Genetic Requirements for Protein Degradation at the Eukaryotic Translocon

描述（由申请方提供）：通过内质网相关降解（ERAD）的几个分支之一破坏在内质网（ER）处积累的错误折叠和潜在有害蛋白质。最近在酿酒酵母中鉴定的ERAD-T（translocon）途径催化持续或异常地接合ER translocon（负责将蛋白质移动穿过ER膜的通道）的蛋白质的破坏。ERAD-T由Hrd 1泛素连接酶介导，在ER应激条件下（ER中未折叠蛋白的负荷升高）通过未表征的ER应激反应特异性受损。最近的证据有力地表明，低密度脂蛋白（LDL，即“坏胆固醇”）的蛋白质组分通过ERAD-T降解时，其易位到ER已经停滞。此外，许多疾病与长期的ER应激有关（包括某些癌症，神经退行性疾病和免疫疾病）。从药理学上操纵ER的蛋白质折叠和质量控制能力可能是此类疾病的常见治疗策略。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提高对ERAD-T的理解，ERAD-T是一种细胞质量控制机制，几乎一无所知。关于这一途径的见解很可能有助于了解LDL生理学，并为胆固醇和ER应激相关病理学制定改进的治疗策略。拟议的研究将解决两个假设：（1）Hrd 1泛素连接酶与转位子的相互作用是降解ERAD-T底物所必需的，以及（2）新的ER应激传感机制的组分参与ERAD-T并在ER应激条件下限制ERAD-T。该项目的具体目标是（1）鉴定特异性损害ERAD-T的突变， (2)探讨内质网应激与ERAD-T的关系。将进行基于酵母细胞生长指示ERAD-T是否具有功能的新型遗传测定，并将进行生化分析以鉴定和表征ERAD-T的遗传要求。这项工作将使本科生和硕士生接触到有意义的生物医学相关研究。学生将参与该项目的各个方面，包括实验设计和执行，结果解释和数据演示。学生将获得生物医学研究的宝贵第一手经验，并有机会对具有重要医学意义的基础生物学领域进行新的发现。

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.