REGULATION OF UBIQUITINATION BY DUB-E2 UBIQUITIN CONJUGATING ENZYME COMPLEXES

DUB-E2 泛素结合酶复合物对泛素化的调节

• 批准号: 8916798
• 负责人: Cynthia Wolberger
• 金额: $ 48.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8916798
• 关键词: Address; BRCA1 gene; Binding; Biochemical; Biological; Biological Assay; Breast; Charge; Cleaved cell; Complex; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Defect; Deubiquitinating Enzyme; Ensure; Enzymes; Equilibrium; Eukaryota; Event; Goals; Health; Human; Immunologic Deficiency Syndromes; Inflammation; Lead; Learning; Light; Link; Malignant Neoplasms; Multienzyme Complexes; Ovarian; Pathway interactions; Pharmacotherapy; Play; Polyubiquitin; Polyubiquitination; Process; Property; Proteins; Regulation; Relative (related person); Repression; Roentgen Rays; Role; Structure; System; Testing; Time; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Work; base; design; drug discovery; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; p53-binding protein 1; prevent; response; thioester; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Covalent attachment of ubiquitin regulates a broad range of processes in eukaryotes. A major challenge understands how ubiquitination is regulated by the large network of enzymes that conjugate and remove ubiquitin from substrates, therefore controlling their fate. Many of the more than 90 human deubiquitinating enzymes (DUBs) are found in complexes containing E2 ubiquitin conjugating or E3 ubiquitin ligases enzymes, suggesting cross-regulation of opposing activities. Our long-term goal is to understand the underlying mechanism by which interactions between deubiquitinating and ubiquitin conjugating enzymes cooperate to control ubiquitination. This proposal focuses on a DUB-E2 complex that regulates key ubiquitination events in the response to DNA double strand breaks. OTUB1 is a DUB that specifically cleaves K48-linked polyubiquitin chains, yet binds to E2 enzymes and non-catalytically inhibits synthesis polyubiquitin. Preliminary results show that OTUB1-E2 interactions play an additional role in stimulating OTUB1 to cleave K48-linked polyubiquitin, and that the balance between the catalytic and non-catalytic OTUB1 activities is regulated by availability of uncharged E2 versus charged E2~Ub thioester as well as free ubiquitin. We will use a combination of biochemical, biophysical and structural approaches to investigate the mechanism underlying the dual functions of OTUB1-E2 complexes (Aim 1) and to dissect how the balance between catalytic and non-catalytic functions of OTUB1 is regulated by E2 charging, free ubiquitin and polyubiquitin chains (Aim 2). These results will provide a basis for investigating the contributions of OTUB1 cross-regulation to the temporal regulation of ubiquitination after DNA damage.

描述（由申请人提供）：泛素的共价附着调节真核生物的广泛过程。一个主要的挑战是了解泛素化是如何由结合和从底物中去除泛素的酶的大网络调节的，从而控制它们的命运。在90多种人类去泛素化酶（DUBs）中，有许多存在于含有E2泛素偶联酶或E3泛素连接酶的复合物中，表明它们具有相互作用的交叉调控作用。我们的长期目标是了解去泛素化和泛素偶联酶之间相互作用共同控制泛素化的潜在机制。本研究的重点是DUB-E2复合物，该复合物在DNA双链断裂反应中调节关键的泛素化事件。OTUB1是一种DUB，它特异性地切割k48连接的多泛素链，但与E2酶结合，非催化抑制多泛素的合成。初步结果表明，OTUB1-E2相互作用在刺激OTUB1切割k48连接的多泛素中起着额外的作用，并且OTUB1的催化和非催化活性的平衡受未带电E2与带电E2~Ub硫酯的可用性以及游离泛素的调节。我们将结合生物化学、生物物理和结构方法来研究OTUB1-E2复合物双重功能的机制（目的1），并剖析OTUB1的催化和非催化功能之间的平衡是如何通过E2充电、游离泛素和多泛素链来调节的（目的2）。这些结果将为探讨OTUB1交叉调控对DNA损伤后泛素化时间调控的贡献提供基础。