ASSEMBLY PATHWAY OF VLDL--A THREE STEP PROCESS

描述（改编自申请人摘要）：研究者希望 建立一个肝脏组装VLDL的模型。 他们的研究是 旨在定义VLDL组装的步骤。 这是他们 假设这个过程有三个步骤。 两个步骤发生在 粗面内质网（ER），产生富含磷脂酰肌醇的 脂蛋白 第三步包括加入甘油三酯和 磷脂的成熟颗粒和发生的颗粒是在路线 到高尔基体或高尔基体内部。 本提案中的研究将测试 以下假设与这些组装步骤相关：1）酰基CoA 胆固醇酰基转移酶（ACAT）和二酰基甘油酰基转移酶（DGAT） 为粗ER中的前两个组装步骤提供脂质。 （二） 甘油三酯和胆固醇酯被转移到粗糙的管腔 ER通过不需要微粒体甘油三酯转移的过程 蛋白（MTP）。 MTP将脂质从该内腔池转移。 道歉 的 池用于第二组装步骤中的大量核心脂质转移。 第三章 MTP以脂质形式与粗糙ER中小而致密颗粒上的apoB结合 合成得到加强。 4)甘油三酯和胆固醇的形成 酯促进载脂蛋白B从粗糙的内质网膜转移到 流明 5)来自光滑ER的甘油三酯和磷脂是 在不同的第三步中加入到形成的VLDL颗粒中， 从粗糙的内质网转移到高尔基体 6)ApoB-48对小脂蛋白的影响 还没有经历第二次或可能第三次的粒子， 磷酸化的高尔基体，保护它免受降解。 这些假设将通过以下具体目标进行检验：1） 定义了含有VLDL的apoB-100和apoB-48的组装步骤。 2)定义ACAT、DGAT和MTP在VLDL前两个步骤中的角色 组装件. 3)为了确定和阐明apoB磷酸化的作用， 含载脂蛋白B的脂蛋白的组装和分泌。 与人类疾病的相关性是明确的，因为它与VLDL的关系 和LDL代谢。 这些实验将进行，为特定的目的 1，大鼠将注射35 S-蛋氨酸或3 H-甘油。 他们 也可以使用标记的棕榈酸作为前体。 他们都将完成 用这些大鼠肝脏和分离的肝灌注研究 脂蛋白 将通过SDS-PAGE评估载脂蛋白。 的作用 ACAT、DGAT和MTP在装配过程中的作用将利用 粗糙的ER组分，可能来自大鼠肝细胞，尽管它不是 阐述 他们将使用MTP抑制剂BMS 200150进行额外的研究。 在具体的子目标2.1中，他们还将使用粗略的ER分数，因为他们 将针对次级目标2.2和2.3，审查中期计划是否与 在活性脂质合成过程中与特定脂蛋白类中的apoB结合， 脂质转移 在具体目标3中，他们将审查 apoB的磷酸化和apoB脂蛋白的分泌将使用大鼠 肝高尔基体富集组分。 他们将审查这些问题 apoB被高尔基体磷酸化的关键特征是什么， 磷酸化位点到底是什么 他们会问 apoB的磷酸化发生在什么样的脂蛋白上， 它出现在高尔基体的哪个部分。 他们会问 高尔基体中磷酸化的490 kD蛋白质是什么？ 这与apoB降解的关系。 他们将评估 apoB的磷酸化与其分泌之间的关系和/或 降解 他们将使用MCA细胞进行这些研究， 研究cAMP依赖性蛋白激酶抑制剂的作用， 双吲哚马来酰亚胺以及冈田酸，其抑制蛋白质 磷酸酶1A和2A。 具体的方法在更大的 在关于具体方法的单独章节中详细说明。 老鼠和细胞 培养实验是非常明确的，因为是拟议的时间表， 研究。

DESCRIPTION (Adapted from applicant's abstract): The investigators wish to develop a model for the assembly of VLDL by the liver. Their studies are designed to define the steps in the assembly of VLDL. It is their hypothesis that there are three steps in this process. Two steps occur in the rough endoplasmic reticulum (ER), producing triglyceride-rich lipoproteins. The third step involves addition of triglyceride and phospholipid to the maturing particle and occurs as the particle is in route to the Golgi or within the Golgi. The studies in this proposal will test the following hypotheses related to these assembly steps: 1) Acyl CoA cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) provide lipid for the first two assembly steps in the rough ER. 2) Triglyceride and cholesterol ester are transferred to the lumen of the rough ER by a process that does not require microsomal triglyceride transfer protein (MTP). MTP transfers lipid from this lumenal pool. to apoB. The pool is used for bulk core lipid transfer in the second assembly step. 3) MTP associates with apoB on small dense particles in the rough ER as lipid synthesis is augmented. 4) The formation of triglyceride and cholesterol ester promotes translocation of apoB from the rough ER membrane to the lumen. 5) Triglyceride and phospholipid derived from the smooth ER are added to the forming VLDL particle in a distinct third step as the particle moves from the rough ER to the Golgi. 6) ApoB-48 on small lipoprotein particles that have not yet undergone second or possibly third is phosphorylated in the Golgi, protecting it from degradation. These hypotheses will be tested with the following specific aims: 1) To define the steps in the assembly of apoB-100 and apoB-48 containing VLDL. 2) To define the roles of ACAT, DGAT, and MTP in the first two steps of VLDL assembly. 3) To determine and elucidate the role of apoB phosphorylation in the assembly and secretion of apoB-containing lipoproteins. The relevance to human disease is clear because of its relationships to VLDL and LDL metabolism. These experiments will be carried out, for specific aim 1, with rats that will be injected with 35S-methionine or 3H-glycerol. They may also use labeled palmitic acid as a precursor. They will carry out isolated liver profusion studies with these rat livers and isolate lipoproteins. Apolipoproteins will be assessed by SDS-PAGE. The role of ACAT, DGAT, and MTP in the assembly process will be investigated utilizing rough ER fractions, presumably from rat hepatocytes, although it is not stated. They will do additional studies using the MTP inhibitor BMS200150. In specific sub-aim 2.1, they will also use the rough ER fractions as they will for sub-aim 2.2 and 2.3, examining issues of whether MTP associates with apoB in a specific lipoprotein class during active lipid synthesis and lipid transfer. In specific aim 3, where they will examining the role of phosphorylation of apoB and the secretion of apoB lipoproteins will use rat hepatic Golgi apparatus-rich fractions. They will examine the questions about what are the key features of apoB phosphorylation by the Golgi and what the phosphorylation sites actually are. They will ask the question of on what lipoprotein species does this phosphorylation of apoB occur and in which compartment of the Golgi does it occur. They will ask the question of what is the 490 kD protein that is phosphorylated in the Golgi and what is the relationship of this to apoB degradation. They will assess the relationship between phosphorylation of apoB and its secretion and/or degradation. They will use MCA cells for these studies, and they will examine the effects of cAMP-dependent protein kinase inhibitor and look at bisindolylmaleimide as well as okadaic acid, which inhibits protein phosphatase 1A and 2A. Specific methods are spelled out in much greater detail in a separate section on specific methods. Both the rat and the cell culture experiments are very well defined as is the proposed time table for the studies.