ATP Binding Cassette (ABC) Transporters in Fungal Drug Tolerance

三磷酸腺苷结合盒多药耐药蛋白家族成员 转运蛋白在包括念珠菌在内的主要真菌病原体中对药物耐受性有贡献 方法：1)它们使细胞中的细胞毒性分子解毒，如抗真菌药物和亲电剂/氧化剂 通过将它们隔离在液泡中，以及2)它们引起复杂的形态变化，如菌丝 细菌的延伸和生物膜的形成与药物耐受性有关。因为他们在这些生存中所扮演的角色 在这些机制中，MRP家族成员往往是感染所必需的，并受到细胞的严格调控。 这项提议的总体目标是弥合我们对这些运输者如何 导致真菌病原体的耐药性威胁日益增加。机械论模型 解释这种耐药性尤其缺乏，限制了抗真菌治疗的效果。长期目标是 以了解抗真菌治疗是如何失败的。为此，我们特别关注早期的抗压力 两种重要的空泡MRP的酶、结构和细胞研究反应 转运蛋白：Ycf1和Mlt1。我们的理由是，这些洞察力将为利用 传输器架构的独特方面，以产生更有效的疗法，克服 真菌感染的药物耐受性。 我们的初步工作确定了真菌胁迫过程中MRP家族转运蛋白调控的关键特征 回应。这些都是复杂的底物结合部位和调节的不同状态的一个 通过磷酸化，内在的无序区域被称为调节域(R-域)。我们的中央 假设MRP转运蛋白是由保守序列外的结构域插入控制的 调节多段底物结合位点的转运蛋白折叠。我们的具体目标是测试这一点 MPR转运体的假说将定义：(1)跨转运体采用的监管架构 循环，(2)R结构域磷酸化在催化调节中的功能和结构作用，以及(3) 膜动态平衡中底物选择和脂质转运的分子基础。 这项应用使用了一种创新的多学科方法，将膜方面的进展应用于 蛋白质生物化学和电子冷冻显微镜对结构和生物物理特性的不足 真菌整体膜蛋白。鉴于念珠菌感染的威胁越来越大，这一意义 我们的建议有两个方面。首先，我们将建立一个机制框架，以理解 重要念珠菌毒力因子及其酵母菌治疗失败的分子基础 同系物。第二，我们将为发展变构疗法奠定基础。 与真菌MRP家族转运蛋白相关的药物耐受性，普遍适用于所有MRP转运蛋白。

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.