HUMAN GAMMA GLUTAMYL HYDROLASE

描述（改编自研究者摘要）：叶酸是 这是合成细胞生长所需的代谢物所必需的。 由于这个原因，叶酸代谢途径一直是治疗的目标 旨在用抗叶酸剂阻止细胞分裂。的细胞摄取 抗叶酸单谷氨酸盐通过特异性转运蛋白产生。细胞内 典型的抗叶酸剂通过聚谷氨酸盐转化为聚-γ-谷氨酸盐 合成酶聚-γ-谷氨酸衍生物不能穿过细胞 膜，在细胞内浓缩，并且可以是更好的底物或 叶酸依赖酶的抑制剂。安替比林的形成 聚-γ-谷氨酸盐和细胞滞留通过去除 聚-γ-谷氨酸尾部由一种知之甚少的酶催化， γ-谷氨酰水解酶。细胞内叶酸稳态和叶酸的功效 某些抗叶酸剂如甲氨蝶呤是通过以下平衡来确定的 这两种酶的活性。拟议研究的目标是 定义人γ-谷氨酰作用机制的分子细节 水解酶（hGH），并确定结构特征，有助于 这种酶的特殊作用方式hGH的机制将是 通过使用位点定向鉴定活性位点残基来研究 诱变以测试酶的催化折叠的分子模型。 结合底物的聚-γ-谷氨酸链的氨基酸将被 通过定点诱变鉴定。的三维结构 将通过X射线晶体学测定酶。用cDNA或 反义构建体将用于改变癌细胞中的hGH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GH是否是调制电流的潜在位点 治疗这些药物。鉴定的细胞的表征 还将进行人γ-谷氨酰水解酶抑制剂。的 对hGH的特异性抑制对于确定其在叶酸中的作用是必要的 体内平衡和抗叶酸作用。研究结果可能会导致 涉及人γ-谷氨酰水解酶的特异性抑制剂的开发， 其可与已建立的抗叶酸剂结合使用， 氟嘧啶在治疗人类癌症中的应用。

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.