Discovering the machinery and mechanism of ERAD-M retrotranslocation

描述（由申请人提供）：通过内质网相关蛋白降解（ERAD）消除错误折叠的蛋白质，确保进入分泌途径的蛋白质被正确折叠，并将内质网应激维持在可接受的低水平。ERAD的所有变异都包括一个被称为逆转录的蛋白质易位过程，在此过程中，泛素化的ERAD底物在被胞质26S蛋白酶体降解之前，通过专用机器从内质网选择性地提取出来。该项目的总体目标是揭示逆转录易位的未知机制。在发现保守的HRD ERAD通路的过程中，我们的实验室开发了各种独特的工具、技术和专业知识，这些工具、技术和专业知识将用于解决逆转录易位这个紧迫而开放的问题。具体来说，我将1)对典型ERAD底物HMG-CoA还原酶进行生化和蛋白质组学分析，以了解其从内质网逆转录的机制，并确定参与该过程的关键蛋白。此外，我将2)采用遗传方法作为一种独立和互补的方法来识别逆转录易位因素。它们将包括运行传统的和基于阵列的筛选，并结合使用直接体内和体外分析对候选基因进行验证。最后，我将在ERAD的每个已知分支中识别所有已发现的因素的作用。综上所述，这些研究将揭示ERAD核心的一个关键、普遍和保守的过程，该过程涉及管理细胞应激和各种临床疾病。我在酵母中的研究将为ERAD的逆转录过程提供新的基础知识。ERAD与衰老、阿尔茨海默病、亨廷顿病、帕金森病等疾病以及正常的固醇调节有关，我的研究有助于了解ERAD缺陷如何与这些过程和疾病相关。

DESCRIPTION (provided by applicant): Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptable low levels. All variations of ERAD includes a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD subsrates are selectively extracted by dedicated machinery from the ER before degradation by the cytosolic 26S proteasome. The overall goal of this project is to unravel the unknown mechanism of retrotranslocation. In the course of discovering the conserved HRD ERAD pathway, our laboratory has developed a variety of unique tools, techniques and expertise that will be brought to bear on the pressing and open question of retrotranslocation. Specifically, I will 1) perform biochemical and proteomic analyses on the prototypical ERAD substrate HMG-CoA reductase to understand the mechanism of its retrotranslocation from the ER and identify the key proteins involved in this process. Furthermore, I will 2) employ a genetic approach as an independent and complementary approach to identify retrotranslocation factors. They will include running traditional and array-based screens, combined with validation of candidate genes using direct in vivo and in vitro assays. Finally, 3) I will discern the roles of all discoveed factors in each known branch of ERAD. Taken together, these studies will reveal a key, universal and conserved process at the heart of ERAD involved in managing cell stress and a variety of clinical maladies. My studies in yeast will provide a new fundamental knowledge of the retrotranslocation process of ERAD. ERAD has been implicated in diseases such as aging, Alzheimer's disease, Huntington's disease, Parkinson's disease and in normal sterol regulation, my studies are pertinent for understanding how defects in ERAD are associated with these processes and maladies.