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

项目概要/摘要 在过去的20年里，蛋白ASCC 2已被证明有助于许多肿瘤的定位。 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物 反应这些途径的一个共同特征是它们组装K63连接的多聚泛素链 其被ASCC 2识别以募集适当的多蛋白复合物。ASCC 2使用CUE域 然而，为了结合K63连接的多聚泛素链，CUE结构域通常是混杂的泛素结合剂， 目前还不清楚ASCC 2如何特异性识别K63连接的多聚泛素链， 存在于细胞中的多聚泛素链。拟议实验的首要目标是阐明 ASCC 2特异性结合K63连接的多聚泛素链的结构基础，具有以下特异性 目的：1）鉴定介导ASCC 2和K63连接的K63之间结合的分子间相互作用。 多聚泛素链，2）使用核磁共振（NMR）光谱， 创建ASCC 2和K63连接的多聚泛素链之间相互作用的表示， 我们的实验确定的NMR，诱变，和小角X射线散射数据，和3）量化的 ASCC 2和K63连接的多聚泛素链之间预测的相互作用的功能意义， 体外和基于细胞的测定。这些研究是更广泛的努力的一部分，以更好地了解生物学 依赖于ASCC 2的泛素结合能力的途径，并诊断与ASCC 2相关的疾病。 这些路径的故障。例如，在DNA烷基化损伤修复途径中，抑制 募集ASCC 2的K63连接的多聚泛素链的形成，以及抑制相互作用的突变 ASCC 2和ALKBH 3-ASCC DNA修复复合物的其他成员之间的相互作用，最近已经被关联到 遗传性疾病，因为在这方面的研究。此外，拟议的研究将提供有价值的 为圣玛丽山玛丽大学（MSMU）的学生提供研究机会。与科学家合作， 约翰霍普金斯大学、圣路易斯的华盛顿大学和劳伦斯河的SIBYLS光束线 伯克利国家实验室将允许MSMU本科研究人员获得科学 在他们的家乡校园里没有的仪器。整体而言，建议的研究将有助我们 理解ASCC 2如何将多蛋白复合物靶向K63连接的多聚泛素链标记的位点 同时大大提高了MSMU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.