HUMAN MITOCHONDRIAL FOLATE/ANTIFOLATE TRANSPORT

描述（由申请人提供）：线粒体的内膜具有几种嵌入的转运蛋白，其促进代谢物和辅因子易位到线粒体中。这些蛋白质与外膜中的机制协同工作，将阳离子和阴离子代谢中间体从胞质溶胶带到哺乳动物细胞的基质。在酵母基因组中有35种线粒体内壁转运蛋白，只有13种的功能是已知的。我们最近克隆了一个编码线粒体内蛋白的cDNA，该蛋白促进叶酸辅因子转运到线粒体中。有数据表明，在人体中观察到的四氢叶酸抗代谢物毒性的一个组成部分可能是由于抑制该转运蛋白。缺乏这种转运蛋白的哺乳动物细胞在没有甘氨酸的情况下不能存活。 在本申请中，我们建议研究这种转运蛋白的机制，定义细胞中发现的各种形式的叶酸以及已用于治疗人类癌症的各种抗叶酸剂的底物特异性。运输过程将在分离的线粒体和重组蛋白重组成蛋白脂质双层进行研究。将定义线粒体外膜和内膜中结合叶酸的蛋白质，以及参与通过线粒体内膜或外膜转运的任何结合配偶体。基因工程改造缺乏这种转运蛋白的小鼠的表型将作为线粒体叶酸转运的人类遗传缺陷的模型进行研究。叶酸在该转运蛋白中的结合位点和跨膜结构域的方向将通过定点诱变、肽的随机诱变和表位作图来确定。 这些研究将导致对这种运输过程的基本生物化学的理解，并将导致对内膜叶酸转运蛋白在抗叶酸毒性和叶酸代谢的人类遗传疾病中的作用的理解。

DESCRIPTION (provided by applicant): The inner membrane of the mitochondria has several embedded transport proteins that facilitate the translocation of metabolites and cofactors into the mitochondria. These proteins work in concert with mechanisms in the outer membrane to bring cationic and anionic metabolic intermediates from the cytosol to the matrix of mammalian cells. There are 35 inner mitochondrial wall transporters recognizable in the yeast genome; the function of only 13 are known. We have recently cloned a cDNA that encodes an inner mitochondrial protein that facilitates the transport of folate cofactors into the mitochondria. There are data suggesting that a component of the toxicity of the tetrahydrofolate antimetabolites seen in man may be due to inhibition of this transporter. Mammalian cells deficient in this transporter cannot survive in the absence of glycine. In this application, we propose to study the mechanism of this transporter, defining the substrate specificity for the various forms of the folates found in cells and also for the various antifolates that have been used to treat human cancers. Transport process will be studied in isolated mitochondria and in recombinant protein reconstituted into proteolipid bilayers. The proteins in the outer and inner mitochondrial membrane that bind folates will be defined, as will any binding partners involved in the transport through inner or outer membrane of the mitochondria. The phenotype of mice genetically engineered to lack this transporter will be studied as a model for human genetic deficiencies of mitochondrial folate transport. The binding site for folates in this transporter and the orientation of the transmembrane domains will be determined by site-directed mutagenesis, random mutagenesis of peptides, and epitope mapping. These studies will lead to an understanding of the basic biochemistry of this transport process and will lead to an understanding of the role of the inner membrane folate transporter in antifolate toxicity and in human genetic disorders of folate metabolism.