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

• 批准号: 7073770
• 负责人: JOHN E GOLIN
• 金额: $ 19.56万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073770
• 关键词: Saccharomyces cerevisiae; binding sites; bioinformatics; fungal genetics; fungal proteins; hydrogen bond; membrane transport proteins; molecular genetics; multidrug resistance; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): Yeast cells contain several ABC (ATP-binding cassette) transmembrane proteins that mediate broad- spectrum drug resistance. The Pdr5p multidrug transporter is required for resistance to a large range of chemically and mechanistically distinct substrates. Previous work by our group used several transport assays to establish that substrate size is critical for this interaction. We also demonstrated that Pdr5p has three substrate-binding sites and that the number and organization of electronegative groups is partly responsible for site specificity. Pdr5p-substrate specificity is different from its mammalian counterparts such as P-glycoprotein. In consultation with Dr. Di Xia, we used bioinformatics to define the core residues of the transmembrane helical domains (TMHs). The major goal of this proposal is to use site-directed mutagenesis to identify amino acids present in the TMHs that serve as hydrogen-bond donors at the Pdr5p substrate- binding sites. Very little is known about the molecular basis of substrate / multidrug-transporter interaction in eukaryotes. Such information is of considerable clinical relevance, however, as multidrug resistance is a major problem in the treatment of fungal and parasitic pathogens. An understanding of the molecular basis of substrate specificity may help in the design of better chemotherapeutic agents or inhibitors of transporter function. Successful implementation of this proposal will have impact beyond the training of graduate students. As described further, we have strong collaboration with Dr. Suresh V. Ambudkar and are now consulting with Dr. Di Xia (NIH/NCI/Laboratory of Cell Biology). We also train numerous undergraduates and summer interns.

描述（由申请人提供）：酵母细胞含有几种介导广谱耐药的ABC （atp结合盒）跨膜蛋白。Pdr5p多药转运体是对大范围化学和机械不同底物的抗性所必需的。我们小组以前的工作使用了几种传输分析来确定底物大小对这种相互作用至关重要。我们还证明Pdr5p有三个底物结合位点，并且电负性基团的数量和组织部分负责位点特异性。pdr5p底物特异性不同于其在哺乳动物中的对应物，如p糖蛋白。在与Dr. Di Xia的咨询下，我们使用生物信息学来定义跨膜螺旋结构域（TMHs）的核心残基。本研究的主要目标是利用位点定向诱变技术来鉴定存在于TMHs中的氨基酸，这些氨基酸在Pdr5p底物结合位点上充当氢键供体。我们对真核生物中底物/多药物转运体相互作用的分子基础知之甚少。然而，这些信息具有相当大的临床相关性，因为多药耐药是治疗真菌和寄生虫病原体的主要问题。了解底物特异性的分子基础可能有助于设计更好的化疗药物或转运蛋白功能抑制剂。这一建议的成功实施将产生超出研究生培养的影响。正如进一步描述的那样，我们与Suresh V. Ambudkar博士有强有力的合作，现在正在与Di Xia博士（NIH/NCI/细胞生物学实验室）进行磋商。我们还培养了大量的本科生和暑期实习生。