A multidisciplinary approach to understanding the of the role of E4

采用多学科方法来理解 E4 的作用

• 批准号: 9386664
• 负责人: Brian Andrew Maxwell
• 金额: $ 5.71万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9386664
• 关键词: ATP phosphohydrolase; Address; Automobile Driving; Binding; Biochemical; Biological Assay; Biological Process; CDC48 protein; Cardiac development; Cell physiology; Cellular biology; Chromatin; Complex; Critical Pathways; Data; Defect; Degenerative Disorder; Disease; Drosophila genus; Electron Microscopy; Enzymatic Biochemistry; Enzymes; Equation; Eukaryota; Eukaryotic Cell; Experimental Designs; Future; Genetic; Genetic Screening; Goals; Health; Homeostasis; Human; Knowledge; Laboratories; Ligase; Link; Lysine; Malignant Neoplasms; Mediating; Mentors; Modeling; Molecular; Monoubiquitination; Nerve Degeneration; Neuromuscular Diseases; Pathology; Pathway interactions; Phenotype; Play; Polyubiquitin; Polyubiquitination; Primary carcinoma of the liver cells; Process; Prognostic Marker; Proteins; Proteomics; Quality Control; Reaction; Regulation; Research; Research Personnel; Role; Signal Transduction; Site; Structure; Syndrome; Techniques; Testing; Ubiquitin; Ubiquitination; Work; X-Ray Crystallography; Yeasts; adverse outcome; cancer therapy; design; disease-causing mutation; genetic association; insight; interdisciplinary approach; malignant breast neoplasm; man; neuromuscular; novel therapeutics; protein complex; protein degradation; protein transport; public health relevance; structural biology; therapy design/development; therapy development; trafficking; tumor

 DESCRIPTION (provided by applicant): Background: Ubiquitin mediated degradation and trafficking is critical for protein quality control in eukaryotic cells. Significant efforts by man investigators have elucidated many mechanistic details of E1-E2-E3 ubiquitin transfer cascades. However, the structural mechanism by which a fourth type of "E4" ligase catalyzes polyubiquitin chain elongation is unknown. Furthermore, unpublished data from the co-sponsor's laboratory has implicated that an interaction between the human E4, UbE4B, and a ubiquitinated protein processing enzyme, VCP, is critical to the degenerative phenotype observed in the disease multi-system proteinopathy (MSP). Despite its role in MSP, cancer, and other neurodegenerative processes, the pathways and protein substrates regulated by UBE4B-mediated polyubiquitination remain elusive. Long-term Objectives: The long-term goals of the PI are to understand how the processing of polyubiquitination targets contributes to cellular homeostasis, particularly in pathways mediated by UBE4B and VCP. Specific Aims: This proposal aims to (1) Determine the detailed molecular basis for the yeast E4's activity and (2) Elucidate mechanisms and pathways of E4 from higher eukaryotes. Experimental Design: In aim 1 this proposal will elucidate the structural mechanism of the better-characterized yeast E4 enzyme, Ufd2p. In order to facilitate this aim, both x-ray crystallography and cyro-electron microscopy will be used to obtain a structure of catalytic intermediate of the E4 elongating a Ub chain in complex with an E2 bound to a donor Ub. Quantitative binding assays and ubiquitin transfer assays will then be used to further test hypotheses inferred from structural data on the mechanism of E4 polyubiquitination. In aim 2, this proposal will use a multi-faceted approach involving genetic screening in a Drosophila model of MSP, quantitative biochemical assays and proteomics to identify the E2 and E3 enzymes and ultimately the protein substrates that comprise the pathways regulated by UbE4B and VCP. Relevence to health: The proposed research will provide new insight into the mechanism of protein trafficking and degradation mediated by VCP and UBE4B, and thus facilitate further development of therapy for cancer, MSP and related diseases using either or both proteins as targets or prognostic indicators for treatment.

 描述(申请人提供)：背景：泛素介导的降解和运输是真核细胞中蛋白质质量控制的关键。MAN研究人员的重大努力已经阐明了E1-E2-E3泛素转运级联的许多机制细节。然而，第四类“E4”连接酶催化多泛素链延长的结构机制尚不清楚。此外，来自联合发起人实验室的未发表的数据表明，人类E4、UbE4B和泛素化蛋白质处理酶VCP之间的相互作用对于在疾病多系统蛋白病(MSP)中观察到的退行性表型至关重要。尽管它在MSP、癌症和其他神经退化过程中发挥作用，但UBE4B介导的多泛素化调节的通路和蛋白质底物仍然难以捉摸。长期目标：PI的长期目标是了解多泛素化靶标的处理如何有助于细胞内稳态，特别是在UBE4B和VCP介导的途径中。具体目的：本研究旨在(1)确定酵母E4‘S活性的详细分子基础；(2)从高等真核生物中阐明E4的作用机制和途径。实验设计：在目标1中，本方案将阐明特性更好的酵母E4酶Ufd2p的结构机制。为了促进这一目的，将使用X射线结晶学和循环电子显微镜来获得E4催化中间体的结构，该结构的E4在与供体Ub结合的络合物中延长Ub链。然后将使用定量结合分析和泛素转移分析来进一步检验从结构数据中推断的关于E4多泛素化机制的假说。在目标2中，这项建议将使用多方面的方法，包括在MSP的果蝇模型中进行遗传筛选，定量生化分析和蛋白质组学，以确定E2和E3酶，并最终确定组成UbE4B和VCP调控途径的蛋白质底物。与健康的关系：拟议的研究将为VCP和UBE4B介导的蛋白质运输和降解机制提供新的见解，从而促进进一步发展以其中一种或两种蛋白质作为治疗靶点或预后指标的癌症、MSP和相关疾病的治疗方法。