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Molecular Genetic Analysis of Pdr5p, a Major Yeast Multidrug Transporter

主要酵母多药转运蛋白 Pdr5p 的分子遗传学分析

基本信息

批准号：
9376625
负责人：
John Golin
金额：
$ 47万
依托单位：
CATHOLIC UNIVERSITY OF AMERICA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9376625
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Affinity Alanine Antibiotic Resistance Antibiotics Antigens Back Binding Biochemical Biochemical Genetics Biochemical Process Bioinformatics Biological Assay Cells Clinical Collection Communication Conserved Sequence Coupled Disease Drug Binding Site Drug Hypersensitivity Drug Transport Drug resistance Eukaryota Frequencies Hydrolysis Laboratories Malignant Neoplasms Medical Molecular Molecular Conformation Molecular Genetics Mutate Mutation Nature Pharmaceutical Preparations Phenotype Process Property Reflux Reporting Research Support Role Scanning Series Signal Transduction Site Site-Directed Mutagenesis Structure Substrate Specificity Transmembrane Domain United States National Institutes of Health Work Xenobiotics Yeasts chemotherapeutic agent design efflux pump extracellular genetic analysis genetic approach member multi drug transporter mutant novel operation overexpression pathogen prevent transmission process

项目摘要

Project Summary Overexpression of multidrug transporters leading to increased, broad-spectrum drug resistance is a major medical problem in the treatment of pathogens and malignant tumors. We use biochemical and genetic approaches to study Pdr5, a major yeast efflux pump that is the founding member of a large subfamily of clinically important fungal ABC transporters. This proposal focuses on a central issue common to all ABC exporters. Once a toxic drug is expelled from the cell, how is it prevented from reentry via the drug-binding sites which are now facing in an extracellular direction? A simple 10-30 fold reduction in affinity which is typically seen during the transport cycle is not sufficient to preclude reflux. Work in our laboratory established that Pdr5 transport is unidirectional and that this efflux pump is therefore a molecular diode. We also identified one residue, Ser-1368 that is essential for diode function. A substitution creating a S1368A mutant shows considerable reflux when a novel diode assay is performed. The present proposal has two aims: First, using a combination of mutant suppression and site-directed mutagenesis, we will identify additional residues that make up the Pdr5 diode. These mutants will be carefully characterized using several biochemical assays including one that is specific for diode function. A central issue is whether both halves of this transporter participate in diode formation or whether like several other biochemical processes diode function is asymmetric. The second aim of the proposal is to biochemically characterize the diode. For instance, we will determine whether there is any substrate specificity to diode operation. The role of ATP hydrolysis and the well-established Pdr5 transmission interface in diode function will be evaluated.

项目概要多药转运蛋白的过度表达导致广谱耐药性增加治疗病原体和恶性肿瘤的重大医学问题。我们使用生化和研究 Pdr5 的遗传方法，Pdr5 是一种主要的酵母外排泵，是大型酵母菌的创始成员临床上重要的真菌 ABC 转运蛋白亚家族。该提案重点关注所有 ABC 出口商共同的核心问题。一旦有毒药物被排出体外细胞，如何防止它通过目前面临的药物结合位点重新进入细胞外方向？亲和力简单地降低 10-30 倍，通常在运输周期不足以防止回流。我们实验室的工作证实 Pdr5 传输是单向的，因此该外排泵是分子二极管。我们还确定了 Ser-1368 是二极管功能所必需的一个残基。产生 S1368A 突变体的替代当进行新型二极管测定时显示出相当大的回流。目前的提案有两个目标：首先，结合使用突变抑制和定点突变诱变后，我们将鉴定构成 Pdr5 二极管的其他残基。这些突变体将是使用多种生化分析仔细表征，其中包括一种特定于二极管功能的分析。一个中心问题是该转运蛋白的两半是否都参与二极管的形成，或者是否像其他几个生化过程中二极管的功能是不对称的。该提案的第二个目标是对二极管进行生化表征。例如，我们将确定是否有任何基材二极管操作的特殊性。 ATP 水解的作用和成熟的 Pdr5 传输将评估二极管功能中的接口。