Elucidating role of UBIAD1 in sterol-accelerated ERAD of HMG CoA reductase

描述(申请人提供)：HMG CoA还原酶催化HMG CoA转化为甲氧丙戊酸，这是合成胆固醇和必需的非甾醇异戊二烯的限速步骤。还原酶通过N-末端结构域固定在内质网(ER)膜上，N-末端结构域包含8个跨膜螺旋；C-末端结构域投射到胞浆中并发挥催化活性。加速的内质网相关降解(ERAD)是还原酶反馈控制的几种机制之一，它是由类固醇诱导的还原酶与内质网膜蛋白Insig-1和Insig-2结合的结果。还原酶被INSIG相关的泛素连接酶泛素化，标志着该酶可以逆转位穿过内质网，并错位到胞浆中进行蛋白酶体降解。非甾醇类异戊二烯香叶基香叶醇通过一种完全未知的机制来调节这些后激素化步骤。在初步研究中，我们发现，甾醇还可以触发还原酶与含UbiA戊烯基转移酶结构域蛋白-1(UBIAD1)的结合，从而催化细菌和植物维生素K衍生物的香叶基香叶化反应，产生孟喹酮-4(MK-4)。UBIAD1基因突变导致一种罕见的常染色体显性遗传性眼病，称为施奈德角膜营养不良(SCD)，其特征是胆固醇在角膜中进行性积累。香叶醇抑制了固醇诱导的还原酶与UBIAD1的结合，RNA干扰研究表明，还原酶与UBIAD1的结合导致了还原酶的Insig募集和ERAD。因此，我们假设1)UBIAD1介导了还原酶-INSIG结合所需的甾醇敏感反应；2)香叶醇介导的UBIAD1置换是还原酶ERAD中启动后素化步骤所必需的。为了验证这些假说，我们提出了三个具体的目标：1)阐明UBIAD1介导固醇加速的还原酶ERAD的机制；2)探索固醇调节UBIAD1功能的机制；3)确定UBIAD1在整个动物的还原酶ERAD和胆固醇稳态调节中的作用。总而言之，这些研究将提供有关从内质网膜上去除多聚蛋白以降解蛋白酶体的关键信息。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标，他汀类药物是广泛使用的处方药 这可以降低血浆低密度脂蛋白-胆固醇，降低心血管疾病的发生率。他汀类药物引发的反应会导致还原酶的积累，从而削弱其临床效果。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的机制有望开发出新的治疗方法，提高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的机制可能导致延缓或防止与SCD相关的角膜胆固醇积聚的治疗干预。

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.