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

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还原为甲羟戊酸，这是胆固醇和非固醇类异戊二烯合成中的速率决定步骤。还原酶通过含有八个跨膜螺旋的N-末端结构域整合到ER膜中。还原酶的C-末端突出到细胞质中并发挥催化活性。甲羟戊酸代谢的终产物加速还原酶的ER相关降解（ERAD），作为确保胆固醇稳态的复杂反馈系统的一部分。过量的甾醇导致还原酶的膜结构域与称为Insig-1和Insig-2的ER膜蛋白结合，导致还原酶的多聚泛素化。这种泛素化对于还原酶的识别和递送到胞质26 S蛋白酶体进行降解是必需的。通过过表达哺乳动物还原酶和Insig-1或Insig-2的膜结构域，在果蝇S2细胞中重建了该反应。作为研究生物学基本问题的模型系统，S2细胞具有许多优势。例如，转基因可以很容易地在S2细胞中过表达以研究其功能，并且RNAi在S2细胞中比在哺乳动物细胞中更简单且更有效。为了进一步了解Insig介导的还原酶降解机制，我们提出了三个具体目标：1）确定哺乳动物HMG CoA还原酶在S2细胞中的甾醇加速降解机制; 2）确定Hrd 1泛素连接酶复合物组分在哺乳动物细胞中甾醇加速还原酶降解中的作用;和3）在S2细胞中通过全基因组RNAi筛选鉴定还原酶降解所需的新基因。总的来说，这些研究将提供有关还原酶和其他多位蛋白从ER降解机制的重要信息。此外，这些研究具有重要的临床意义。还原酶是他汀类药物的靶点，他汀类药物是一种广泛使用的处方药，可以降低血液中的LDL-胆固醇，降低冠状动脉疾病的发病率。他汀类药物引发的反应，导致活性还原酶蛋白的积累，从而削弱其作用。这种增加的部分原因是由于还原酶的降解减缓。因此，阐明还原酶ERAD的机制可能会导致新的治疗方法，增加他汀类药物的有效性，并最终降低心脏病发作的发生率。 公共卫生相关性：胆固醇合成的关键酶是HMG CoA还原酶，它通过多种机制（包括调节蛋白质稳定性）受到严格控制。HMG CoA还原酶的竞争性抑制剂称为他汀类药物，通常用于降低血液胆固醇，但它们会引发导致还原酶蛋白积累的调节反应。这项资助将研究还原酶降解的机制，其阐明将为开发治疗方法提供深入了解，以抵消他汀类药物诱导的还原酶积累并提高药物的有效性。

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. PUBLIC HEALTH RELEVANCE: The key enzyme in cholesterol synthesis is HMG CoA reductase, which is tightly controlled through multiple mechanisms that includes regulation of protein stability. Competitive inhibitors of HMG CoA reductase called statins are routinely used to lower blood cholesterol, but they trigger regulatory responses that lead to the accumulation of reductase protein. This grant will investigate the mechanism for the degradation of reductase, the elucidation of which will provide insight into development of therapies to counteract statin-induced accumulation of reductase and improve the effectiveness of the drugs.