体运输过程中对极低密度脂蛋白组装的贡献。我们的假设是，在新生粒子从内质网向高尔基体运输的过程中，组装仍在继续。这些研究将利用体外囊泡生成系统来评估运输过程中的组装。明确载脂蛋白E在极低密度脂蛋白组装中的作用。载脂蛋白E缺乏使肝脏甘油三酯分泌减少50%，这与我们对ER后增加形成极低密度脂蛋白的甘油三酯质量的估计相一致。这些研究将检验载脂蛋白E调节甘油三酯在细胞内的分布并影响新生脂蛋白脂化的假设。血浆低密度脂蛋白(LDL)浓度升高是动脉粥样硬化性心脏病的主要危险因素。由于DL仅通过极低密度脂蛋白的血管内分解代谢在血浆中形成，了解极低密度脂蛋白组装途径中的细胞内事件可能被证明对于开发新的治疗策略对低密度脂蛋白水平升高和有动脉粥样硬化性心脏病风险的患者很重要。

DESCRIPTION (provided by applicant): The long term objective of the proposed studies is to develop a model for the assembly of very low density lipoproteins (VLDL) by the liver. The prevailing model for VLDL assembly is a two step model in which the formation of a small, dense, phospholipid- rich, apoB-containing particle in the rough endoplasmic reticulum (ER) (step 1) is followed by the addition of bulk triglyceride within the ER (step 2). Studies from our laboratory have suggested that assembly is not complete within the ER, and have led to our working hypothesis: VLDL is assembled in the liver in a three-step process with the first two steps occurring in the ER and the final step, the addition of triglyceride and phospholipid, occurring in route to or within the Golgi apparatus. To test this working hypothesis we propose the following specific aims: Specific Aim #1. To define the role of the Golgi apparatus in the third step of VLDL assembly. These studies will utilize both in vivo and in vitro approaches to define further the mechanism of lipidation of nascent VLDL within the Golgi apparatus and to evaluate the role of microsomal triglyceride transfer protein (MTP) in this process. Specific Aim #2. To evaluate the contributions to VLDL assembly during vesicular transport from the endoplasmic reticulum to the Golgi apparatus. It is our hypothesis that assembly continues during the trafficking of nascent particles from the ER to the Golgi apparatus. These studies will utilize an in vitro vesicle generation system to assess assembly during transport. Specific Aim #3. To define the role of apoE in the assembly of VLDL. ApoE deficiency decreases hepatic triglyceride secretion by 50%, which corresponds to our estimates of the mass of triglyceride added to forming VLDL post ER. These studies will test the hypothesis that apoE modulates the intracellular distribution of triglyceride and affects the lipidation of nascent lipoproteins. Elevated concentrations of low density lipoproteins (LDL) in the plasma represent a primary risk factor for atherosclerotic heart disease. Since DL are formed in the plasma compartment exclusively through the intravascular catabolism of VLDL, an understanding of the intracellular events in the assembly pathway of VLDL may prove important in developing new treatment strategies for patients with elevated levels of LDL and at risk for atherosclerotic heart disease.