HUMAN GAMMA GLUTAMYL HYDROLASE

描述(改编自《调查者摘要》)：叶酸 合成细胞生长所需的代谢物所必需的。 因此，叶酸代谢途径一直是治疗的靶点。 目的是使用反叶酸来阻止细胞分裂。细胞摄取 抗叶酸单谷氨酸盐通过特定的转运蛋白发生。细胞内 经典的抗叶酸被聚谷氨酸转化为聚γ-谷氨酸 合成酶。多聚伽马-谷氨酸衍生物不能通过细胞 膜，在细胞内浓缩，可以是更好的底物或 叶酸依赖酶的抑制剂。反叶的形成 多聚-γ-谷氨酸和细胞滞留通过去除 聚γ-谷氨酸尾巴由一种鲜为人知的酶催化， 谷氨酰水解酶。细胞叶酸动态平衡及其疗效 某些抗叶酸，如甲氨蝶呤，是由 这两种酶的活性。拟议研究的目标是 明确人类γ-谷氨酰基作用机制的分子细节 水解酶(HGH)，并确定促成 这种酶的特殊作用方式。HGH的作用机制将是 通过使用位点定向识别活性位点残基进行调查 诱变以测试该酶催化折叠的分子模型。 结合底物聚γ-谷氨酸链的氨基酸将是 通过定点突变进行鉴定。建筑的三维结构 酶将通过X射线结晶学进行测定。用cDNAs或 将使用反义构建物来改变癌细胞中hGH的表达 确定这种酶在抗叶酸和氟尿嘧啶作用中的作用。这些 研究将确定生长激素是否为电流调制的潜在部位 用这些药物进行治疗。已鉴定的细胞的特征 人类γ-谷氨酰水解酶的抑制剂也将进行。这个 HGH的特异性抑制是确定其在叶酸中的作用所必需的 动态平衡和抗叶酸作用。拟议的研究结果可能会导致 对于人类伽马-谷氨酰水解酶特异性抑制剂的开发， 它可以与已建立的抗叶酸或 氟嘧啶类药物在人类癌症治疗中的应用。

DESCRIPTION (As Adapted From the Investigator's Abstract): Folic acid is necessary for the synthesis of metabolites that are required for cell growth. For this reason, the folate metabolic pathways have been targets for therapies aimed at blocking cell division using antifolates. Cellular uptake of antifolate monoglutamate occurs via specific transporters. Intracellular classical antifolates are converted to poly-gamma-glutamates by polyglutamate synthetase. The poly-gamma-glutamate derivatives cannot cross the cell membrane, concentrate intracellularly, and can be better substrates or inhibitors for the folate dependent enzymes. Formation of antifolyl poly-gamma-glutamates and cellular retention are countered by removal of the poly-gamma-glutamate tail catalyzed by a poorly understood enzyme, gamma-glutamyl hydrolase. Cellular folate homeostasis and the efficacy of certain antifolates such as methotrexate are determined by the balance between the activities of these two enzymes. The goal of the proposed research is to define the molecular details of the mechanism of action of human gamma-glutamyl hydrolase (hGH) and to determine the structural features that contributed to the specific mode of action of this enzyme. The mechanism of hGH will be investigated by identifying the active site residues using site directed mutagenesis to test a molecular model for the catalytic fold of the enzyme. Amino acids, which bind the poly-gamma-glutamate chain of substrates will be identified by site directed mutagenesis. The three dimensional structure of the enzyme will be determined by X-ray crystallography. Transfection with cDNA or antisense constructs will be used to alter hGH expression in cancer cells to establish the role of this enzyme in antifolate and fluorouracil action. These studies will determine if GH is a potential site for modulation of current therapy with these drugs. The characterization of an identified cellular inhibitor of human gamma-glutamyl hydrolase will also be carried out. The specific inhibition of hGH is necessary to determine its role in folate homeostasis and antifolate action. The results of the proposed studies may lead to the development of specific inhibitors of human gamma-glutamyl hydrolase, which could be used in conjunction with established antifolates or fluoropyrimidines in the treatment of human cancers.