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至关重要。内质网（ER）负责蛋白质的正确折叠和递送到分泌途径。在内质网内，蛋白质受到一个复杂的校对系统的影响，该系统可以区分正确折叠和末端错误折叠的种类。不通过质量控制标准的蛋白质被转移到ER相关降解（ERAD）途径中。ERAD涉及底物的泛素化和逆转运穿过ER膜进入胞质溶胶，随后被泛素蛋白酶体系统降解。然而，关于这些过程的机制细节仍然不明确。目前还不清楚如何区分“异常”蛋白质与“正常”蛋白质，但泛素连接酶被认为起着核心作用。此外，涉及的机制，所需的组件和能量的基板retrotranslocation的细节是缺乏。鉴于该系统的失调与许多人类疾病有关，包括癌症，神经退行性疾病，囊性纤维化和糖尿病，因此更全面地了解ERAD的机制是促进人类健康的高度优先事项。许多关于ERAD的知识都是通过利用芽殖酵母（Saccharomyces cerevisiae）作为模式生物的开创性研究发现的。Doa 10是酵母中协调ERAD的两种高度保守的ER-驻留泛素连接酶之一。Doa 10与其人类直系同源物TEB 4/MARCH 6具有相似的形态学和底物特异性，使其研究高度相关。拟议的研究培训计划的目的是解决基本的问题，仍然关于底物选择和retrotranslocation从ER使用酵母作为模型系统的机制细节。遗传学和生物化学方法将作为两种互补的方法来映射Doa 10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如何选择ERAD底物降解的机理见解。最后，ER-提取的过程中，在无细胞的生化系统将重建，以解剖详细的分子事件所涉及的。最终，对ERAD的更全面的了解将有望为开发新的治疗方法铺平道路，用于治疗与该途径相关的越来越多的人类疾病。 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功能至关重要。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。该项目的目的是提高我们对蛋白质如何在ER中选择和降解的机制的理解，以期确定用于疾病干预的新型治疗靶点。

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.