Determining the structural basis of polyubiquitin signaling in response to DNA alkylation damage

项目摘要/摘要 在过去的20年里，ASCC2蛋白被证明对许多 参与DNA烷基化、损伤修复和停滞核糖体等途径的多蛋白复合体 回应。其中几条途径的一个共同特征是它们组装K63连接的多泛素链 它们被ASCC2识别来招募适当的多蛋白复合体。ASCC2利用提示域 然而，为了结合K63连接的多泛素链，CUE结构域通常是混杂的泛素结合体， 目前尚不清楚ASCC2如何在多种类型的泛素中具体识别K63连接的多泛素链 细胞内存在多聚泛素链。提议的实验的首要目标是阐明 ASCC2的S结合K63连接的多泛素链与下列特异性结合的结构基础 目的：1)确定介导ASCC2和K63连接的分子间相互作用 使用核磁共振波谱的多泛素链，2)使用集成模型 创建符合以下条件的ASCC2和K63连接的多泛素链之间相互作用的表示法 我们的实验确定了核磁共振、诱变和小角X射线散射数据，并3)量化了 预测ASCC2与K63连接的多泛素链相互作用的功能意义 体外和基于细胞的分析。这些研究是更广泛的努力的一部分，目的是更好地了解 依赖ASCC2的S泛素结合能力和诊断与ASCC2相关疾病的途径 这些通路出现了故障。例如，在DNA烷基化损伤修复途径中，抑制 招募ASCC2的K63连接的多泛素链的形成，以及抑制相互作用的突变 在ASCC2和ALKBH3-ASCC DNA修复复合体的其他成员之间，最近被发现 由于在这一领域的研究而导致的遗传病。此外，拟议的研究将提供有价值的 圣玛丽山大学(MSMU)学生的研究机会。与科学家在 约翰·霍普金斯大学、圣路易斯的华盛顿大学和劳伦斯的SIBYLS光束线 伯克利国家实验室将允许MSMU本科生研究人员获得科学 本校园内没有的仪器设备。总括而言，拟议的研究将进一步推动我们 了解ASCC2如何将多蛋白复合体靶向K63连接的多泛素链标记的位点 同时大大增加了密歇根州立大学学生的研究机会。

Project Summary/Abstract Over the past 20 years, the protein ASCC2 has been shown to contribute to the localization of numerous multiprotein complexes involved in pathways such as DNA alkylation damage repair and the stalled ribosome response. A common feature of several of these pathways is that they assemble K63-linked polyubiquitin chains which are recognized by ASCC2 to recruit the appropriate multiprotein complexes. ASCC2 utilizes a CUE domain to bind the K63-linked polyubiquitin chains, however, CUE domains are typically promiscuous ubiquitin binders, and it is unclear how ASCC2 specifically recognizes K63-linked polyubiquitin chains among the myriad types of polyubiquitin chains present in the cell. The overarching goal of the proposed experiments is to elucidate the structural basis for ASCC2’s specificity for binding K63-linked polyubiquitin chains with the following specific aims: 1) identify the intermolecular interactions that mediate binding between ASCC2 and K63-linked polyubiquitin chains using nuclear magnetic resonance (NMR) spectroscopy, 2) use integrative modeling to create a representation of the interaction between ASCC2 and K63-linked polyubiquitin chains that agrees with our experimentally determined NMR, mutagenesis, and small angle X-ray scattering data, and 3) quantify the functional significance of the predicted interactions between ASCC2 and K63-linked polyubiquitin chains using in vitro and cell-based assays. These studies are part of a broader effort to better understand the biological pathways that depend on ASCC2’s ubiquitin-binding abilities and to diagnose diseases associated with the malfunction of these pathways. For example, in the DNA alkylation damage repair pathway, mutations that inhibit the formation of the K63-linked polyubiquitin chains that recruit ASCC2, and mutations that inhibit the interaction between ASCC2 and other members of the ALKBH3-ASCC DNA repair complex, have recently been associated with genetic diseases due to research in this area. Additionally, the proposed studies will provide valuable research opportunities for students at Mount St. Mary’s University (MSMU). Collaborations with scientists at Johns Hopkins University, Washington University in St. Louis, and the SIBYLS beamline at the Lawrence Berkeley National Laboratory will allow the MSMU undergraduate researchers access to scientific instrumentation that is not available on their home campus. Overall, the proposed studies will further our understanding of how ASCC2 targets multiprotein complexes to sites marked by K63-linked polyubiquitin chains while greatly enhancing the research opportunities available for students at MSMU.