ASSEMBLY PATHWAY OF VLDL--A THREE STEP PROCESS

描述(改编自申请人的摘要)：调查人员希望 建立肝脏组装极低密度脂蛋白的模型。他们的研究是 旨在定义极低密度脂蛋白组装中的步骤。这是他们的 假设这个过程有三个步骤。两个步骤发生在 粗面内质网(ER)，产生富含甘油三酯 脂蛋白。第三步是添加甘油三酯和 磷脂到成熟的颗粒，并在颗粒的路线上发生 到高尔基山脉或在高尔基山脉内。这项提案中的研究将测试 与这些组装步骤相关的下列假设：1)酰基辅酶A 胆固醇酰基转移酶(ACAT)和甘油二酰基转移酶(DGAT) 为粗略ER中的前两个组装步骤提供脂质。2) 甘油三酯和胆固醇酯转移到毛坯的管腔 通过不需要微粒体甘油三酯转移的过程 蛋白质(MTP)。MTP从这个管腔池中转移脂质。致载脂蛋白B。这个 在第二组装步骤中，POOL用于散装核心脂质转移。3) MTP与载脂蛋白B在粗面内质网小而致密的颗粒上以脂质的形式结合 综合能力得到了增强。4)甘油三酯和胆固醇的形成 Ester促进apoB从粗大的内质网膜向 流明。5)来自光滑内质网的甘油三酯和磷脂是 以不同的第三步添加到形成的极低密度脂蛋白颗粒中 从粗糙的急诊室到高尔基。6)载脂蛋白B-48对小脂蛋白的影响 尚未经历第二次或可能第三次的粒子是 在高尔基体中被磷酸化，保护它不被降解。 这些假设将以以下具体目标进行测试：1) 定义含有极低密度脂蛋白的apoB-100和apoB-48的组装步骤。 2)确定ACAT、DGAT和MTP在极低密度脂蛋白前两步中的作用 集合。3)确定和阐明载脂蛋白B磷酸化在 含有载脂蛋白B的脂蛋白的组装和分泌。 与人类疾病的相关性是显而易见的，因为它与极低密度脂蛋白有关 和低密度脂蛋白代谢。这些实验是为了特定的目的而进行的 1，大鼠注射35S-蛋氨酸或~3H-甘油。他们 也可以使用标记棕榈酸作为前体。他们将执行 用这些大鼠肝脏和分离株进行分离的肝血流灌注研究 脂蛋白。用SDS-PAGE分析载脂蛋白。的作用 将使用以下工具调查组装过程中的ACAT、DGAT和MTP 粗略的内质网组分，推测来自大鼠肝细胞，尽管它不是 已述明。他们将使用MTP抑制剂BMS200150进行进一步的研究。 在具体次级目标2.1中，他们还将使用粗略的ER分数，因为它们 将用于次级目标2.2和2.3，审查MTP是否与 在活跃的脂质合成过程中与特定脂蛋白类别的载脂蛋白B结合 脂类转移。在具体目标3中，他们将审查 载脂蛋白B的磷酸化和载脂蛋白的分泌将使用大鼠 肝脏高尔基体富含组分。他们将研究这些问题 关于高尔基人apoB磷酸化的关键特征是什么 真正的磷酸化位点是什么。他们会问这个问题 载脂蛋白B的这种磷酸化是在什么脂蛋白物种上发生的？ 它发生在高尔基的哪个隔间。他们会问这个问题 高尔基体中被磷酸化的490kD蛋白是什么？ 这与载脂蛋白B降解的关系。他们将评估 载脂蛋白B的磷酸化与其分泌和/或 退化。他们将使用MCA细胞进行这些研究，他们将 研究cAMP依赖的蛋白激酶抑制剂的作用 双吲哚马来酰亚胺和冈田酸，抑制蛋白质 磷酸酶1 A和2 A。具体的方法在更大的 在单独的章节中详细介绍具体的方法。无论是老鼠还是细胞 培养实验的定义非常明确，建议的时间表也是如此 这些研究。

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.