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）是固醇途径的关键酶，产生多种必需分子。HMGR是一种完整的膜内质网蛋白，受内质网相关降解（ERAD）介导的调节破坏。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揭示HMGR ERAD的潜在机制及其通过固醇途径的调节。酵母HMGR同工酶Hmg2通过HRD质量控制途径进行泛素介导的内质网降解。HRD依赖的Hmg2降解受甾醇途径分子法尼基焦磷酸（FPP）水平的控制：FPP升高导致进入HRD降解途径的增加。我们已经在了解HRD机制如何识别Hmg2和其他底物，以及甾醇途径如何控制Hmg2（一种正常蛋白）进入HRD质量控制途径方面取得了实质性进展。在过去的资助期内，我们发现酵母和哺乳动物系统之间存在显著的相似之处，包括信号的性质、ERAD作为降解介质的使用、保守基元的参与以及insg参与给予固醇控制。利用我们为这些研究开发的独特可用工具，我们计划推进HRD机制和甾醇途径信号传导的平行研究。我们将继续对HRD E3连接酶复合物的研究，重点了解错误折叠膜蛋白的检测机制和HRD复合物的调控。2)利用我们团队开发的一种新的体外实验，结合遗传学和蛋白质组学方法，分析Hmg2从内质网膜逆转录易位的机制，以识别参与这一仍然神秘的过程的分子。3)研究Hmg2允许甾醇通路信号调控的特征，特别强调Hmg2高度保守的甾醇敏感域（SSD）和我们发现的对Hmg2降解具有甾醇介导控制作用的insg Nsg1。4)发现控制Hmg2 ERAD的甾醇通路信号的性质和作用，验证了GGPP是fpp衍生的实际降解信号的假设，以及GGPP导致Hmg2经过调控的错误折叠触发HRD通路进入的模型。这些研究提供了双重好处，揭示了细胞用来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本质。

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.