Exploration of Molecular Chaperone Complexes During Active Protein Triage

活性蛋白分类过程中分子伴侣复合物的探索

• 批准号: 8853890
• 负责人: Philip C Andrews
• 金额: $ 26.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8853890
• 关键词: Acetylation; Acute; Address; Alzheimer' s Disease; Architecture; Binding; Binding Proteins; Cell physiology; Cells; Chemicals; Client; Complex; Couples; Crosslinker; Cryoelectron Microscopy; Cytosol; Defect; Disease; Event; Goals; Heart; Homeostasis; Huntington Disease; Image; In Vitro; Knowledge; Ligase; Link; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Methodology; Microtubules; Modeling; Molecular; Molecular Chaperones; Molecular Structure; Nature; Nerve Degeneration; Neurons; Phosphorylation; Post-Translational Protein Processing; Process; Property; Proteins; Proteomics; Recruitment Activity; Relative (related person); Research; Resolution; Series; Site; Structural Models; Structure; System; Tauopathies; Technology; Time; Triage; Ubiquitin; Ubiquitination; Variant; Western Blotting; Work; crosslink; design; hyperphosphorylated tau; improved; in vivo; innovation; multicatalytic endopeptidase complex; novel; overexpression; protein complex; protein crosslink; protein misfolding; protein protein interaction; protein structure; public health relevance; reconstitution; stoichiometry; tau Proteins; three dimensional structure; ubiquitin-protein ligase

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.

描述（由申请人提供）：分子伴侣HSP70和HSP90与展开的蛋白质（例如客户）结合，并招募“促折叠”或“促脱糖”或“降解”的副酮，生成一系列不同的多蛋白质复合物，这些蛋白质可以折叠或降级蛋白质。对于某些重要客户，例如微管结合蛋白tau（mapt/tau），“选择”降级的关键步骤似乎是E3泛素偶联的连接酶芯片的动态募集，这构成了HSP70和HSP90均与HSP90伴随ubiquitin-Prototeasemome（UPSS）。然而，对于控制芯片组装到复合物中的分子事件知之甚少，我们尚不理解为什么某些与疾病相关的蛋白质（例如高磷酸化的tau）逃避了这一过程。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发现与细胞功能联系起来的困难，阻碍了了解如何将客户加载到“促进”复合体中的进展。我们假设对蛋白质分类的严格全面理解将从化学交联质质谱法（CXL-MS），冷冻电子显微镜（Cryo-EM）和新的化学探针的组合中出现，这些探针触发了急性切换到“降解”复合物。在强烈的初步结果的指导下，我们将：（SA1）生成Hsp70-Chip-Tau和Hsp90-Chip-Tau复合物的完整CXL-MS特征，并绘制其在体外，体外，完整的神经元细胞中的蛋白质 - 蛋白质接触（SA2），（SA2）使用这些签名和其他效果的效果，并了解如何招募这些复杂的效果。在细胞质和（SA3）中的活性分类阐明了伴侣复合物的大分子结构，这是首次将细胞观测与仔细测量的蛋白质 - 蛋白质接触的仔细测量联系起来。这项工作很重要，因为它将揭示大分子复合物的TH组成变化，该复合物的驱动活性蛋白质分类并且具有创新性，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，以解决细胞蛋白稳态中的关键问题之一。此外，拟议的工作汇集了在结构，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。