Exploration of Molecular Chaperone Complexes During Active Protein Triage

描述(申请人提供)：分子伴侣，热休克蛋白70和热休克蛋白90结合到未折叠的蛋白质(例如客户)，并招募“亲折叠”或“亲降解”共伴侣，产生一系列不同的多蛋白质复合体，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)，选择降解的关键步骤似乎是动态招募E3泛素结合连接酶芯片，它将Hsp70和Hsp90连接到泛素-蛋白酶体系统(UPS)。然而，对控制芯片组装到复合体中的分子事件知之甚少，我们还不知道为什么一些与疾病相关的蛋白质，如过度磷酸化的tau，逃避了这一过程。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，在理解客户如何被加载到“促进降解”复合体方面的进展受到了阻碍。我们假设，通过化学交联质谱(CXL-MS)、低温电子显微镜(Cryo-EM)和新的化学探针的组合，将出现对蛋白质分流的严格和全面的理解，这些化学探针会触发向“促降解”复合体的急性切换。在强劲的初步结果指导下，我们将：(SA1)产生完整的CXL-MS信号，并绘制它们在体外和完整神经细胞中的蛋白质接触图；(SA2)利用这些信号、化学探针和质谱学来了解这些复合体是如何形成的，以及它们在细胞质中的活性摩擦过程中如何招募UPS的CHIP和其他效应物；(SA3)阐明伴侣复合体的大分子结构，首次将细胞观察与仔细测量体外蛋白质间的接触联系起来。这项工作意义重大，因为它将揭示驱动活性蛋白质分流的大分子络合物组成的变化，而且它具有创新性，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，以解决细胞蛋白质动态平衡的关键问题之一。此外，这项拟议的工作汇集了结构、化学和细胞方法方面的连续研究专业知识，以研究动态蛋白质-蛋白质相互作用。

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.