Dissecting ER-Associated Degradation of a Membrane Protein in Drosophila S2 Cells

剖析果蝇 S2 细胞中内质网相关的膜蛋白降解

基本信息

批准号：
8494639
负责人：
Russell Alfred DeBose-Boyd
金额：
$ 29.62万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8494639
关键词：
26S proteasome Address Binding Biochemical Genetics Biological Assay Biological Models Biology Blood Cells Chimeric Proteins Cholesterol Cholesterol Homeostasis Clinical Co-Immunoprecipitations Coenzyme A Complex Coronary Arteriosclerosis Cytosol Drosophila genome Drosophila genus Drug Prescriptions Effectiveness Endoplasmic Reticulum Ensure Enzymes Family Feedback Genes Grant Incidence LDL Cholesterol Lipoproteins Laboratories Lead Luciferases Mammalian Cell Mediating Membrane Membrane Proteins Metabolism Methods Molecular Chaperones Myocardial Infarction N-terminal Oxidoreductase Pharmaceutical Preparations Process Proteasome Inhibition Proteins RNA Interference Reaction Regulation Reporting Role Specificity Sterols System Techniques Transgenes Ubiquitination genome-wide improved inhibitor/antagonist insight isoprenoid mammalian genome mevalonate novel overexpression public health relevance reconstitution response therapy development tool ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): In mammalian cells, the enzyme HMG CoA reductase catalyzes reduction of HMG CoA to mevalonate, a rate-determining step in the synthesis of cholesterol and non-sterol isoprenoids. Reductase is integrated into the ER membrane through an N-terminal domain that contains eight membrane-spanning helices. The C-terminus of reductase projects into the cytosol and exerts catalytic activity. End-products of mevalonate metabolism accelerate ER-associated degradation (ERAD) of reductase as part of a complex feedback system that ensures cholesterol homeostasis. Excess sterols cause binding of the membrane domain of reductase to ER membrane proteins called Insig-1 and Insig-2, resulting in the poly-ubiquitination of reductase. This ubiquitination is obligatory for recognition and delivery of reductase to cytosolic 26S proteasomes for degradation. The reaction has been reconstituted in Drosophila S2 cells by overexpressing the membrane domain of mammalian reductase and Insig-1 or Insig-2. As a model system to study fundamental questions in biology, S2 cells offer a number of advantages. For example, transgenes can be easily overexpressed in S2 cells for study of their function and RNAi is simpler and much more effective in S2 cells than in mammalian cells. To gain further insight into mechanisms for Insig-mediated degradation of reductase, we propose three specific aims: 1) Determine mechanism for sterol-accelerated degradation of mammalian HMG CoA reductase in S2 cells; 2) Define role of Hrd1 ubiquitin ligase complex components in sterol-accelerated degradation of reductase in mammalian cells; and 3) Identify novel genes required for degradation of reductase through a genome-wide RNAi screen in S2 cells. Collectively, these studies will provide crucial information regarding mechanisms for degradation of reductase and other polytopic proteins from the ER. In addition, these studies have significant clinical implications. Reductase is the target of statins, a family of widely prescribed drugs that lower blood LDL-cholesterol and reduce the incidence of coronary artery disease. Statins trigger responses that lead to accumulation of active reductase protein, thereby blunting their effects. Part of this increase is due to slowed degradation of reductase. Thus, elucidating mechanisms for the ERAD of reductase may lead to new therapies that increase the effectiveness of statins and ultimately reduce the incidence of heart attacks.

描述（由申请人提供）：在哺乳动物细胞中，HMG COA还原酶催化HMG COA将HMG COA催化为甲甲酸盐，这是合成胆固醇和非蛋白酶类异on-的速率确定步骤。还通过包含八个膜旋转螺旋的N末端结构域将还原酶整合到ER膜中。还原酶的C末端投射到细胞质中并发挥催化活性。甲谷酸酯代谢的终产物加速了与ER相关的还原酶降解（ERAD），这是确保胆固醇稳态的复杂反馈系统的一部分。过量的固醇会导致还原酶的膜结构域与ER膜蛋白的结合称为Insig-1和Insig-2，从而导致还原酶的多泛素化。这种泛素化是必须识别和递送还原酶至胞质26S蛋白酶体降解的。通过过表达哺乳动物还原酶和Insig-1或Insig-2的膜结构域，该反应已在果蝇S2细胞中重构。作为研究生物学基本问题的模型系统，S2细胞提供了许多优势。例如，可以在S2细胞中轻易表达转基因以研究其功能，而RNAi在S2细胞中比在哺乳动物细胞中更有效。为了进一步了解减少还原酶促进降解的机制，我们提出了三个具体目的：1）确定S2细胞中固醇加速型固醇加速降解的机制； 2）定义HRD1泛素连接酶复合成分在哺乳动物细胞中还原酶还原酶降解中的作用； 3）确定通过S2细胞中的全基因组RNAi筛选降解还原酶所需的新基因。总的来说，这些研究将提供有关从ER降解的还原酶和其他多重蛋白质降解机制的关键信息。此外，这些研究具有显着的临床意义。还原酶是他汀类药物的靶标，他是一个普通处方药的家族，可降低血液LDL-胆固醇并降低冠状动脉疾病的发生率。他汀类药物的反应会导致积累活性还原酶蛋白，从而使它们的作用变钝。这种增加的一部分是由于还原酶的降解减轻了。因此，阐明还原酶省略的机制可能会导致新的疗法，从而提高他汀类药物的有效性并最终降低心脏病发作的发病率。