Relating protein interaction networks to physiology by systematic mutant analyses

通过系统突变分析将蛋白质相互作用网络与生理学联系起来

• 批准号: 8991326
• 负责人: DANIEL N BOLON
• 金额: $ 32.24万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8991326
• 关键词: ATP phosphohydrolase; Address; Adverse effects; Alleles; Allosteric Site; Amino Acid Substitution; Anabolism; Antifungal Agents; Antineoplastic Agents; Azole resistance; Binding Sites; Biological; Calcineurin; Client; Complement; Complex; Dependency; Development; Disease; Drug resistance; Engineering; Ergosterol; Eukaryota; Exhibits; Flow Cytometry; Fluconazole; Frequencies; Fungal Drug Resistance; Gene Deletion; Genes; Genetic; Genetic Epistasis; Genomics; Glucocorticoid Receptor; Goals; Growth; Health; Human; Individual; Industrial fungicide; Knock-out; Lead; Link; Malignant Neoplasms; Molecular Chaperones; Motivation; Mutation; Mycoses; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Physiology; Point Mutation; Property; Proteins; Reporter; Research; Resistance; Role; Saccharomycetales; Series; Signaling Protein; Site; System; Therapeutic; Therapeutic Index; Transcription Coactivator; Ubiquitin; Yeasts; biological systems; calcineurin phosphatase; combat; fitness; fungus; gene function; human disease; inhibitor/antagonist; insight; mutant; protein degradation; research study; resistance mechanism; signal processing

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.

描述（由申请人提供）：Hsp90 是一种重要的真核伴侣，有助于产生和维持一组选定的底物或客户的活性状态，这些底物或客户与信号传导过程密切相关，包括许多促进癌症和真菌耐药性出现的信号传导过程。由于这些原因，操纵 Hsp90 伴侣系统的抑制剂策略有望带来许多潜在的治疗益处。针对 Hsp90 ATP 酶位点的抑制剂可有效限制真菌中耐药性的出现，并显示出作为抗癌药物的前景。然而，ATP 酶抑制剂会阻断 Hsp90 的所有已知功能，从而导致不良副作用。在本提案中，我们将确定对 Hsp90 伴侣系统的数千种特定扰动如何影响真菌中耐药性的出现以及三个特定客户的成熟，以便系统地识别可作为客户选择性抑制剂开发的 Hsp90 辅助伴侣区域。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以及与共伴侣的敲除和/或亚等位基因相结合对酵母生长的影响。我们将增强这些适应性研究，通过使用酵母报告策略对特定客户进行分析，整合 Hsp90 突变体对许多客户功能的影响。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，我们的研究将描绘客户和共伴侣结合位点以及 Hsp90 上的变构位点，这些位点对于特定客户的成熟和真菌耐药性的出现至关重要。