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: 9050117
• 负责人: Brian Andrew Maxwell
• 金额: $ 5.43万
• 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/9050117
• 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; Crystallography; 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; System; Techniques; Testing; Ubiquitin; Ubiquitination; Work; Yeasts; adverse outcome; base; 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 Furthermore, unpublished data from the co-sponsor's laboratory has implicated that an interaction between the human E4, UbE4B, and a ubiquitinated protein processing enzyme, VCP, is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.