Molecular Genetic Analysis of Pdr5p, a Major Yeast Multidrug Transporter

描述（由申请人提供）：酵母细胞含有几种ABC（ATP结合盒）跨膜蛋白，其介导广谱耐药性。Pdr 5 p多药转运蛋白是对大范围的化学和机械上不同的底物具有抗性所必需的。我们小组以前的工作使用了几种转运试验来确定底物大小对这种相互作用至关重要。我们还表明，Pdr 5 p有三个底物结合位点，负电性基团的数量和组织是部分负责位点特异性。Pdr 5 p-底物特异性不同于其哺乳动物对应物如P-糖蛋白。在与夏棣博士的协商中，我们使用生物信息学来定义跨膜螺旋结构域（TMH）的核心残基。该提议的主要目标是使用定点诱变来鉴定TMH中存在的在Pdr 5 p底物结合位点处充当氢键供体的氨基酸。关于真核生物中底物/多药转运蛋白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意义，因为多药耐药性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。这项建议的成功实施将产生超出研究生培训范围的影响。如进一步所述，我们与Suresh V. Ambudkar博士有着密切的合作，现在正在与Di Xia博士（NIH/NCI/细胞生物学实验室）进行咨询。我们还培训了许多本科生和暑期实习生。

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.