Protein degradation and cholesterol regulation

描述(申请人提供)：(蛋白质降解和胆固醇调节)HMG-CoA还原酶(HMGR)是类固醇途径的关键酶，产生各种必要的分子。HMGR是一种完整的膜ER蛋白，受ER相关降解(ERAD)介导的调节性破坏。我们最初的发现是，HMGR调节的降解在酵母中是保守的，这使得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类固醇途径的调节。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。依赖于HRD的Hmg2的降解受固醇途径分子法尼基焦磷酸(FPP)水平的控制：FPP的升高导致更多的进入HRD降解途径。我们在了解HRD机制如何识别Hmg2和其他底物，以及类固醇途径如何控制正常蛋白质Hmg2进入HRD质量控制途径方面取得了实质性进展。在过去的资助期间，我们发现酵母和哺乳动物系统之间有显著的相似之处，包括信号的性质，ERAD作为降解介质的使用，保守基序的参与，以及INSIGs的参与传递甾醇控制。利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机制和类固醇途径信号的平行研究。我们将1)继续我们对HRD E3连接酶复合体的研究，重点了解错误折叠的膜蛋白检测和HRD复合体调控的机制，2)使用我们团队开发的一种新的体外分析方法，结合遗传学和蛋白质组学方法来分析Hmg2从ER膜反向易位的机制，以识别这一仍然神秘的过程中的参与分子，3)研究Hmg2的特性，允许通过类固醇途径信号进行调节-特别是Hmg2的高度保守的类固醇敏感结构域(SSD)和我们发现的部分类固醇介导的Hmg2降解控制，4)发现控制Hmg2 ERAD的类固醇途径信号的性质和作用，检验GGPP是实际的FPP来源的降解信号的假设，以及GGPP导致Hmg2经历调节的错误折叠以触发HRD途径进入的模型。这些研究提供了双重好处，揭示了细胞用来测量和修改固醇合成的策略，以及具有重大基础和生物医学兴趣的蛋白质质量控制途径的性质。

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.