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

确定响应 DNA 烷基化损伤的多聚泛素信号传导的结构基础

基本信息

批准号：
10796099
负责人：
Patrick Lombardi
金额：
$ 44.4万
依托单位：
MOUNT ST. MARY'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10796099
关键词：
Affinity Agreement Alkylating Agents Alkylation Area Binding Binding Sites Biochemistry Biological Biological Assay Biological Process Cell Survival Cell physiology Cells Chemicals Collaborations Complex DNA Alkylation DNA Repair Data Defect Docking Enzymes Funding Genetic Diseases Genetic Transcription Goals Health Home Immunofluorescence Microscopy In Vitro Knock-out Label Laboratories Link Location Maps Mediating Modeling Multiprotein Complexes Mutagenesis Mutate Mutation NMR Spectroscopy Nuclear Nuclear Magnetic Resonance Pathway interactions Polyubiquitin Principal Investigator Process Proteins Relaxation Repair Complex Research Research Personnel Ribosomes Roentgen Rays Scientist Shapes Signal Transduction Site Specificity Structure Students Travel Ubiquitin Universities Washington Work beamline chemical reaction disease diagnosis experimental study improved instrumentation intermolecular interaction member mutant preference protein complex recruit repaired response restraint undergraduate student university student

项目摘要

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.

项目概要/摘要在过去的 20 年里，ASCC2 蛋白已被证明有助于许多细胞的定位。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。被 ASCC2 识别以招募适当的多蛋白复合物，ASCC2 利用 CUE 结构域。结合 K63 连接的多聚泛素链，然而，CUE 结构域通常是混杂的泛素结合物，目前还不清楚 ASCC2 如何特异性识别多种类型的 K63 连接的多聚泛素链。所提出的实验的首要目标是阐明细胞中存在的多聚泛素链。 ASCC2 与 K63 连接的多聚泛素链结合的特异性的结构基础，具有以下特异性目标：1) 确定介导 ASCC2 和 K63 连接之间结合的分子间相互作用使用核磁共振 (NMR) 光谱分析多泛素链，2) 使用综合建模创建 ASCC2 和 K63 连接的多聚泛素链之间相互作用的表示，该表示与我们通过实验确定的 NMR、诱变和小角度 X 射线散射数据，以及 3) 量化使用 ASCC2 和 K63 连接的多聚泛素链预测的相互作用的功能意义这些研究是更好地了解生物学的更广泛努力的一部分。依赖于 ASCC2 泛素结合能力的途径并诊断与 ASCC2 相关的疾病例如，在DNA烷基化损伤修复途径中，突变会抑制这些途径的发生。招募 ASCC2 的 K63 连接的多聚泛素链的形成，以及抑制相互作用的突变 ASCC2 和 ALKBH3-ASCC DNA 修复复合物的其他成员之间的关系最近被关联起来此外，该领域的研究将提供有价值的遗传疾病。为圣玛丽山大学 (MSMU) 的学生提供与科学家合作的研究机会。约翰·霍普金斯大学、圣路易斯华盛顿大学和劳伦斯大学的 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成果总的来说，拟议的研究将进一步推进我们的研究。了解 ASCC2 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极大地增加了 MSMU 学生的研究机会。