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）负责蛋白质的适当折叠和传递到分泌途径。在内质网内，蛋白质受到复杂的校对系统的影响，该系统区分正确折叠和最终错误折叠的物种。未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录，通过内质网膜进入细胞质，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。目前尚不清楚“异常”蛋白与“正常”蛋白的区别，但普遍认为泛素连接酶起着核心作用。此外，还缺乏涉及底物反转位的力学、所需组分和能量学的细节。鉴于该系统的失调与包括癌症、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，因此对ERAD的更完整的机制理解是促进人类健康的重中之重。关于ERAD的许多已知信息是通过利用出芽酵母（酿酒酵母）作为模式生物的开创性研究发现的。Doa10是酵母中协调ERAD的两个保守的er驻留泛素连接酶之一。Doa10与其人类同源物TEB4/MARCH6具有相似的形态和底物特异性，因此其研究具有高度相关性。拟议的研究培训计划旨在解决有关底物选择和内质网逆转录的机制细节的基本问题，这些问题以酵母为模型系统。遗传和生化方法将被用作两种互补的方法来绘制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为如何选择ERAD底物进行降解提供机制见解。最后，将重构无细胞生化系统中er提取的过程，以详细剖析所涉及的分子事件。最终，对ERAD更全面的了解将有望为开发新的治疗方法铺平道路，以治疗与该途径相关的越来越多的人类疾病。

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.