Decoding the molecular logic of TPR cochaperones

项目摘要/摘要 四肽重复序列(TPR)辅伴蛋白是一个不同的接头蛋白家族，与 热休克蛋白(HSP)70和HSP90分子伴侣系统。这些模块化蛋白由它们的 同名的TPR结构域，它介导与HSPs形成复合体，以及酶或支架 在客户招募或质量控制方面可以帮助监护人的领域。这些配饰的重要性 TPR辅伴蛋白在广泛的意义上证明了维持蛋白质动态平衡的功能 从神经退化到癌症的一系列疾病。然而，我们对分子机制的理解 这些蛋白质对底物的识别是不完整的。特别是，有越来越多的证据表明 TPR结构域可以招募超越其典型伴侣结合伙伴的蛋白质，及其意义 目前还不知道这条替代途径的具体情况。此外，通常很难建立底物 在蛋白质动态平衡中可能发生的补偿情况下，TPR辅伴蛋白之间的关系 遗传扰动后的网络。特异性抑制TPR的化学探针的研究进展 因此，具有高时间分辨率的辅伴侣络合物是非常理想的。努力开发这样的产品 通过了解TPR结构域的哪些分子特征可以极大地增强工具 被利用来实现高亲和力和选择性结合。这项提议的目标是开发一种化学物质 通过解码TPR之间相互作用的分子逻辑来帮助解决这两个问题的工具包 辅伴侣及其底物。在我的第一个目标中，我将开发化学蛋白质组学工具来描述结合 并测量底物结合对不同刺激的反应变化。这些 探针还将使TPR抑制剂的特异性能够以快速方式进行评估。在我的第二个目标中，我 将改进预测评分函数，以便全面识别自主的底物 被E3连接酶芯片识别。在探索芯片与其相互作用的分子决定因素方面 底物，我还将确定使单个TPR辅助伴侣能够结合不同配体的特征 从它的相关家庭成员那里。这项工作意义重大，因为它将提供对 管理TPR辅伴侣的生物学规则，并将成为未来底物发现的基石 并探索这个蛋白质家族的开发努力。拟议的研究还将提供培训 一个非常适合我成为一名翻译化学生物学家的目标的环境，有机会 获得最先进的技术经验，同时培养我进行研究的能力 独立的。

PROJECT SUMMARY / ABSTRACT Tetratricopeptide repeat (TPR) cochaperones are a diverse family of adaptor proteins that cooperate with the heat shock protein (Hsp) 70 and Hsp90 chaperone systems. These modular proteins are composed of their eponymous TPR domain, which mediates complex formation with the Hsps, and an enzymatic or scaffolding domain that can aid the chaperone in client recruitment or quality control. The importance of these accessory functionalities in maintaining protein homeostasis is evidenced by the implication of TPR cochaperones in a wide range of diseases from neurodegeneration to cancer. However, our understanding of the molecular mechanisms that underpin substrate recognition by these proteins is incomplete. In particular, there is increasing evidence that TPR domains can recruit proteins beyond their canonical chaperone binding partners, and the significance of this alternative pathway is currently unknown. In addition, it is generally difficult to establish substrate relationships for TPR cochaperones given the compensation that can occur within the protein homeostasis network following genetic perturbations. Development of chemical probes that can specifically inhibit TPR cochaperone complexes with high temporal resolution is therefore highly desirable. The effort to develop such tools would be greatly augmented by an understanding of which molecular features of TPR domains can be exploited to achieve high affinity and selective binding. The objective of this proposal is to develop a chemical toolkit that helps solve both of these problems by decoding the molecular logic of interactions between TPR cochaperones and their substrates. In my first aim, I will develop chemical proteomic tools to profile the binding of TPR cochaperones and measure changes in substrate association in response to different stimuli. These probes will also enable the specificity of TPR inhibitors to be assessed in a rapid fashion. In my second aim, I will refine a predictive scoring function in order to comprehensively identify substrates that are autonomously recognized by the E3 ligase CHIP. In exploring the molecular determinants of CHIP's interactions with its substrates, I will also identify features that enable an individual TPR cochaperone to bind ligands that are distinct from its related family members. This work is significant because it will provide fundamental insights into the rules governing the biology of TPR cochaperones, and will serve as the bedrock for future substrate discovery and probe development efforts across this protein family. The proposed studies will also provide a training environment that is ideally suited to my goal of becoming a translational chemical biologist, with opportunities to gain experience with state-of-the-art techniques while also cultivating my ability to conduct research independently.