Elucidation Role of UBIAD1 in Sterol-Accelerated ERAD of HMG CoA Reductase

描述（由申请人提供）：HMG CoA还原酶催化HMG CoA转化为甲羟戊酸，这是胆固醇和必需非固醇类异戊二烯合成的限速步骤。还原酶通过含有八个跨膜螺旋的N-末端结构域锚定到内质网（ER）膜; C-末端结构域投射到细胞溶质中并发挥催化活性。加速ER相关降解（ERAD）是还原酶反馈控制的几种机制之一，是由甾醇诱导的还原酶与称为Insig-1和Insig-2的ER膜蛋白结合引起的。Insig相关的泛素连接酶对还原酶的泛素化标记了该酶穿过ER膜的逆转位和移位到细胞溶质中以进行蛋白酶体降解。这些postubiquitination步骤是通过一个完全未知的机制由非甾醇类异戊二烯香叶基香叶醇调制。在初步研究中，我们发现甾醇还触发还原酶与UbiA异戊烯基转移酶结构域蛋白1（UBIAD 1）的结合，UBIAD 1催化细菌和植物维生素K衍生物的香叶基香叶基化以产生甲基萘醌-4（MK-4）。UBIAD 1的突变导致一种罕见的常染色体显性眼病，称为施奈德角膜营养不良（SCD），其特征是角膜中胆固醇的进行性积累。甾醇诱导的还原酶与UBIAD 1的结合被香叶基香叶醇抑制，RNA干扰研究表明还原酶-UBIAD 1结合导致还原酶的Insig募集和ERAD。因此，我们假设：1）UBIAD 1介导还原酶-Insig结合所需的甾醇敏感反应; 2）还原酶ERAD中启动后泛素化步骤需要香叶基香叶醇介导的UBIAD 1置换。为了评价这些假设，我们提出了三个具体的目的：1）阐明UBIAD 1介导还原酶的甾醇加速ERAD的机制; 2）探索甾醇调节UBIAD 1功能的机制; 3）确定UBIAD 1在整个动物中还原酶的ERAD和胆固醇稳态调节中的作用。总的来说，这些研究将提供关于从ER膜中去除蛋白酶体降解的多位蛋白的关键信息。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， 降低血浆低密度脂蛋白胆固醇和降低心血管疾病的发病率。他汀类药物引发的反应，导致还原酶的积累，这削弱了他们的临床效果。这种增加的部分原因是还原酶的ERAD减慢。因此，阐明还原酶ERAD的机制有望开发新的治疗方法，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。此外，深入了解还原酶ERAD的机制可能会导致治疗干预，延缓或防止与SCD相关的胆固醇的角膜积聚。

DESCRIPTION (provided by applicant): The enzyme HMG CoA reductase catalyzes conversion of HMG CoA to mevalonate, a rate-limiting step in synthesis of cholesterol and essential nonsterol isoprenoids. Reductase is anchored to endoplasmic reticulum (ER) membranes through an N-terminal domain that contains eight membrane-spanning helices; the C- terminal domain projects into the cytosol and exerts catalytic activity. Accelerated ER-associated degradation (ERAD), one of several mechanisms for feedback control of reductase, results from sterol-induced binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Ubiquitination of reductase by Insig-associated ubiquitin ligases marks the enzyme for retrotranslocation across ER membranes and dislocation into the cytosol for proteasomal degradation. These postubiquitination steps are modulated by the nonsterol isoprenoid geranylgeraniol through a completely unknown mechanism. In preliminary studies, we find that sterols also trigger binding of reductase to UbiA prenyltransferase domain containing protein-1 (UBIAD1), which catalyzes geranylgeranylation of bacterial and plant vitamin K derivatives to produce menaquinone-4 (MK-4). Mutations in UBIAD1 cause a rare autosomal dominant eye disease called Schnyder corneal dystrophy (SCD), which is characterized by progressive accumulation of cholesterol in the cornea. Sterol-induced binding of reductase to UBIAD1 is inhibited by geranylgeraniol and RNA interference studies reveal that reductase-UBIAD1 binding leads to Insig recruitment and ERAD of reductase. Thus, we hypothesize that 1) UBIAD1 mediates the sterol-sensing reaction required for reductase-Insig binding; and 2) geranylgeraniol-mediated displacement of UBIAD1 is required for initiation of postubiquitination steps in reductase ERAD. To appraise these hypotheses, we propose three Specific Aims: 1) delineate mechanisms by which UBIAD1 mediates sterol-accelerated ERAD of reductase; 2) explore mechanism through which sterols modulate function of UBIAD1; and 3) determine the role of UBIAD1 in ERAD of reductase and regulation of cholesterol homeostasis in whole animals. Collectively, these studies will provide key information regarding the removal of polytopic proteins from ER membranes for proteasomal degradation. In addition, these studies have significant clinical implications. Reductase is the target of statins, widely prescribed drugs that lower plasma LDL-cholesterol and reduce the incidence of cardiovascular disease. Statins trigger responses that cause accumulation of reductase, which blunts their clinical effects. Part of this increase results from slowed ERAD of reductase. Thus, elucidating mechanisms for reductase ERAD holds promise for development of new therapies that increase the effectiveness of statins and ultimately reduce the incidence of heart attacks. Moreover, insight into mechanisms for reductase ERAD may lead to therapeutic interventions that retard or prevent corneal accumulation of cholesterol associated with SCD.