ATP Binding Cassette (ABC) Transporters in Fungal Drug Tolerance

真菌耐药性中的 ATP 结合盒 (ABC) 转运蛋白

• 批准号: 10434954
• 负责人: Thomas Michael Tomasiak
• 金额: $ 38.06万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10434954
• 关键词: ABCB1 gene; ATP-Binding Cassette Transporters; Adopted; Allosteric Regulation; Amino Acids; Antifungal Agents; Architecture; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biology; Biophysical Process; Biophysics; Cadmium; Candida; Candida albicans; Carrier Proteins; Cell Line; Cell Survival; Cells; Cellular biology; Cessation of life; Charge; Clinical; Complex; Cryoelectron Microscopy; Data; Defect; Disease; Docking; Drug Metabolic Detoxication; Drug Tolerance; Drug resistance; Electron Spin Resonance Spectroscopy; Electrostatics; Elements; Failure; Family member; Foundations; Future; Glutathione; Goals; HIV; Health; Homeostasis; Hybrids; In Vitro; Infection; Integral Membrane Protein; Investigation; Knock-out; Knowledge; Light; Link; Lipids; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular; Molecular Conformation; Molecular Mimicry; Monitor; Morphology; Multi-Drug Resistance; Mycoses; Natural Killer Cells; Nature; Nucleotides; Oxidants; Oxides; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Play; Protein Biochemistry; Protein Family; Proteins; Publishing; Regulation; Resistance; Role; Saccharomyces cerevisiae; Safety; Site; Structure; System; Testing; Therapeutic; Treatment Efficacy; Tuberculosis; Vacuole; Virulence; Virulence Factors; Work; Yeasts; amphiphilicity; base; biological adaptation to stress; crosslink; drug development; effective therapy; flexibility; glutathione transporter; improved; in vivo; innovation; insight; interdisciplinary approach; lipid transport; member; mortality; multi drug transporter; mutant; pathogen; pathogenic fungus; prevent; stress tolerance; therapeutic development; therapeutic target; thermostability

Members of the Multi-drug Resistance Protein (MRP) family of ATP Binding Cassette (ABC) transporters contribute to drug tolerance in major fungal pathogens, including Candida species, in 2 main ways: 1) they detoxify the cell of cytotoxic molecules such as antifungal drugs and electrophiles/oxidants by sequestering them in the vacuole, and 2) they cause complex morphological changes such as hyphal extension and biofilm formation linked to drug tolerance. Because of their role in these survival mechanisms, MRP family members are often required for infection and are tightly regulated by the cell. The overall objective of this proposal is to bridge gaps in our understanding of how these transporters contribute to the increasing threat of drug resistance in fungal pathogens. Mechanistic models that explain this resistance are especially lacking, limiting antifungal treatment efficacy. The long-term goal is to understand how anti-fungal treatments fail. To this end, we specifically focus on early stage anti-stress responses through enzymatic, structural, and cellular investigations of two prominent vacuolar MRP transporters: Ycf1 and Mlt1. Our rationale is that these insights will provide a foundation for exploiting unique aspects of transporter architecture in order to generate more effective therapies that overcome drug tolerance in fungal infections. Our preliminary work identifies key features of MRP family transporter regulation during fungal stress responses. These are complex substrate binding sites and the regulation of different states of an intrinsically disordered region called the Regulatory-domain (R- domain) by phosphorylation. Our central hypothesis is that MRP transporters are governed by domain insertions outside of the conserved transporter fold that regulate multi-segmented substrate binding sites. Our specific aims testing this hypothesis in MPR transporters are to define: (1) the regulatory architectures adopted across transport cycles, (2) the functional and structural roles of R-domain phosphorylation on catalytic regulation, and (3) the molecular basis of substrate selection and lipid transport in membrane homeostasis. This application uses an innovative multidisciplinary approach that applies advances in membrane protein biochemistry and electron cryo-microscopy to structurally and biophysically undercharacterized fungal integral membrane proteins. In light of the growing threat of Candida infections, the significance of our proposal is twofold. First, we will establish a mechanistic framework for understanding the molecular basis of therapeutic failure driven by important Candida virulence factors and their yeast homologs. Second, we will establish a foundation useful for developing allosteric therapeutics against drug-tolerance linked to fungal MRP family transporters, with general applicability to all MRP transporters.

ATP结合盒（ABC）的多药耐药蛋白（MRP）家族成员 转运蛋白有助于主要真菌病原体（包括念珠菌属）的药物耐受性，主要表现在2个方面 途径：1）它们对细胞毒性分子如抗真菌药物和亲电体/氧化剂进行解毒 2）它们引起复杂的形态学变化，如菌丝 延伸和生物膜形成与药物耐受性有关。因为它们在这些生存中的作用 在这种机制中，MRP家族成员通常是感染所必需的，并且受到细胞的严格调控。 本提案的总体目标是弥合我们对这些运输者如何 导致真菌病原体耐药性的威胁日益增加。机械模型， 解释这种耐药性特别缺乏，限制了抗真菌治疗的效果。长期目标是 了解抗真菌治疗是如何失败的为此，我们特别关注早期抗压 通过对两个突出的液泡MRP的酶促、结构和细胞研究的反应 转运蛋白：Ycf 1和Mlt 1。我们的理由是，这些见解将为开发 独特的方面，以产生更有效的治疗，克服 真菌感染的耐药性。 我们的初步工作确定了真菌胁迫下MRP家族转运蛋白调控的关键特征 应答这些都是复杂的底物结合位点，调节不同的状态， 内在无序的区域称为调节结构域（R-结构域）的磷酸化。我们的中央 一种假说是MRP转运蛋白是由保守区外的结构域插入控制的， 调节多节段底物结合位点转运蛋白折叠。我们的具体目标是测试这个 MPR转运蛋白的假设是定义：（1）跨转运蛋白采用的调控结构 循环，（2）R结构域磷酸化对催化调节的功能和结构作用，以及（3） 膜稳态中底物选择和脂质转运的分子基础。 该应用采用创新的多学科方法， 蛋白质生物化学和电子冷冻显微镜在结构上和生物学上表征不足 真菌整合膜蛋白。鉴于念珠菌感染的威胁越来越大， 我们的建议是双重的。首先，我们将建立一个机制框架， 重要念珠菌毒力因子及其酵母菌导致治疗失败的分子基础 同系物。其次，我们将建立一个基础，为发展变构疗法， 与真菌MRP家族转运蛋白相关的药物耐受性，对所有MRP转运蛋白具有普遍适用性。