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: 10297184
• 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/10297184
• 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）的多药抗性蛋白（MRP）家族的成员 转运蛋白在2个主要的主要真菌病原体（包括念珠菌物种）中有助于药物耐受性 方法：1）他们对细胞毒性分子的细胞排毒，例如抗真菌药物和电力药物/氧化剂 通过将它们隔离在液泡中，2）它们引起复杂的形态变化，例如菌丝 延伸和生物膜形成与药物耐受性有关。由于它们在这些生存中的作用 机理，MRP家族成员通常需要感染，并且受细胞严格调节。 该提案的总体目的是弥合我们对这些运输者的理解的理解 导致真菌病原体耐药性威胁的日益增加。机械模型 解释这种抗药性尤其缺乏，限制了抗真菌治疗功效。长期目标是 了解抗真菌治疗的失败。为此，我们特别关注早期的反压力 通过两种突出的液泡MRP的酶，结构和细胞研究的反应 转运蛋白：YCF1和MLT1。我们的理由是，这些见解将为利用 运输架构的独特方面，以生成克服的更有效的疗法 真菌感染中的药物耐受性。 我们的初步工作确定了在真菌应力期间MRP家族转运蛋白调节的关键特征 回答。这些是复杂的底物结合位点，并且调节了 本质上无序的区域称为调节域（R-域）通过磷酸化。我们的中心 假设是MRP转运蛋白受保守之外的域插入的控制 调节多段底物结合位点的转运蛋白折叠。我们的特定目标测试 MPR转运蛋白中的假设是定义的：（1）运输跨运输所采用的监管架构 循环，（2）R域磷酸化在催化调节中的功能和结构作用，以及（3） 膜稳态中底物选择和脂质转运的分子基础。 该应用程序采用一种创新的多学科方法，该方法适用于膜上的进步 蛋白质的生物化学和电子冷冻微观显微镜，以结构和生物物理不足 真菌整合膜蛋白。鉴于念珠菌感染的威胁日益增长 我们的提议是双重的。首先，我们将建立一个理解的机械框架 由重要念珠菌毒力因子及其酵母菌驱动的治疗衰竭的分子基础 同源物。其次，我们将建立一个针对开发反对变构治疗剂有用的基础 与真菌MRP家族转运蛋白相关的药物耐受性，对所有MRP转运蛋白具有一般适用性。