REGULATION OF UBIQUITINATION BY DUB-E2 UBIQUITIN CONJUGATING ENZYME COMPLEXES

描述(由申请人提供)：泛素的共价连接调节真核生物中广泛的过程。一个主要的挑战是了解泛素化是如何由酶的大型网络调节的，这些酶结合并从底物中移除泛素，从而控制它们的命运。人类90多种脱泛素酶(DUB)中的许多都存在于包含E2泛素结合酶或E3泛素连接酶的复合体中，这表明相反的活性存在交叉调节。我们的长期目标是了解去泛素化和泛素结合酶之间相互作用控制泛素化的潜在机制。这项建议的重点是一个Dub-E2复合体，它调节DNA双链断裂反应中的关键泛素化事件。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，并非催化地抑制多泛素的合成。初步结果表明，OTUB1-E2相互作用在刺激OTUB1裂解K48连接的多聚泛素方面起着额外的作用，催化和非催化OTUB1活性之间的平衡受到未荷电的E2与荷电的E2~Ub硫酸酯以及游离泛素的调节。我们将使用生化、生物物理和结构方法相结合的方法来研究OTUB1-E2复合体双重功能的潜在机制(目标1)，并剖析OTUB1的催化和非催化功能之间的平衡是如何受到E2充电、游离泛素和多泛素链的调节(目标2)。这些结果将为研究OTUB1交叉调节在DNA损伤后泛素化的时间调控中的作用提供基础。

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.