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

描述(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，这是合成胆固醇和非甾醇异戊二烯的速率决定步骤。还原酶通过一个N-末端结构域整合到内质网膜上，该结构域包含八个跨膜螺旋。还原酶的C末端投射到胞浆中，发挥催化活性。甲羟戊酸代谢的最终产物加速了还原酶的内质网相关降解(ERAD)，这是确保胆固醇稳态的复杂反馈系统的一部分。过量的甾醇导致还原酶的膜域与内质网膜蛋白Insig-1和Insig-2结合，导致还原酶的多泛素化。这种泛素化是识别还原酶并将其传递到胞浆26S蛋白酶体以进行降解所必需的。该反应已在果蝇S2细胞中通过过表达哺乳动物还原酶的膜域和Insig-1或Insig-2而重新构建。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，转基因很容易在S2细胞中过表达，以研究其功能，而RNAi在S2细胞中比在哺乳动物细胞中更简单、更有效。为了进一步了解Insig介导的还原酶降解的机制，我们提出了三个具体的目标：1)确定S2细胞中类固醇加速降解哺乳动物HMG CoA还原酶的机制；2)确定Hrd1泛素连接酶复合体在哺乳动物细胞中类固醇加速还原酶降解中的作用；以及3)通过全基因组RNAi筛选在S2细胞中寻找降解还原酶所需的新基因。总而言之，这些研究将提供有关内质网中还原酶和其他多聚体蛋白降解机制的关键信息。此外，这些研究还具有重要的临床意义。还原酶是他汀类药物的靶点，他汀类药物是一类广泛使用的处方药，可以降低血液中的低密度脂蛋白-胆固醇，减少冠状动脉疾病的发生率。他汀类药物触发的反应导致活性还原酶蛋白的积累，从而削弱了它们的作用。这一增长的部分原因是还原酶降解缓慢。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。 与公众健康相关：胆固醇合成中的关键酶是HMG CoA还原酶，它通过包括调节蛋白质稳定性在内的多种机制受到严格控制。HMG CoA还原酶的竞争性抑制剂他汀类药物通常用于降低血液胆固醇，但它们会触发调节反应，导致还原酶蛋白的积累。这笔赠款将研究还原酶降解的机制，阐明这一机制将为开发抗他汀类药物诱导的还原酶积聚的治疗方法和提高药物的有效性提供洞察力。

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.