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的广泛过程。一个主要的挑战是了解泛素化是如何被酶的大网络调节的，这些酶结合并从底物中去除泛素，从而控制它们的命运。在超过90种人类去泛素化酶（DUB）中，有许多存在于含有E2泛素缀合酶或E3泛素连接酶的复合物中，这表明了相反活性的交叉调节。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的潜在机制，以合作控制泛素化。这项提案的重点是DUB-E2复合物，调节关键的泛素化事件在响应DNA双链断裂。OTUB 1是特异性切割K48连接的多聚泛素链的DUB，但结合E2酶并非催化性抑制多聚泛素的合成。初步结果表明，OTUB 1-E2相互作用在刺激OTUB 1切割K48连接的多聚泛素中起额外的作用，并且催化和非催化OTUB 1活性之间的平衡由不带电荷的E2相对于带电荷的E2-Ub硫酯以及游离泛素的可用性调节。我们将使用生物化学，生物物理学和结构的方法相结合，以调查OTUB 1-E2复合物（目标1）的双重功能的机制，并剖析OTUB 1的催化和非催化功能之间的平衡是如何调节E2充电，游离泛素和多聚泛素链（目标2）。这些结果将为研究OTUB 1交叉调节在DNA损伤后泛素化时间调节中的作用提供基础。

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.