Structure and Regulation of hsp90 Chaperones

hsp90 分子伴侣的结构和调控

基本信息

批准号：
7417452
负责人：
DANIEL T GEWIRTH
金额：
$ 30万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7417452
关键词：
ATP Hydrolysis Achievement Address Adenine Adenosine Adenosine-5&apos -(N-ethylcarboxamide)Affect Affinity Antigens Behavior Binding Binding Sites Biochemical Biological Assay Biology C-terminal Cell surface Cells Client Complex Cytoplasmic Tail DNA Gyrase DNA Sequence Rearrangement Deletion Mutagenesis Development Dimerization Disease Elements Event Exhibits Family GRP94 Goals Heat-Shock Proteins 90 Heavy-Chain Immunoglobulins Immune In Vitro Individual Lead Length Libraries Ligand Binding Ligand Binding Domain Ligands Malignant Neoplasms Measurement Molecular Chaperones Molecular Conformation N Domain N-terminal NMR Spectroscopy Natural Immunity Neoplasm Metastasis Nucleotides Oncogenic Peptides Phosphotransferases Physiological Play Production Property Proteins Range Regulation Research Personnel Role Role playing therapy Signal Transduction Specificity Stimulus Stress Structure Substrate Interaction System Testing Therapeutic Toll-like receptors Variant Work X-Ray Crystallography base cancer therapy design in vitro Assay in vivo inhibitor/antagonist insight member microbial monorden mutant novel paralogous gene programs research study response small molecule stereochemistry steroid hormone receptor tumor tumor growth

项目摘要

DESCRIPTION (provided by applicant): The hsp90 chaperones participate in the maturation of proteins involved in diverse cellular activities ranging from signaling to bacterial recognition. They are also key players in the response to cell stress events. Cytoplasmic Hsp90s guide the maturation of steroid hormone receptors and proto-oncogenic kinases, and hsp90 inhibitors disrupt this maturation and display potent anti-cancer activity. GRP94, the ER hsp90 paralog, chaperones proteins destined for transport to the cell surface, including the Toll-like receptors. Cells deficient in GRP94 are unresponsive to microbial stimuli. GRP94 has also been identified as a tumor rejection antigen and elicits suppression of tumor growth and metastasis. The activity of hsp90 chaperones is regulated by small molecule ligands such as ATP that bind to the N-terminal domain, as well as by their conformational state. Key questions that remain unanswered include how ligands exert their inhibitory or activation effects, how different hsp90 paralogs are selectively regulated, how changes in conformation lead to altered activity, and how client proteins interact with the chaperone. An understanding of the stereochemistry that underlies hsp90 biology and hsp90-targeted therapies requires the structural analysis of the relevant macromolecular species. Using X-ray crystallography, NMR spectroscopy, and biochemical- and cell-based assays, the experiments in this proposal will address how GRP94 is regulated by small molecule ligands and interacts with client proteins. We will solve and analyze the structure of the N-terminal domain of GRP94 alone and in complex with activators, broad-spectrum antagonists, and selective inhibitors. Novel selective inhibitors of GRP94 will be designed and synthesized based on these structures. We also propose to examine the interaction of GRP94 with peptides, building on recent work that identified a peptide-binding site in the N-domain. We also plan to investigate the structural basis for N-domain dimerization and oligomerization by constructing GRP94 and Hsp90 molecules lacking candidate interacting elements and determining their functional properties. Finally, the structures of multi-domain GRP94 constructs, including full-length variants, alone or in complex with client proteins will be determined in order to understand the interplay between the domains and the mechanism of substrate interaction. Structural findings will be correlated with measurements of ligand affinity and chaperone activity in both wild-type and mutationally altered molecules.

描述（由申请人提供）：HSP90伴侣参与参与从信号传导到细菌识别的各种细胞活性的蛋白质的成熟。他们也是对细胞压力事件的反应的关键参与者。细胞质HSP90S指导类固醇受体和原癌激酶的成熟，HSP90抑制剂破坏了这种成熟并表现出有效的抗癌活性。 GRP94，ER HSP90旁系同源物，伴侣蛋白注定要运输到细胞表面，包括收费受体。缺乏GRP94的细胞对微生物刺激没有反应。 GRP94也被确定为肿瘤排斥抗原，并引起抑制肿瘤生长和转移的抑制。 Hsp90伴侣的活性受小分子配体（例如与N末端结构域结合的ATP及其构象状态结合的ATP）调节。尚未得到答复的关键问题包括配体如何施加其抑制作用或激活效应，如何选择性地调节不同的HSP90旁系同源物，构象的变化如何导致活性改变以及客户蛋白如何与伴侣蛋白相互作用。对基于HSP90生物学和HSP90靶向疗法的立体化学的理解需要对相关的大分子物种进行结构分析。使用X射线晶体学，NMR光谱和基于生化和细胞的测定法，该提案中的实验将解决GRP94如何受小分子配体调节并与客户蛋白相互作用。我们将单独求解和分析GRP94的N末端结构域的结构，并与激活剂，广谱拮抗剂和选择性抑制剂进行复杂化。 GRP94的新型选择性抑制剂将根据这些结构进行设计和合成。我们还建议检查GRP94与肽的相互作用，这是基于鉴定N域中肽结合位点的最新工作的基础。我们还计划通过构造缺乏候选元素并确定其功能特性的GRP94和HSP90分子来研究N域二聚化和低聚的结构基础。最后，将确定多域GRP94构建体的结构，包括全长变体，或与客户端蛋白质中的复杂性，以了解域之间的相互作用和底物相互作用的机制。结构发现将与野生型和突变改变的分子中配体亲和力和伴侣活性的测量相关。