Assembly Pathway of VLDL-A Three Step Process

描述（由申请人提供）：拟议研究的长期目标是开发肝脏组装极低密度脂蛋白（VLDL）的模型。VLDL组装的主流模型是一个两步模型，其中粗内质网（ER）中形成一个小的、致密的、富含磷脂的、含载脂蛋白的颗粒（步骤1），然后在内质网中添加大块甘油三酯（步骤2）。我们实验室的研究表明，内质网内的组装并不完成，并导致了我们的工作假设：VLDL在肝脏中的组装分为三个步骤，前两个步骤发生在内质网，最后一步，甘油三酯和磷脂的添加，发生在到达或进入高尔基体的途中。为了验证这一假设，我们提出了以下具体目标：确定高尔基体在VLDL组装第三步中的作用。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VLDL脂化的机制，并评估微粒体甘油三酯转移蛋白（MTP）在这一过程中的作用。具体目标2。评估从内质网到高尔基体的水泡运输过程中VLDL组装的贡献。我们的假设是，在将新生粒子从内质网运送到高尔基体的过程中，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。具体目标#3。明确apoE在VLDL组装中的作用。ApoE缺乏使肝脏甘油三酯分泌减少50%，这与我们对ER后加入形成VLDL的甘油三酯质量的估计相符。这些研究将验证apoE调节甘油三酯细胞内分布并影响新生脂蛋白脂化的假设。血浆中低密度脂蛋白（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VLDL的血管内分解代谢在血浆室中形成，因此了解VLDL组装途径中的细胞内事件可能对LDL水平升高和有动脉粥样硬化性心脏病风险的患者制定新的治疗策略具有重要意义。

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.