A chemical strategy to unravel tumor suppression through the ubiquitin pathway

通过泛素途径揭示肿瘤抑制的化学策略

• 批准号: 10213787
• 负责人: Rebecca Scheck
• 金额: $ 29.58万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-05 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213787
• 关键词: Biological Assay; Cancerous; Cell Cycle Arrest; Cell Death; Cell Line; Cells; Charge; Chemicals; Clinical Trials; DNA Damage; Data; Defect; Deubiquitinating Enzyme; Development; Disease; Enzymes; Foundations; Gene Expression; Goals; Human; Human Papillomavirus; In Vitro; Knowledge; Lead; Malignant Neoplasms; Mediating; Methods; Molecular; Monitor; Nature; Pathologic; Pathway interactions; Pharmacology; Phenotype; Proteins; Regulation; Reporting; Resistance; Specificity; System; TP53 gene; Testing; Time; Toxic effect; Tumor Suppression; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Variant; Work; anticancer activity; cancer therapy; cell type; combinatorial; experimental study; fighting; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; new therapeutic target; next generation; novel; novel strategies; novel therapeutics; protein protein interaction; response; side effect; targeted cancer therapy; tool; ubiquitin-protein ligase

PROJECT SUMMARY Much remains unknown about the network that controls the ubiquitin-dependent regulation of the most frequently inactivated protein in all human cancer: the tumor suppressor p53. A complete understanding remains elusive because current methods are unable to fully unravel how ubiquitin is led through the sequence of E1-E2-E3 enzymes to its final target. Mounting evidence suggests that E2s have a tremendous impact on both E3 ligase activity and the resulting ubiquitinated products. One gap in our knowledge is the identity of the E2 ubiquitin-conjugating enzyme (or enzymes) responsible for p53 ubiquitination in vivo. Given the combinatorial nature of interactions between ~40 human E2s and >600 E3s, this network is extraordinarily difficult to study. As a result, there is a striking need for tools that can follow ubiquitin through the enzymatic cascade to its target protein. The long-term goal of this proposal is to isolate and examine the network of protein-protein interactions that regulate p53 ubiquitination in living cells. This goal will be accomplished using a new method developed in the applicant’s lab, called targeted Charging of Ubiquitin to E2 (tCUbE), that can track a tagged ubiquitin as it moves from an E2 enzyme to its target protein. This strategy is unique in its ability to follow ubiquitin through the sequential E1-E2-E3 cascade to its ultimate target, and can therefore be used to provide a systems-level view of the ubiquitin-dependent regulation of p53. This application focuses on the questions surrounding the ubiquitin-dependent regulation of p53 activity. In Aim 1, tCUbE is used to discover the E2 network responsible for p53 ubiquitination in vivo. This method will identify the E2(s) that lead to p53 ubiquitination in vivo, and will enable experiments in which this network is perturbed using known molecules that inhibit p53 ubiquitination by the E3 ligases Mdm2 or MdmX. These studies will validate tCUbE, identify previously unknown interactions that guide p53 ubiquitination, and evaluate their impact on cancer phenotype. In Aim 2, tCUbE is used to interrogate a unique mode of p53 rescue mediated by an E2, UbcH7, which has been shown to protect p53 from degradation in certain cell types. Using tCUbE, it will be possible to evaluate the hypothesis that UbcH7 catalyzes the attachment different types of ubiquitin chains on substrates, like p53, that are protected from degradation. Finally, in Aim 3, tCUbE is combined with variants of ubiquitin that are resistant to deubiquitinase activity in order to profile the effects of deubiquitinases on the regulation of p53 function. These studies will test the hypothesis that distinct deubiquitinases disassemble specific ubiquitinated p53-species, and will evaluate how p53 activity can be differentially controlled through this mechanism. This approach is unmatched in its ability to illuminate the subset of ubiquitinated products that arise from the activity of a single E2 in living cells, thereby revealing the diverse network of interactions that mediate p53 ubiquitination. Such unique insight will enable the discovery of new potential targets for cancer therapy, and facilitate the development of new therapies that can disrupt specific steps within the ubiquitin path.

项目概要 关于控制大多数细胞的泛素依赖调节的网络仍然未知。 所有人类癌症中经常失活的蛋白质：肿瘤抑制因子 p53。完整的了解 仍然难以捉摸，因为当前的方法无法完全阐明泛素是如何通过序列引导的 E1-E2-E3 酶到达其最终目标。越来越多的证据表明 E2 对 E3 连接酶活性和产生的泛素化产物。我们知识中的一个空白是其身份 E2 泛素结合酶（或多种酶）负责体内 p53 泛素化。鉴于 约 40 个人类 E2 和 > 600 个 E3 之间相互作用的组合性质，该网络非常出色 很难学习。因此，迫切需要能够通过酶促作用追踪泛素的工具。 级联至其靶蛋白。该提案的长期目标是隔离和检查网络 调节活细胞中 p53 泛素化的蛋白质-蛋白质相互作用。这个目标将通过使用来实现 申请人实验室开发的一种新方法，称为泛素向 E2 定向充电 (tCUbE)，可以 跟踪标记的泛素从 E2 酶移动到其目标蛋白的过程。该策略的能力是独一无二的 跟随泛素通过顺序 E1-E2-E3 级联到达其最终目标，因此可用于 提供 p53 泛素依赖性调节的系统级视图。 该应用重点关注围绕 p53 活性的泛素依赖性调节的问题。瞄准 1、tCUbE用于发现体内负责p53泛素化的E2网络。该方法将识别 导致体内 p53 泛素化的 E2(s)，并将使该网络受到干扰的实验成为可能 使用通过 E3 连接酶 Mdm2 或 MdmX 抑制 p53 泛素化的已知分子。这些研究将 验证 tCUbE，识别先前未知的指导 p53 泛素化的相互作用，并评估其 对癌症表型的影响。在目标 2 中，tCUbE 用于询问 p53 救援介导的独特模式 E2，UbcH7，已被证明可以保护 p53 在某些细胞类型中免遭降解。使用 tCUbE，它将 可以评估 UbcH7 催化不同类型泛素链附着的假设 在 p53 等底物上，可防止降解。最后，在目标 3 中，tCUbE 与变体相结合 抗去泛素酶活性的泛素，以分析去泛素酶对 p53 功能的调节。这些研究将检验不同的去泛素酶分解的假设 特定泛素化 p53 种类，并将评估如何通过以下方式差异控制 p53 活性 这个机制。这种方法在阐明泛素化产品子集方面的能力是无与伦比的， 由活细胞中单个 E2 的活性产生，从而揭示了不同的相互作用网络 介导 p53 泛素化。这种独特的见解将有助于发现新的潜在癌症靶标 疗法，并促进新疗法的开发，这些新疗法可以破坏泛素路径中的特定步骤。