REGULATION OF UBIQUITINATION BY DUB-E2 UBIQUITIN CONJUGATING ENZYME COMPLEXES

泛素的共价连接调控着真核生物中广泛的过程。一项重大挑战 是理解泛素化是如何由结合和结合的酶的大型网络来调节的 从底物中去除泛素，从而控制它们的命运。90多个人类中的许多人 去泛素酶(DUB)存在于含有E2泛素结合或E3的复合体中 泛素连接酶，暗示相反活动的交叉调节。我们的长期目标是 了解去泛素化和泛素之间相互作用的潜在机制 结合酶相互作用来控制泛素化。这项提议的重点是DUB-E2复合体 这调节了DNA双链断裂反应中的关键泛素化事件。OTUB1是一个配音 它特异性地切割K48连接的多泛素链，但与E2酶和非催化结合 抑制合成多泛素。初步结果表明，OTUB1-E2相互作用起到了额外的作用 在刺激OTUB1裂解K48连接的多聚泛素方面的作用，以及催化之间的平衡 而非催化的OTUB1活性受未荷电的E2与荷电的E2~Ub的可用性的调节 硫代酯以及游离泛素。我们将使用生化、生物物理和结构的组合 研究OTUB1-E2复合体双重功能机制的方法(目标1) 并剖析了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(目标2)。这些结果将为今后的研究提供依据 研究OTUB1交叉调节在泛素化后时间调节中的作用 DNA损伤。

Covalent attachment of ubiquitin regulates a broad range of processes in eukaryotes. A major challenge is understanding 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.