Genetic Requirements for Protein Degradation at the Eukaryotic Translocon

描述（由申请人提供）：积聚在内质网（ER）的错误折叠和潜在有害蛋白质通过内质网相关降解（ERAD）的几个分支之一被破坏。最近在酿酒酵母中发现的ERAD-T（转座子）途径可以催化破坏持续或异常地参与内质网转座子（负责将蛋白质穿过内质网膜的通道）的蛋白质。ERAD-T是由Hrd1泛素连接酶介导的，在内质网应激（内质网中未折叠蛋白的负担升高）条件下，通过未表征的内质网应激反应特异性受损。最近的证据强烈表明，低密度脂蛋白（LDL）的蛋白质成分，即。“坏胆固醇”)在其转运到内质网的过程中受阻，就会通过erd - t进行降解。此外，许多疾病都与内质网长期应激有关（包括某些癌症、神经退行性疾病和免疫疾病）。药理学上操纵蛋白折叠和内质网的质量控制能力可能是这种情况的常见治疗策略。与国家普通医学科学研究所的使命一致，拟议工作的长期目标是增进对根除淋巴细胞白血病的了解，这是一种几乎一无所知的细胞质量控制机制。对这一途径的深入了解极有可能为理解低密度脂蛋白生理学和改进胆固醇和内质网应激相关病理的治疗策略的发展提供信息。这些研究将解决两个假设：(1)Hrd1泛素连接酶与转座子的相互作用是ERAD-T底物降解所必需的；(2)一种新型内质网应激传感机制的组成部分参与了ERAD-T，并在ERAD-T应激条件下限制了ERAD-T。这个项目的具体目标是：(1)识别特异性损害根除- t和

DESCRIPTION (provided by applicant): Misfolded and potentially harmful proteins that accumulate at the endoplasmic reticulum (ER) are destroyed via one of several branches of ER-associated degradation (ERAD). The recently identified ERAD-T (translocon) pathway in Saccharomyces cerevisiae catalyzes the destruction of proteins that persistently or aberrantly engage the ER translocon (the channel responsible for moving proteins across the ER membrane). ERAD-T, which is mediated by the Hrd1 ubiquitin ligase, is specifically impaired under conditions of ER stress (an elevated burden of unfolded proteins in the ER) via an uncharacterized ER stress response. Recent evidence strongly suggests that the protein component of low-density lipoproteins (LDL, i.e. "bad cholesterol") is degraded via ERAD-T when its translocation into the ER has stalled. In addition, many diseases are associated with protracted ER stress (including certain cancers, neurodegenerative conditions, and immune disorders). Pharmacologically manipulating the protein folding and quality control capacity of the ER may be a common therapeutic strategy for such conditions. Consistent with the mission of the National Institute of General Medical Sciences, the long-term objective of the proposed work is an improved understanding of ERAD-T, a cellular quality control mechanism about which virtually nothing is known. Insights about this pathway are highly likely to inform an understanding of LDL physiology and the development of improved therapeutic strategies for cholesterol- and ER-stress-related pathologies. The proposed studies will address two hypotheses: (1) interaction of the Hrd1 ubiquitin ligase with the translocon is required for degradation of ERAD- T substrates, and (2) components of a novel ER stress-sensing mechanism participate in ERAD-T and become limiting for ERAD-T under ER stress conditions. The specific aims of this project are to (1) identify mutations that specifically impair ERAD-T and (2) investigate the relationship between ER stress and ERAD-T. Novel, genetic assays that indicate whether ERAD-T is functional on the basis of yeast cell growth will be conducted and biochemical analyses will be performed to identify and characterize the genetic requirements for ERAD-T. This work will expose undergraduates and master's students to meaningful, biomedically relevant research. Students will participate in every aspect of this project, includin experimental design and execution, interpretation of results, and data presentation. Students will gain valuable firsthand experience in biomedical research and have the chance to make new discoveries about an area of basic biology with important medical implications.