Relating protein interaction networks to physiology by systematic mutant analyses

描述（由申请人提供）：Hsp90是一种重要的真核生物伴侣，有助于产生和维持一组选择的底物或客户的活性状态，这些底物或客户与信号过程有不相称的联系，包括许多促进真菌癌症和耐药性出现的信号过程。由于这些原因，操纵Hsp90伴侣系统的抑制剂策略承诺了许多潜在的治疗益处。靶向Hsp90三磷酸腺苷酶位点的抑制剂有效地限制了真菌耐药的出现，有望成为抗癌药物。然而，atp酶抑制剂阻断了Hsp90的所有已知功能，导致不良的副作用。在这一提议中，我们将确定数千种对Hsp90伴侣系统的特定扰动如何影响真菌耐药的出现和三种特定客户的成熟，以便系统地确定Hsp90合作伴侣的区域，这些区域可以作为开发客户选择性抑制剂的目标。为了实现这些目标，我们将使用我们开发的经验方法来确定Hsp90中所有可能的点突变在存在和不存在抗真菌药物的情况下对酵母生长的影响，并结合敲除和/或共伴侣的次形等位基因。我们将扩大这些适应性研究，整合Hsp90突变体对许多客户功能的影响，并使用酵母报告策略对特定客户进行分析。这些对环境和遗传上位性的分析将为真核生物信号传导过程中Hsp90伴侣系统的网络特性提供重要的机制见解。此外，我们的研究将描述客户端和共同伴侣结合位点以及Hsp90上的变构位点，这些位点对特定客户端成熟和真菌耐药的出现至关重要。

DESCRIPTION (provided by applicant): Hsp90 is an essential eukaryotic chaperone that helps to produce and maintain the active state of a select set of substrates or clients that are disproportionately linked to signaling processes including many that promote cancer and the emergence of drug resistance in fungi. For these reasons, inhibitor strategies to manipulate the Hsp90 chaperone system promise many potential therapeutic benefits. Inhibitors targeting the ATPase site of Hsp90 effectively limit the emergence of drug resistance in fungi and show promise as anti-cancer agents. However, ATPase inhibitors block all known functions of Hsp90, leading to undesirable side effects. In this proposal, we will determine how thousands of specific perturbations to the Hsp90 chaperone system impact the emergence of drug resistance in fungi and the maturation of three specific clients in order to systematically identify regions of Hsp90 o co-chaperones that could be targeted for the development of client selective inhibitors. To accomplish these goals, we will use the EMPIRIC approach that we developed to determine the effects of all possible point mutations in Hsp90 on yeast growth in the presence and absence of an anti-fungal drug and in combination with knockouts and/or hypomorphic alleles of co-chaperones. We will augment these fitness studies that integrate over the effects of Hsp90 mutants on the function of many clients with analyses of specific clients using yeast reporter strategies. These analyses of environmental and genetic epistasis will provide important mechanistic insights into the network properties of the Hsp90 chaperone system that underlies signaling processes in eukaryotes. In addition, our studies will delineate client and co-chaperone binding sites as well as allosteric sites on Hsp90 that are critical for the maturation o specific clients and for the emergence of drug resistance in fungi.