A chemical strategy to unravel tumor suppression through the ubiquitin pathway

项目摘要 关于控制大多数神经元的泛素依赖性调节的网络，还有很多未知之处。 在所有人类癌症中经常失活的蛋白质：肿瘤抑制因子p53。的完整理解 仍然是难以捉摸的，因为目前的方法无法完全解开泛素是如何通过序列引导的， E1-E2-E3酶的最终目标。越来越多的证据表明，E2对人类有着巨大的影响。 E3连接酶活性和所产生的泛素化产物。我们知识上的一个空白是 在体内负责p53泛素化的E2泛素结合酶。鉴于 由于约40个人类E2和>600个E3之间相互作用的组合性质，该网络是非常重要的。 很难研究。因此，迫切需要能够通过酶促降解来跟踪泛素的工具。 级联到其靶蛋白。该提案的长期目标是隔离和检查 在活细胞中调节p53泛素化的蛋白-蛋白相互作用。这一目标将通过使用 在申请人的实验室中开发的一种新方法，称为靶向泛素充电到E2（tCUbE）， 当标记的泛素从E2酶移动到它的靶蛋白时，跟踪它。这种策略是独一无二的， 通过E1-E2-E3级联反应，跟随泛素到达其最终靶点，因此可以用于 提供了一个系统水平的观点，泛素依赖的调节p53。 这个应用程序的重点是周围的问题，p53活性的泛素依赖性调节。在Aim中 1，tCUbE用于发现体内负责p53泛素化的E2网络。该方法将识别 E2（s）在体内导致p53泛素化，并将使该网络被干扰的实验成为可能 使用已知的通过E3连接酶Mdm 2或MdmX抑制p53泛素化的分子。这些研究将 验证tCUbE，鉴定以前未知的指导p53泛素化的相互作用，并评估它们的 对癌症表型的影响。在目的2中，tCUbE用于询问由以下介导的p53拯救的独特模式： 一种E2，UbcH 7，已显示在某些细胞类型中保护p53免于降解。使用tCUbE，它将 可以评估UbcH 7催化不同类型的泛素链的附着的假设 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 为了描述去泛素化酶对细胞凋亡的影响， p53功能的调节。这些研究将检验不同的去泛素化酶分解的假设 具体的泛素化p53-物种，并将评估如何p53活性可以通过差异控制， 这个机制。这种方法在阐明泛素化产物的子集方面是无与伦比的， 由活细胞中单个E2的活性产生，从而揭示了多种相互作用网络， 介导p53泛素化。这种独特的洞察力将有助于发现新的潜在癌症靶点 治疗，并促进新疗法的发展，可以破坏泛素途径中的特定步骤。

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.