Protein degradation and cholesterol regulation

描述（由申请人提供）：（蛋白质降解和胆固醇调节）HMG-CoA还原酶（HMGR）是产生多种必需分子的固醇途径的关键酶。HMGR是一种整合的膜ER蛋白，并受到ER相关降解（ERAD）介导的调节性破坏。我们最初发现HMGR调节的降解在酵母中是保守的，这使我们能够使用独特的简便方法来解开HMGR ERAD及其通过固醇途径调节的潜在机制。酵母HMGR同工酶Hmg 2通过HRD质量控制途径经历泛素介导的ER降解。HRD依赖性Hmg 2降解受固醇途径分子焦磷酸法呢酯（FPP）水平控制：FPP升高导致进入HRD降解途径的增加。我们已经取得了实质性的进展，了解HRD机制如何识别Hmg 2和其他底物，以及甾醇途径如何控制Hmg 2（一种正常蛋白质）进入HRD质量控制途径。在过去的资助期间，我们发现了酵母和哺乳动物系统之间的显着相似之处，包括信号的性质，使用ERAD作为降解介质，保守基序的参与，以及INSIG的参与，以赋予甾醇控制。使用我们为这些研究开发的独特可用的工具，我们计划推进我们对HRD机制和甾醇途径信号传导的平行研究路径。我们将1）继续我们对HRD E3连接酶复合物的研究，重点是理解错误折叠膜蛋白检测的机制，以及HRD复合物的调节，2）使用我们小组开发的一种新的体外测定法分析Hmg 2从ER膜逆转位的机制，结合遗传和蛋白质组学方法来识别参与这个仍然神秘的过程的分子，3）研究Hmg 2允许通过固醇途径信号调节的特征-特别强调Hmg 2的高度保守的固醇传感结构域（SSD）和我们发现的赋予固醇介导的Hmg 2降解控制的INSIG Nsg 1，和4）发现控制Hmg 2 ERAD的固醇途径信号的性质和作用，测试GGPP是实际FPP的假设。推导的降解信号和模型，GGPP导致Hmg 2进行调节的错误折叠，触发HRD途径进入。这些研究提供了双重好处，揭示了细胞测量和修饰甾醇合成的策略，以及具有重大基础和生物医学意义的蛋白质质量控制途径的性质。 公共卫生相关性：HMG-CoA还原酶（HMGR）是胆固醇途径中的关键酶，从胆固醇途径产生固醇和多种其他必需分子。我们正在研究受调节的HMGR，通过该HMGR，来自固醇途径的信号控制HMGR的稳定性，从而控制HMGR的量。使用强大的酵母方法，我们发现HMGR是通过去除受损蛋白质的重要途径降解的，这是衰老和一般细胞应激综合征的关键过程。因此，我们的工作是教我们关于细胞用来破坏错误折叠蛋白质的途径，以及固醇途径如何通过这条途径控制HMGR的破坏。

DESCRIPTION (provided by applicant): (Protein Degradation and Cholesterol Regulation) HMG-CoA reductase (HMGR) is a key enzyme of the sterol pathway that produces a variety of essential molecules. HMGR is an integral membrane ER protein and is subject to regulated destruction mediated by ER-associated degradation (ERAD). Our initial discovery that HMGR regulated degradation is conserved in yeast has allowed us uses the uniquely facile approaches to unravel the underlying mechanisms of HMGR ERAD and its regulation by the sterol pathway. The yeast HMGR isozyme Hmg2 undergoes ubiquitin-mediated ER degradation by the HRD quality control pathway. HRD- dependent Hmg2 degradation is controlled by levels of the sterol pathway molecule farnesyl pyrophosphate (FPP): elevated FPP leads to increased entry into the HRD degradation pathway. We have made substantial progress towards understanding how the HRD machinery recognizes Hmg2 and other substrates, and how the sterol pathway controls entry of Hmg2, a normal protein, into the HRD quality control pathway. In the past funding period we have found remarkable similarities between the yeast and mammalian systems, including the nature of the signals, the use of ERAD as the degradative mediator, the involvement of conserved motifs, and the participation of INSIGs to impart sterol control. Using uniquely available tools we developed for these studies we plan to push forward our parallel paths of study on HRD mechanisms and sterol pathway signaling. We will 1) Continue our study of the HRD E3 ligase complex, focusing on understanding the mechanism of misfolded membrane protein detection, and HRD complex regulation, 2) Analyze the mechanism of Hmg2 retrotranslocation from the ER membrane using a new in vitro assay developed by our group, in conjunction with genetic and proteomic approaches to discern the participating molecules in this still-mysterious process, 3) Study the features of Hmg2 allowing regulation by sterol pathway signals - with a particular emphasis on the highly conserved sterol sensing domain (SSD) of Hmg2 and the INSIG Nsg1 that we have discovered imparts sterol-mediated control on Hmg2 degradation, and 4) Discover the nature and action of the sterol pathway signals that control Hmg2 ERAD testing the hypothesis that GGPP is the actual FPP-derived degradation signal and the model that GGPP causes Hmg2 to undergo regulated misfolding to trigger HRD pathway entry. These studies provides the double benefit of revealing the tactics employed by cells to measure and modify sterol synthesis, and the nature of a protein quality control pathway of great basic and biomedical interest. PUBLIC HEALTH RELEVANCE: HMG-CoA reductase (HMGR) is a key enzyme in the cholesterol pathway, from which sterols and a wide variety of other essential molecules are produced. We are studying the regulated HMGR, by which signals from the sterol pathway control the stability and thus the amount of HMGR. Using the powerful methods of yeast, we have discovered that HMGR is degraded by an important pathway for removal of damaged proteins, a critical process in syndromes of aging and general cell stress. Thus, our work is teaching us about both the pathways cells use to destroy misfolded proteins, and how sterol pathway control HMGR destruction by this route.