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首页> 外文期刊>Advances in enzyme regulation >Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity.
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Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity.

机译：Folylpoly-γ-谷氨酸合成酶：同工酶的产生以及在一个碳代谢和抗叶酸细胞毒性中的作用。

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A single human gene encodes both mitochondrial and cytosolic isoforms of the enzyme. The major mRNA species in human cells encodes the mitochondrial isoform but alternate translation initiation at a downstream in-frame ATG also generates the cytosolic isoform. Cytosolic FPGS may also be generated by use of alternate transcription initiation start sites 3' to the start ATG of the mitochondrial FPGS. Three additional human FPGS mRNAs differing in exon 1 have been identified. One of these is a major species in HEP-G2 cells and other tissue culture cells, and can encode a protein lacking the first 8 amino acids of cytosolic FPGS. A protein of the predicted size is observed in coupled transcription/translation systems. However, expression of this protein in E. coli does not generate an active enzyme. Mutagenesis studies indicate that Tyr-3 of the missing N terminal residues is required for enzyme activity. The major cellular folate pools are in the cytosol and mitochondria and FPGS activity is normally distributed in both compartments. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, and cells lacking this isozyme are auxotrophic for glycine. Overexpression of cytosolic FPGS does not complement the lack of mitochondrial activity. Cells expressing FPGS activity solely in the mitochondria are glycine prototrophs, but also possess cytosolic folylpolyglutamates and are prototrophic for thymidine and purines, products of cytosolic one carbon metabolism. Although cytosolic folylpolyglutamates cannot enter the mitochondrion, mitochondrial folylpolyglutamates are released intact into the cytosolic compartment. Cellular accumulation of some antifolates and their cytotoxic efficacy is highly responsive to the level of FPGS activity. Polyglutamylation of methotrexate (MTX) has little affect on its affinity for dihydrofolate reductase, its target enzyme, but does affect the cellular accumulation of the drug. The sensitivity of model cells, expressing a range of FPGS activities similar to that observed in leukemia blasts, to MTX varied over four orders of magnitude. MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX. The cytotoxic efficacy of other folate antagonists that are transported into the mitochondria was enhanced by mitochondrial FPGS activity, even when their loci of inhibition was a cytosolic enzyme. Mitochondrial metabolism of these drugs increased cytosolic drug levels. Compartmentalization of antifolate metabolism has to be considered in evaluating mechanisms for increased drug cytotoxicity and for the development of acquired resistance to these agents.
机译：单个人类基因编码该酶的线粒体和胞质异构体。人细胞中的主要mRNA种类编码线粒体同工型，但在下游读框ATG处的交替翻译起始也产生了胞质同工型。胞质FPGS也可以通过使用与线粒体FPGS的起始ATG交替的转录起始起始位点3'来产生。已经鉴定出在外显子1上不同的三个其他人FPGS mRNA。其中之一是HEP-G2细胞和其他组织培养细胞中的主要物种，可以编码缺少胞质FPGS的前8个氨基酸的蛋白质。在偶联的转录/翻译系统中观察到了预测大小的蛋白质。但是，这种蛋白质在大肠杆菌中的表达不会产生活性酶。诱变研究表明，缺少N末端残基的Tyr-3是酶活性所必需的。主要的细胞叶酸池在细胞质和线粒体中，FPGS活性通常分布在两个区室中。线粒体叶酸积累需要线粒体FPGS活性，而缺乏这种同工酶的细胞对甘氨酸营养缺陷。胞浆FPGS的过表达不能弥补线粒体活性的缺乏。仅在线粒体中表达FPGS活性的细胞是甘氨酸原养型，但也具有胞质叶酰聚谷氨酸盐，并且对于胸苷和嘌呤是胞质一碳代谢产物。尽管胞质叶酰聚谷氨酸盐不能进入线粒体，但线粒体叶酰聚谷氨酸盐会完整地释放到胞质区室中。一些抗叶酸药物的细胞蓄积及其细胞毒性功效对FPGS活性水平高度敏感。甲氨蝶呤（MTX）的聚谷氨酰化对其靶酶二氢叶酸还原酶的亲和力影响很小，但确实会影响药物的细胞蓄积。表达一系列FPGS活性（类似于白血病母细胞中观察到的）的模型细胞对MTX的敏感性变化了四个数量级。 MTX的毒性取决于胞质FPGS的活性，因为该药物不会进入线粒体，仅在线粒体中表达非常高水平的FPGS的细胞对MTX耐药。转运至线粒体的其他叶酸拮抗剂的细胞毒功效通过线粒体FPGS活性得以增强，即使它们的抑制位点是胞质酶。这些药物的线粒体代谢增加了胞质药物水平。在评估增加药物细胞毒性和发展对这些药物的耐药性的机制时，必须考虑抗叶酸代谢的分区。

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