HUMAN MITOCHONDRIAL FOLATE/ANTIFOLATE TRANSPORT

描述(申请人提供)：线粒体的内膜有几种嵌入的转运蛋白，有助于代谢物和辅因子转移到线粒体中。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浆输送到哺乳动物细胞的基质中。酵母基因组中可识别的线粒体内壁转运体有35个，其中只有13个的功能已知。我们最近克隆了一个编码内部线粒体蛋白的cDNAs，该蛋白有助于叶酸辅助因子向线粒体的运输。有数据表明，在人类中看到的四氢叶酸抗代谢药物的毒性的一个组成部分可能是由于抑制了这种转运蛋白。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。 在这一应用中，我们建议研究这种转运蛋白的机制，确定在细胞中发现的各种形式的叶酸以及用于治疗人类癌症的各种抗叶酸的底物特异性。转运过程将在分离的线粒体和重组成蛋白脂双层的重组蛋白中进行研究。线粒体膜内外结合叶酸的蛋白质以及参与线粒体内膜或外膜运输的任何结合伙伴都将被定义。缺乏这种转运蛋白的基因工程小鼠的表型将作为人类线粒体叶酸运输遗传缺陷的模型进行研究。叶酸在该转运体中的结合位置和跨膜区的方向将通过定点突变、多肽的随机突变和表位映射来确定。 这些研究将有助于了解叶酸转运过程的基本生物化学，并有助于了解叶酸内膜转运体在抗叶酸毒性和人类叶酸代谢遗传性疾病中的作用。

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.