Structure and Regulation of hsp90 Chaperones

描述(由申请人提供)：HSP90伴侣参与蛋白质的后期成熟，涉及从信号到细菌识别的各种细胞活动。细胞质Hsp90指导类固醇激素受体、原癌基因蛋白、G蛋白和其他肿瘤转化的关键介质的成熟。破坏这种成熟的Hsp90抑制剂显示出强大的抗癌活性。GRP94是ER HSP90的副蛋白，是一种伴侣蛋白，目的是运输到细胞表面和分泌，在内质网输出有毒的错误折叠蛋白方面发挥关键作用，当靶向时会导致肿瘤细胞死亡。HSP90伴侣的活性受ATP和其他配体与N-末端结构域的结合调节，该结构域的反应是HSP90伴侣循环的中心。然而，尽管它们在序列和结构上具有高度的同源性，但细胞质和ER HSP90对调节配体的反应显示出根本的不同。细胞质Hsp90 N-末端结构域LID的配体依赖性构象变化一直难以证实，任何变化的可能触发因素也尚未确定。另一方面，在GRP94中，LID上的保守插入启动了该区域进行构象重排，这种重排根据进入的配体的身份而有很大不同。这些重排改变了伴侣的四级结构。这项建议的研究将利用X射线结晶学来确定GRP94可用的配体依赖的构象状态，以可视化配体驱动的GRP94络合物的结构。体内试验将用于评估这些结构重排的生物学重要性。GRP94盖对进入的配体的敏感反应允许GRP94结合不能被细胞质Hsp90容纳的抑制剂，因此对GRP94具有选择性。由于选择性和非选择性配体是密切相关的，我们将使用X射线结晶学来可视化这两类配体与GRP94和Hsp90的结合，并推测它们的选择性机制。因为每个HSP90平行蛋白负责伴随不同的客户蛋白集合，所以用选择性抑制剂特异性地靶向一个HSP90平行蛋白可能会导致更高的疗效和治疗控制。最后，新的发展为表征新的客户和辅助伴侣与Hsp90和GRP94的相互作用提供了前景：最小客户蛋白负载系统为分离客户-Hsp90复合体用于生化和生物物理研究打开了道路，最近发现的第一个GRP94辅助因子OS9首次为表征蛋白质-蛋白质相互作用在内质网副对数中发生的方式打开了大门。

DESCRIPTION (provided by applicant): The hsp90 chaperones participate in the late stage maturation of proteins involved in diverse cellular activities ranging from signaling to bacterial recognition. Cytoplasmic Hsp90 guides the maturation of steroid hormone receptors, proto-oncogenic kinases, G-proteins, and other key mediators of neoplastic transformation. Inhibitors of Hsp90 that disrupt this maturation display potent anti-cancer activity. GRP94, the ER hsp90 paralog, chaperones proteins destined for transport to the cell surface and secretion, plays a key role in the export of toxic malfolded proteins from the ER, and when targeted leads to the death of tumor cells. The activity of hsp90 chaperones is regulated by the binding of ATP and other ligands to the N-terminal domain, and the response of this domain is central to the hsp90 chaperone cycle. Yet despite their high degree of sequence and structural homology, the cytoplasmic and ER hsp90 paralogs exhibit fundamental differences in their response to regulatory ligands. Ligand dependent conformational changes in the N-terminal domain lid of cytoplasmic Hsp90 have been difficult to demonstrate, and a plausible trigger for any changes has not been identified. In GRP94, on the other hand, a conserved insertion in the lid primes this region to undergo conformational rearrangements that differ dramatically depending on the identity of the incoming ligand. These rearrangements alter the quaternary structure of the chaperone. The research in this proposal will identify the ligand dependent conformational states available to GRP94 using X-ray crystallography to visualize the structures of ligand-driven GRP94 complexes. In vivo assays will be used to assess the biological importance of these structural rearrangements. The sensitive response of the GRP94 lid to incoming ligands allows GRP94 to bind inhibitors that cannot be accommodated in cytoplasmic Hsp90 and are therefore selective for GRP94. Because selective and non-selective ligands are closely related, we will use X-ray crystallography to visualize the both classes of ligands bound to GRP94 and Hsp90 and infer their mechanism of selectivity. Because each of the hsp90 paralogs is responsible for chaperoning distinct sets of client proteins, specific targeting of one hsp90 paralog with selective inhibitors may result in higher efficacy and therapeutic control. Finally, new developments offer the prospect of characterizing novel client and co-chaperone interactions with both Hsp90 and GRP94: minimal client protein loading systems open the way to isolating client-Hsp90 complexes for biochemical and biophysical study, and the recent identification of the first GRP94 accessory factor, os9, has, for the first time, opened the door to characterizing how protein-protein interactions occur in the ER paralog.