Regulation of the extracellular HSP90 chaperone machinery

项目总结 尽管分泌型分子伴侣热休克蛋白90(EHSP90)与肿瘤细胞有关 通过结合和稳定细胞外基质蛋白酶客户端MMP2侵袭，我们对 控制eHSP90伴侣系统的调节机制是设计的一大缺陷 成功的HSP90靶向治疗。我们的长期目标是开发治疗策略，防止 靶向细胞外HSP90分子伴侣机制的肿瘤细胞侵袭。我们的总体目标是 阐明eHSP90及其调节子是如何被单独和集体以及在 它们的胞外抑制物的存在。我们的中心假设是细胞外的三磷酸腺苷和酪氨酸 EHSP90辅助伴侣TIMP2的磷酸化，调节客户MMP2的伴侣，并影响 HSP90与细胞外抑制物结合。这一假设是基于我们最近发表的工作和 初步研究表明eHSP90结合并水解三磷酸腺苷，辅助伴侣TIMP2增强 HSP90与三磷酸腺苷及其药物抑制剂的结合。我们的理论基础是，通过阐明 EHSP90伴侣调节，我们将为涉及eHSP90的疾病设计更好的治疗方法 机械设备。我们计划通过追求以下三个具体目标来检验我们的中心假设：1)调查 ATPase活性在细胞外热休克蛋白90(EHSP90)调节中的作用；2)确定 TIMP2的酪氨酸磷酸化对其辅助伴侣功能的影响；3)阐明TIMP2在 增强eHSP90与细胞外抑制物的结合。在第一个目标下，将使用的方法已经 在Pi的实验室中建立，包括从人细胞中纯化细胞内和细胞外HSP90 培养，等温量热法测定对三磷酸腺苷类似物的亲和力，三磷酸腺苷酶分析测定 ATPase活性，确定蛋白分解活性的酶动力学，以及体外和体内蛋白质-蛋白质 基因敲除细胞培养中细胞外ATP存在和耗尽时的相互作用。 在第二个目标下，在PI中设计的拟磷或非磷酸化TIMP2突变体 将使用实验室，将使用CRISPR基因敲除c-Src激酶、TIMP2和MMP2的细胞株用于 蛋白质之间的相互作用，并确定对MMP2活性的影响。在第三个目标下，TIMP的作用， 增强eHSP90与胞外结合的非磷酸化和仿磷酸化突变体 抑制剂将在体外和体内细胞外药物结合试验中进行评估，使用的是人 细胞系和siRNA技术。TIMP2和eHSP90抑制剂对MMP2介导的蛋白降解的影响 将在基质降解研究中进行评估。研究方法是创新的，在国际刑警组织看来， 因为它代表着一种新的、实质性的偏离现状的做法，将重点转向目标 伴侣调节的细胞外途径。这项拟议的研究意义重大，因为预计它将 提供对支持肿瘤细胞侵袭机制的基本细胞外调节元件的洞察。

PROJECT SUMMARY Although secreted molecular chaperone heat shock protein 90 (eHSP90) has been implicated in tumor cell invasion through binding and stabilizing extracellular matrix protease client MMP2, our limited understanding of the regulatory mechanisms that control the eHSP90 chaperone system is a major drawback for designing successful HSP90-targeted therapies. Our long-term goal is to develop therapeutic strategies that prevent tumor cell invasion by targeting the extracellular HSP90 chaperone machinery. Our overall objective is to elucidate how eHSP90 and its regulators are mechanistically modulated, individually and collectively, and in the presence of their extracellular inhibitors. Our central hypothesis is that extracellular ATP, and tyrosine phosphorylation of eHSP90 co-chaperone TIMP2, modulate the chaperoning of client MMP2, and affect HSP90 binding to extracellular inhibitors. This hypothesis is based on our recent published work and preliminary studies showing that eHSP90 binds and hydrolyzes ATP, and that co-chaperone TIMP2 enhances HSP90 binding to ATP and its pharmacologic inhibitors. Our rationale is that by elucidating mechanisms of eHSP90 chaperone regulation, we will design better therapies for diseases that implicate the eHSP90 machinery. We plan to test our central hypothesis by pursuing the following three specific aims: 1) Investigate the role of ATPase activity in the regulation of extracellular HSP90 (eHSP90); 2) Determine the impact of tyrosine phosphorylation of TIMP2 on its co-chaperone function; and 3) Elucidate the role of TIMP2 in enhancing eHSP90 binding to extracellular inhibitors. Under the first aim, approaches to be used are already established in PI's laboratory and include purification of intracellular and extracellular HSP90 from human cell cultures, isothermal calorimetry to determine affinity towards an ATP analog, ATPase assays to measure ATPase activity, enzyme kinetics to determine proteolytic activity, and in vitro and in vivo protein-protein interactions in the presence and following depletion of extracellular ATP in genetic knock out cell cultures. Under the second aim, phosphomimetic or non-phosphorylatable TIMP2 mutants designed in the PI's laboratory will be used, CRISPR knock out cell lines for c-Src kinase, TIMP2 and MMP2 will be employed for protein-protein interactions and to determine impact on MMP2 activity. Under the third aim, the role of TIMP2, non-phosphorylatable and phophomimetic mutants in enhanced binding of eHSP90 to its extracellular inhibitors will be evaluated in drug binding assays performed in vitro and extracellularly in vivo, using human cell lines and siRNA technology. The impact of TIMP2, and eHSP90 inhibitors on MMP2 mediated proteolysis will be evaluated in matrix degradation studies. The research approach is innovative, in the PI's opinion, because it represents a new and substantive departure from the status quo by shifting focus towards targeting chaperone-regulated extracellular pathways. The proposed research is significant because, it is expected to provide insight on the basic extracellular regulatory elements that support mechanisms of tumor cell invasion.