A chemical strategy to unravel tumor suppression through the ubiquitin pathway

项目总结 对于控制大多数依赖泛素的调控的网络，人们仍然知之甚少 所有人类癌症中常见的失活蛋白：肿瘤抑制因子P53。完全了解 仍然难以捉摸，因为目前的方法无法完全揭开泛素是如何在序列中被引导的 E1-E2-E3酶的最终目标。越来越多的证据表明，雌二醇对 E3连接酶的活性和由此产生的泛素化产物。我们知识中的一个缺口是 E2泛素结合酶(或酶)，负责体内的P53泛素化。给定 ~40个人类E2和600个E3之间相互作用的组合性质，这个网络非同寻常 很难研究。因此，对能够跟随泛素通过酶促反应的工具的需求非常迫切。 级联到它的目标蛋白。该提案的长期目标是隔离和检查 调节活细胞中P53泛素化的蛋白质-蛋白质相互作用。这一目标将通过以下方式实现 申请人的实验室开发了一种新方法，称为将泛素靶向充电到E2(TCUbE)，它可以 跟踪标记的泛素从E2酶到其目标蛋白的运动。这一战略在能力上是独一无二的 以跟随泛素通过顺序的E1-E2-E3级联到达其最终目标，因此可用于 提供了泛素依赖的p53调控的系统水平的观点。 本申请的重点是围绕泛素依赖的P53活性调节的问题。在AIM 1、tCUbE用于发现体内参与P53泛素化的E2网络。此方法将标识 导致体内p53泛素化的E2(S)，并将使这一网络被扰乱的实验成为可能 使用已知的分子，通过E3连接酶MDM2或MDMX抑制p53泛素化。这些研究将 验证tCUbE，确定指导p53泛素化的先前未知的相互作用，并评估其 对癌症表型的影响。在目标2中，tCUbE被用来询问一种独特的p53救援模式，该模式通过 一种E2，UbcH7，已被证明在某些细胞类型中保护P53不被降解。使用tCUbE，它将 可以评估UbcH7催化不同类型泛素链附着的假设 在底物上，如P53，被保护不被降解。最后，在目标3中，tCUbE与变体相结合 对脱泛素酶活性具有抵抗力的泛素，以描述脱泛素酶对 P53功能的调控。这些研究将检验不同的去泛素酶分解的假设 特定泛素化的p53-物种，并将评估如何通过 这个机制。这种方法在照亮泛素化产品子集方面是无与伦比的， 产生于活细胞中单个E2的活动，从而揭示了 介导P53泛素化。这种独特的洞察力将使发现新的潜在癌症靶点成为可能 并促进新疗法的开发，这些疗法可以扰乱泛素途径中的具体步骤。

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.