Molecular mechanisms of ubiquitin-dependent Er-associated degradation

泛素依赖性 Er 相关降解的分子机制

• 批准号: 7545108
• 负责人: JAMES A OLZMANN
• 金额: $ 4.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545108
• 关键词: 3-Dimensional; Affect; Area; Biochemical; Biological Assay; Biology; Cells; Class; Classification; Complex; Cystic Fibrosis Transmembrane Conductance Regulator; Development; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Flow Cytometry; Gap Junctions; Goals; Homeostasis; Human; Individual; Integral Membrane Protein; Kinetics; Liver diseases; Lung diseases; Malignant Neoplasms; Mediating; Membrane; Molecular; Molecular Conformation; Mutation; Neurodegenerative Disorders; Nexus (resin cement); Pathway interactions; Post-Translational Protein Processing; Prealbumin; Process; Proteins; Proteome; Proteomics; Quality Control; RNA Interference; RNA library; Regulation; Research; Role; Site; Specificity; Substrate Specificity; System; Therapeutic; Triage; Ubiquitin; Ubiquitination; Validation; Variant; base; clinically relevant; cytotoxic; functional genomics; glycosylation; high throughput analysis; human disease; insight; multicatalytic endopeptidase complex; mutant; novel; protein folding; protein misfolding; research study; small hairpin RNA; tool; ubiquitin-protein ligase

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.

描述（由申请人提供）：蛋白质折叠是一个复杂的过程，许多疾病相关突变会损害蛋白质获得适当三维天然构象的能力。末端错误折叠的蛋白质必须分类降解，以避免错误折叠蛋白质的细胞毒性积累。ER相关降解（ERAD）是识别和降解末端错误折叠的分泌和跨膜蛋白的重要质量控制过程，对细胞稳态至关重要。所涉及的蛋白质机制和分子机制现在才开始出现。该提案的长期目标是确定底物特异性ERAD的顺式和反式因子。本项目的近期目标是：1）表征已知ERAD组分的底物特异性的分子特征; 2）鉴定ERAD中涉及的新型UPS组分; 3）表征ERAD中鉴定的UPS组分的分子作用。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途径中涉及的组分，将使用RNA干扰消除已知的ERAD组分，并使用一组拓扑学上不同的荧光标记ERAD底物分析该效应。为了系统地鉴定新的ERAD机制，将使用高通量流式细胞术，采用靶向UPS的所有已知和预测组分的小发夹RNA（shRNA）文库进行功能性基因组筛选。将使用一系列细胞和生物化学测定对阳性命中进行广泛验证和表征，以确定其在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ERAD中介导特异性的底物特征和ERAD中涉及的组分的身份。ERAD的缺陷已经被认为是许多人类疾病的潜在原因，包括肺病、肝病、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，代表了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ERAD过程的见解，并可能产生临床相关的目标和策略，用于开发基于机制的治疗方法。