Exploration of Molecular Chaperone Complexes During Active Protein Triage

描述（由申请人提供）：分子伴侣Hsp 70和Hsp 90与未折叠的蛋白质（例如客户）结合，并招募“促折叠”或“促降解”辅伴侣，产生一系列不同的多蛋白复合物，折叠或降解结合的蛋白质。对于一些重要的客户，如微管结合蛋白tau（MAPT/tau），降解的“选择”的关键步骤似乎是E3泛素缀合连接酶CHIP的动态募集，它将Hsp 70和Hsp 90偶联到泛素-蛋白酶体系统（UPS）。然而，我们对控制CHIP组装成复合物的分子事件知之甚少，我们还不明白为什么一些疾病相关蛋白质，如过度磷酸化的tau蛋白，会逃避这一过程。由于缺乏结构信息、蛋白质-蛋白质相互作用的动态性质以及将体外研究结果与细胞功能联系起来的困难，在了解客户如何加载到“促降解”复合物方面的进展受到阻碍。我们假设，一个严格的和全面的理解蛋白质分类将出现从化学交联质谱（CXL-MS），低温电子显微镜（cryo-EM）和新的化学探针，触发急性开关的组合“促降解”复杂。在强有力的初步结果的指导下，我们将：（SA 1）产生Hsp 70-CHIP-tau和Hsp 90-CHIP-tau复合物的完整CXL-MS特征，并在体外和完整神经元细胞中绘制它们的蛋白质-蛋白质接触，（SA 2）使用这些特征，化学探针和质谱分析，以了解复合物如何形成，以及它们如何在胞质溶胶中的活性分类过程中招募CHIP和UPS的其他效应物，SA 3）阐明了分子伴侣复合物的大分子结构，首次将细胞观察与体外蛋白质-蛋白质接触的仔细测量联系起来。这项工作是重要的，因为它将揭示驱动活性蛋白分类的大分子复合物组成的变化，并且它是创新的，因为它将强大的体外方法与新的化学探针和CXL-MS联系起来，以解决细胞蛋白质稳态中的关键问题之一。此外，拟议的工作汇集了结构，化学和细胞方法研究动态蛋白质-蛋白质相互作用的连续研究专业知识。

DESCRIPTION (provided by applicant): The molecular chaperones, Hsp70 and Hsp90 bind to unfolded proteins (e.g. clients) and recruit either "pro-folding" or "pro-degradation" co-chaperones, generating a series of distinct multi- protein complexes that either fold or degrade the bound protein. For some important clients, such as microtubule-binding protein tau (MAPT/tau), a key step in the "choice" to degrade seems to be the dynamic recruitment of the E3 ubiquitin conjugating ligase CHIP, which couples both Hsp70 and Hsp90 to the ubiquitin-proteasome system (UPS). However, little is known about the molecular events that govern CHIP assembly into the complex and we do not yet understand why some disease-associated proteins, such as hyper-phosphorylated tau, evade this process. Progress towards understanding how clients are loaded into the "pro-degradation" complex has been hindered by a lack of structural information, the dynamic nature of the protein-protein interactions and the difficulties of linking in vitro findings with cellular functions. We hypothesize that a rigorous ad comprehensive understanding of protein triage will emerge from a combination of chemical crosslinking-mass spectrometry (CXL-MS), cryo-electron microscopy (cryo-EM) and new chemical probes that trigger an acute switch to the "pro- degradation" complex. Guided by strong preliminary results, we will: (SA1) generate a complete CXL-MS signature of the Hsp70-CHIP-tau and Hsp90-CHIP-tau complexes and map their protein-protein contacts in vitro and in intact neuronal cells, (SA2) use these signatures, chemical probes and mass spectrometry to understand how the complexes form and how they recruit CHIP and other effectors of the UPS during active triage in the cytosol and (SA3) elucidate the macromolecular architecture of the chaperone complexes to, for the first time, link cellular observations with careful measurements of protein-protein contacts in vitro. This work is significant because it will reveal changes in th composition of macromolecular complexes that drive active protein triage and it is innovative because it links powerful in vitro approaches with new chemical probes and CXL-MS to address one of the key questions in cellular protein homeostasis. Moreover, the proposed work brings together a continuum of research expertise in structural, chemical and cellular approaches to the study of dynamic protein-protein interactions.