Regulation of the extracellular HSP90 chaperone machinery

项目摘要 虽然分泌型分子伴侣热休克蛋白90（eHSP 90）与肿瘤细胞的增殖和分化有关， 通过结合和稳定细胞外基质蛋白酶客户MMP 2入侵，我们对 控制eHSP 90分子伴侣系统的调节机制是设计 成功的HSP 90靶向治疗。我们的长期目标是开发治疗策略， 通过靶向细胞外HSP 90分子伴侣机制抑制肿瘤细胞侵袭。我们的总体目标是 阐明eHSP 90及其调节因子是如何单独和共同地进行机械调节的， 细胞外抑制剂的存在。我们的中心假设是细胞外ATP和酪氨酸 eHSP 90共伴侣TIMP 2的磷酸化，调节客户MMP 2的伴侣作用，并影响 HSP 90与细胞外抑制剂的结合。这个假设是基于我们最近发表的工作， 初步研究表明，eHSP 90结合并水解ATP，辅助伴侣TIMP 2增强了 HSP 90与ATP的结合及其药理学抑制剂。我们的基本原理是，通过阐明 eHSP 90伴侣调节，我们将设计更好的治疗方法，涉及eHSP 90 机械.我们计划通过追求以下三个具体目标来测试我们的中心假设：1）调查 ATP酶活性在细胞外热休克蛋白90（eHSP 90）调节中的作用; 2）确定 TIMP 2的酪氨酸磷酸化对其共伴侣功能的影响;和3）阐明TIMP 2在 增强eHSP 90与细胞外抑制剂的结合。在第一个目标下，已经采用了各种方法， 包括从人细胞中纯化细胞内和细胞外HSP 90 培养物，等温量热法以确定对ATP类似物的亲和力，ATP酶测定以测量 ATP酶活性、测定蛋白水解活性的酶动力学以及体外和体内蛋白质-蛋白质 在遗传敲除细胞培养物中，在细胞外ATP存在下和耗尽后的相互作用。 在第二个目标下，在PI中设计磷酸化模拟物或不可磷酸化的TIMP 2突变体， 将使用CRISPR敲除c-Src激酶、TIMP 2和MMP 2的细胞系， 蛋白质-蛋白质相互作用并确定对MMP 2活性的影响。在第三个目标下，TIMP 2的作用， 非磷酸化和拟磷酸化突变体增强eHSP 90与其细胞外 抑制剂将在体外和细胞外体内进行的药物结合试验中进行评价，使用人 细胞系和siRNA技术。TIMP 2和eHSP 90抑制剂对MMP 2介导的蛋白水解的影响 将在基质降解研究中进行评价。PI认为，研究方法具有创新性， 因为它代表了一种新的、实质性的脱离现状的做法，将重点转向瞄准目标， 分子伴侣调节的细胞外途径。这项研究意义重大，因为预计 提供对支持肿瘤细胞侵袭机制的基本细胞外调节元件的见解。

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.