Functional Characterization of an ER-Resident Ubiquitin Ligase in Yeast

描述(由申请人提供)：蛋白质组的结构完整性对所有活细胞都是极其重要的。内质网(ER)负责蛋白质的正确折叠和传递到分泌途径。在内质网中，蛋白质受到一个复杂的校对系统的影响，该系统区分正确折叠和末端错误折叠的物种。没有通过质量控制标准的蛋白质被转移到内质网相关降解(ERAD)途径。ERAD涉及底物的泛素化和逆转位，穿过内质网进入胞浆，随后被泛素蛋白酶体系统降解。然而，关于这些过程的机械细节仍然定义不清。目前尚不清楚“异常”蛋白质与“正常”蛋白质的区别，但泛素连接酶被认为起着核心作用。此外，关于底物逆转移位的机制、所需成分和能量的细节还缺乏。鉴于这一系统的错误调节与包括癌症、神经退行性疾病、囊性纤维化和糖尿病在内的许多人类疾病有关，对ERAD的更完整的机制了解是促进人类健康的高度优先事项。关于ERAD的许多已知知识是通过利用萌芽酵母酿酒酵母作为模式生物的开创性研究发现的。Doa10是两个保守的内质网驻留泛素连接酶之一，在酵母中协调ERAD。Doa10与其人类同源物TEB4/MARCH6具有相似的形态和底物特异性，使其研究具有很高的相关性。拟议的研究培训计划旨在解决基本问题，这些问题仍然存在，涉及底物选择和从ER回转的机制细节，使用酵母作为模型系统。遗传和生化方法将被用作两种互补的方法来定位Doa10泛素连接酶和ERAD底物之间的相互作用界面。这一目标的成功完成将提供对如何选择ERAD底物进行降解的机械性见解。最后，将重构无细胞生化系统中的内质网提取过程，以剖析所涉及的详细分子事件。最终，对ERAD的更全面的了解将有望为开发治疗与这一途径相关的越来越多的人类疾病的新疗法铺平道路。 公共卫生相关性：内质网(ER)中错误折叠和异常蛋白质的选择性降解对于细胞的正常运作是必不可少的。这一过程的失调与越来越多的人类疾病有关，包括癌症、神经退行性疾病、囊性纤维化和糖尿病，这使得对其的详细了解成为促进人类健康的高度优先事项。拟议项目的目的是提高我们对蛋白质在内质网中如何选择和降解的机械理解，希望为疾病干预确定新的治疗靶点。

DESCRIPTION (provided by applicant): The structural integrity of the proteome is of utmost importance to all living cells. The endoplasmic reticulum (ER) is responsible for the proper folding and delivery of proteins to the secretory pathway. Within the ER, proteins are subjected to a sophisticated proofreading system that discriminates between properly folded and terminally misfolded species. Proteins which do not pass quality control standards are diverted into the ER- associated degradation (ERAD) pathway. ERAD involves ubiquitination and retrotranslocation of substrates across the ER membrane into the cytosol for subsequent degradation by the ubiquitin proteasome system. However, the mechanistic details regarding these processes remain ill defined. It is not understood how "aberrant" proteins are distinguished from "normal" ones but ubiquitin ligases are believed to play a central role. Furthermore, the details involving the mechanics, required components and energetics of substrate retrotranslocation are lacking. Given that the misregulation of this system is linked to a number of human ailments which include cancer, neurodegenerative disorders, cystic fibrosis and diabetes, a more complete mechanistic understanding of ERAD is a high priority for the advancement of human health. Much of what is known about ERAD was discovered by pioneering studies utilizing the budding yeast, Saccharomyces cerevisiae, as a model organism. Doa10 is one of two well-conserved ER-resident ubiquitin ligases that coordinate ERAD in yeast. Doa10 shares similar morphology and substrate specificity with its human orthologue, TEB4/MARCH6, making its study highly relevant. The proposed research training program is designed to address fundamental questions which remain regarding the mechanistic details of substrate selection and retrotranslocation from the ER using yeast as a model system. Both genetic and biochemical methods will be utilized as two complementary approaches to map the interaction interface between the Doa10 ubiquitin ligase and an ERAD substrate. Successful completion of this aim will provide mechanistic insights into how ERAD substrates are selected for degradation. Finally, the process of ER-extraction in a cell free biochemical system will be reconstituted in order to dissect the detailed molecular events involved. Ultimately, a more comprehensive understanding of ERAD will hopefully pave the way for the development of novel therapeutics for treatment of the expanding number of human disorders associated with this pathway. PUBLIC HEALTH RELEVANCE: The selective degradation of mis-folded and aberrant proteins at the endoplasmic reticulum (ER) is essential for proper functioning of the cell. Dysregulation of this process is associated with an expanding number of human ailments which include cancer, neurodegenerative disorders, cystic fibrosis and diabetes, which makes its detailed understanding a high priority for the advancement of human health. The objective of the proposed project is to improve our mechanistic understanding of how proteins are selected and degraded at the ER with the hope of identifying novel therapeutic targets for disease intervention.