Molecular mechanisms of ubiquitin-dependent Er-associated degradation

描述(申请人提供)：蛋白质折叠是一个复杂的过程，许多与疾病相关的突变会削弱蛋白质获得适当的三维天然构象的能力。末端错误折叠的蛋白质必须进行分类以进行降解，以避免错误折叠蛋白质的细胞毒性积累。内质网相关降解(ERAD)是识别和降解最终错误折叠的分泌和跨膜蛋白的重要质量控制过程，对细胞内稳态至关重要。其中涉及的蛋白质机制和分子机制现在才刚刚开始出现。这项建议的长期目标是确定底物特异性ERAD的顺式和反式因素。该项目的近期目标是：1)确定导致已知ERAD组件底物专一性的分子特征；2)确定参与ERAD的新型UPS组件；3)确定已确定的UPS组件在ERAD中的分子作用。为了表征介导底物特异性降解的分子特征并确定底物特异性ERAD途径中涉及的成分，将使用RNA干扰来耗尽已知的ERAD成分，并将使用一组拓扑上不同的、荧光标记的ERAD底物来分析其影响。为了系统地鉴定新的ERAD机制，将使用高通量流式细胞术进行针对所有已知和预测的UPS组件的小发夹RNA(ShRNA)文库的功能基因组筛选。阳性HITS将接受广泛的验证，并使用一系列细胞和生化分析来确定它们在ERAD中的作用，包括评估底物降解动力学、泛素化、糖基化、易位和聚集。总之，这些研究将深入了解介导ERAD特异性的底物特征以及ERAD中涉及的成分的身份。ERAD的缺陷被认为是许多人类疾病的潜在原因，包括肺部疾病、肝脏疾病、癌症、糖尿病和几种神经退行性疾病。调节这一途径的蛋白质和分子机制知之甚少，是一个重要的研究领域，将直接影响我们对广泛疾病的理解。该项目的完成将确定新的蛋白质和对ERAD过程的见解，并可能为基于机制的疗法的发展产生临床相关的目标和战略。

DESCRIPTION (provided by applicant): Protein folding is a complex process, and many disease-associated mutations impair the ability of the protein to attain the proper 3-dimensional native conformation. Terminally misfolded proteins must be triaged for degradation to avoid cytotoxic accumulation of misfolded proteins. ER-associated degradation (ERAD) is an essential quality control process that recognizes and degrades terminally misfolded secretory and transmembrane proteins, and is critical for cellular homeostasis. The protein machinery and molecular mechanisms that are involved are only now beginning to emerge. The long-term objective of this proposal is to determine the cis- and trans- factors underlying substrate specific ERAD. The immediate goals of this project are to 1) Characterize the molecular features responsible for substrate specificity of known ERAD components; 2) Identify novel UPS components involved in ERAD; 3) Characterize the molecular role of the identified UPS components in ERAD. To characterize the molecular features mediating substrate-specific degradation and to define the components involved in substrate-specific ERAD pathways, known ERAD components will be depleted using RNA interference and the effect will be analyzed using a panel of topologically distinct, fluorescently tagged ERAD substrates. In order to systematically identify novel ERAD machinery, a functional genomic screen employing a small hairpin RNA (shRNA) library targeting all known and predicted components of the UPS will be performed using high-throughput flow cytometry. Positive hits will be subjected to extensive validation and characterized using a battery of cellular and biochemical assays to determine their role in ERAD, including assessment of substrate degradation kinetics, ubiquitination, glycosylation, translocation, and aggregation. Together these studies will yield insight into the substrate features mediating specificity in ERAD and the identity of the components involved in ERAD. Deficits in ERAD have been implicated as a underlying cause for many human diseases, including lung disease, liver disease, cancer, diabetes, and several neurodegenerative diseases. The proteins and molecular mechanisms that regulate this pathway are poorly understood and represent an important area of research that will directly impact our understanding of a broad spectrum of diseases. Completion of this project will identify new proteins and insights into the ERAD process, and may yield clinically relevant targets and strategies for the development of mechanism-based therapeutics.