A LYMPHOBLAST MODEL FOR DISEASES OF PURINE METABOLISM

我们已经确定了含NAD的酶S-腺苷高半胱氨酸的作用， 水解酶（cAMP cyase），也是腺苷（Ado）和cAMP结合酶 蛋白，介导Ado和2'-dAdo对 S-腺苷甲硫氨酸（SNAMet）依赖性转甲基化反应。 的 这项建议的目的是扩大我们的研究纯化的人 评估腺苷脱氨酶中RNA甲基化的抑制 用2'-dAdo（其不可逆地失活 Hcyase）;并评估Hcyase对免疫缺陷的贡献。 遗传或药物诱导的ADA缺乏症。 具体研究计划在3 a）使用有限的蛋白水解作图技术，以进一步 定义核苷、cAMP和NAD结合的组织和功能 的结构域。 蛋白水解图谱和结构域特异性单克隆抗体 抗体将用于确定这些区域是否与 蛋白激酶的cAMP结合亚基或NAD的类似结构域 其他含NAD酶的结合域。 B）研究 2 ′-dAdo对核糖体甲基化作用对胞内腺苷酸酶的失活 有丝分裂原刺激的人外周血淋巴细胞中的RNA前体， 从接受ADA抑制剂治疗的患者获得的细胞 脱氧共福霉素，或与抗病毒剂阿糖腺苷。 我们将 评估在不同细胞类型中谷胱甘肽半胱氨酸酶合成的速率， 淋巴样细胞中选择性失活谷胱甘肽半胱氨酸酶的基础。 替代 还将研究各种细胞类型中β-Hcy催化剂的途径， 的选择性作用的基础。 c）突变淋巴样细胞 将使用或选择细胞系来确定腺嘌呤毒性的基础， 5'-甲硫基腺苷和其他嘌呤类似物;以及用于确定 T细胞中负责磷酸化2'-dAdo的酶的身份。 我们将 尝试分离具有改变的谷胱甘肽半胱氨酸酶活性或产量的突变体。

We have identified a role for the NAD-containing enzyme S-adenosylhomocysteine hydrolase (AdoHcyase), which is also an adenosine (Ado) and cAMP binding protein, in mediating inhibitory effects of Ado and 2'-dAdo on S-adenosylmethionine (AdoMet)-dependent transmethylation reactions. The objectives of this proposal are to extend our studies of purified human AdoHcyase; to assess inhibition of RNA methylation in Ado deaminase (ADA)-inhibited cells treated with 2'-dAdo (which irreversibly inactivates AdoHcyase); and to assess the contribution of AdoHcyase to the immune defect in genetic or drug induced ADA deficiency. Specific studies are planned in 3 areas: a) To use limited proteolytic mapping techniques in order to further define the organization and function of the nucleoside, cAMP, and NAD binding domains of AdoHcyase. Proteolytic mapping and domain-specific monoclonal antibodies will be used to determine whether these regions are related to analogous domains of the cAMP binding subunit of protein kinase, or the NAD binding domains of other NAD containing enzymes. b) To study the effect of inactivation of intracellular AdoHcyase by 2'-dAdo on methylation of ribosomal RNA precursor in mitogen-stimulated human peripheral blood lymphocytes, and in cells obtained from patients undergoing treatment with the ADA inhibitor deoxycoformycin, or with the antiviral agent adenine arabinoside. We will assess the rates of AdoHcyase synthesis in different cell types as a possible basis for selective inactivation of AdoHcyase in lymphoid cells. Alternative pathways of AdoHcy catabolism in various cell types will also be studied as a basis for selective effects of AdoHcyase inactivation. c) Mutant lymphoid cell lines will be used or selected to define the basis for toxicity of adenine, 5'-methylthioadenosine, and other purine analogs; and for determining the identity of enzymes in T cells responsible for phosphorylating 2'-dAdo. We will attempt to isolate a mutant with altered activity or production of AdoHcyase.