Interactions between Hsp90, Co-chaperones and Client Proteins

Hsp90、共伴侣和客户蛋白之间的相互作用

基本信息

批准号：
9269592
负责人：
HELEN JANE DYSON
金额：
$ 38.5万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9269592
关键词：
Address Adverse reactions Affinity Alpha Cell Back Binding Binding Proteins Binding Sites Biological Biophysics C-terminal Cell Extracts Cell physiology Cells Client Complex Consensus Consensus Sequence Crowding Cytoplasm DNA Binding Domain Data Dimerization Disease Electron Microscopy Environment Eukaryota Eukaryotic Cell Fluorescence Glucocorticoid Receptor Goals Histidine Homeostasis Human Hydrophobicity In Vitro Incentives Integration Host Factors Interruption Length Ligand Binding Domain Ligands Literature Location Mammalian Cell Measurable Measurement Mediating Membrane Methods Molecular Chaperones Molecular Conformation Movement Mutate NMR Spectroscopy Nature Organelles Pathway interactions Peptides Phosphotransferases Play Preparation Process Protein Binding Domain Protein Conformation Proteins Reporting Research Roentgen Rays Role Sampling Series Site Stretching Structure System TP53 gene Tail Temperature Thinking Work Yeasts Zinc chaperonin dimer experimental study innovation insight novel operation overexpression physical state prevent protein complex protein folding protein function protein p23 protein protein interaction public health relevance steroid hormone receptor tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): In eukaryotes, the chaperone Hsp90 and its co-chaperones interact with and stabilize a number of crucially important cellular factors, including steroid hormone receptors, cellular kinases and transcription factors. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. Part of the difficulty with obtaining data on these systems is that the client proteins ar generally extremely difficult to prepare in quantities suitable for structural analysis. Doubtless this is a major incentive for the cell to employ Hsp90 in their stabilization. We propose innovative methods of preparation of client protein-Hsp90 complexes, by addition of other necessary components of the chaperone cascade of the eukaryotic cell and by re-thinking the likely form of a high-affinity client protein. Our first aim will be to characterize interactions between a zinc-free form of the transcription factor p53 and the full-length Hsp90 dimer, using a set of structural measurements that can interrogate such a large complex. These measurements include small-angle X-ray scattering, room- temperature EPR, fluorescence methods and electron microscopy. The second specific aim will be concerned with the effect of co-chaperones on the system. Preliminary data show that there is a measurable interaction between the p53 DBD client protein and the p23 co-chaperone. NMR spectroscopy and other methods will be used to characterize this binary interaction, as well as the ternary interaction that we observe with the addition of Hsp90. In the third specific aim, we will examine the complex of the glucocorticoid receptor ligand-binding domain (GR-LBD) and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. A new preparation method for the complex includes the over-expression of a histidine-tagged GR-LBD protein together with the incorporation of accessory proteins from the chaperone cascade in the preparation of the complex. The successful conclusion of the proposed work will give important new insights not only into the specific nature of the chaperone-client protein complex, but also into the range of structural states sampled by proteins in their cellular environments: we have only recently begun to appreciate that protein conformational states other than the "fully and stably folded" states typified by traditional structural studies may be present and fully functional in the cell.

描述（由申请人提供）：在真核生物中，伴侣 Hsp90 及其辅助伴侣与许多至关重要的细胞因子相互作用并稳定它们，包括类固醇激素受体、细胞激酶和转录因子。尽管对分子伴侣和共分子伴侣结构了解很多，但 Hsp90 与其客户之间相互作用的结构基础仍然未知。根本问题是，我们仍然不了解客户蛋白与 Hsp90 分子伴侣结合时的物理状态，并且我们对客户蛋白在 Hsp90 分子上的接触位置只有一个粗略的了解。在这些系统上获取数据的部分困难在于，客户蛋白质通常极难制备适合结构分析的数量。毫无疑问，这是细胞使用 Hsp90 来稳定细胞的主要动力。我们提出了制备客户蛋白-Hsp90复合物的创新方法，通过添加真核细胞伴侣级联的其他必要成分，并重新思考高亲和力客户蛋白的可能形式。我们的第一个目标是使用一组可以询问如此大的复合物的结构测量来表征转录因子 p53 的无锌形式和全长 Hsp90 二聚体之间的相互作用。这些测量包括小角 X 射线散射、室温 EPR、荧光方法和电子显微镜。第二个具体目标将涉及辅助伴侣对系统的影响。初步数据表明，p53 DBD 客户蛋白和 p23 共伴侣之间存在可测量的相互作用。核磁共振波谱和其他方法将用于表征这种二元相互作用，以及我们在添加 Hsp90 时观察到的三元相互作用。在第三个具体目标中，我们将检查糖皮质激素受体配体结合结构域 (GR-LBD) 和 Hsp90 的复合物，采用文献中已使用的方法来证明哺乳动物细胞提取物中存在这种相互作用。该复合物的一种新制备方法包括组氨酸标记的 GR-LBD 蛋白的过表达以及在复合物的制备中掺入来自分子伴侣级联的辅助蛋白。这项工作的成功完成不仅将为伴侣-客户蛋白复合物的具体性质提供重要的新见解，而且还将为细胞环境中蛋白质采样的结构状态范围提供重要的新见解：我们最近才开始认识到这一点除传统结构研究所代表的“完全且稳定折叠”状态之外的蛋白质构象状态可能存在于细胞中并具有完全功能。