NitFhit--Structure and Molecular Pharmacology

NitFhit--结构和分子药理学

• 批准号: 6570499
• 负责人: Charles M Brenner
• 金额: $ 15.13万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6570499
• 关键词: X ray crystallography; active sites; aminohydrolases; chimeric proteins; enzyme inhibitors; enzyme substrate; intermolecular interaction; mercury; methionine; mutant; selenium; tumor suppressor proteins

DESCRIPTION: (Applicant's Description) The Fhit homolog in flies and worms is encoded as a natural fusion protein with a homolog of nitrilase, an enzyme that cleaves indole-3-acetonitrile to produce the plant growth and differentiation factor, indole-3-acetic acid. Animal-type nitrilase homologs (Nit proteins) have been cloned from humans, mice and yeast. Though not fused to the Fhit polypeptide in mammals, mammalian Nit and Fhit have nearly identical expression patterns and Project 1 has shown that Nit appears to link Fhit to other interacting proteins. In view of these linkages between Nit and Fhit, understanding the structure and activity of Nit is seen as part of the problem of understanding the function of Fhit. The Aims of Project 3 are as follows. First, we will determine the 3D structure of NitFhit from diffraction data of native, selenomethionyl and mercurated I222 crystals that have already been obtained. Second, we will obtain substrates and inhibitors of NitFhit with biochemical assays we have developed and characterize the nature of the biochemical interactions. Third, we will generate active-site mutants of Nits and use yeast cells that accumulate Nit substrates to identify and purify the authentic substrates of these enzymes. The 3D structure of Nit-Fhit is expected to reveal the nature of communication between Nit and Fhit, structural and chemical features of nitrilase superfamily enzymes, and the nature of the binding pocket of animal-type nitrilase homologs. Discovery of small molecules that interact with Nit and Fhit is expected to clarify the cellular roles for these enzymes and make possible novel chemical means of disrupting, amplifying or bypassing their functions. Expression of substrate-trapping mutants of Nits may provide an additional way to perturb signaling genetically. Structural and pharmacological characterization of Nit and Fhit are parts of a long-term plan to define molecular targets for tumors with inactivated FHIT genes for genotype-specific chemotherapeutic elimination.

描述：（申请人的描述）苍蝇和蠕虫中的 Fhit 同源物 被编码为与腈水解酶同源物的天然融合蛋白，腈水解酶是一种酶 裂解吲哚-3-乙腈以促进植物生长和 分化因子，吲哚-3-乙酸。动物型腈水解酶同系物 （Nit 蛋白）已从人类、小鼠和酵母中克隆出来。虽然没有融合 对于哺乳动物中的 Fhit 多肽，哺乳动物 Nit 和 Fhit 几乎 相同的表达模式和项目 1 表明 Nit 似乎链接 F 与其他相互作用的蛋白质结合。鉴于 Nit 和 Fhit，了解 Nit 的结构和活性被视为 理解Fhit功能的问题。项目 3 的目标是 接下来。首先，我们将确定 NitFhit 的 3D 结构 天然、硒代甲硫氨酰和汞化 I222 晶体的衍射数据 已经获得。其次，我们将获得底物和抑制剂 我们开发了 NitFhit 的生化检测方法并对其进行了表征 生化相互作用的本质。第三，我们将生成活动站点 Nits 突变体并使用积累 Nit 底物的酵母细胞来识别 并纯化这些酶的真实底物。的 3D 结构 Nit-Fhit 有望揭示 Nit 和 Fhit，腈水解酶超家族酶的结构和化学特征，以及 动物型腈水解酶同系物的结合口袋的性质。发现 与 Nit 和 Fhit 相互作用的小分子有望阐明 这些酶的细胞作用并使新的化学方法成为可能 破坏、放大或绕过其功能。表达 Nits 的底物捕获突变体可能提供另一种干扰方式 遗传信号。 Nit 的结构和药理学特征 和 Fhit 是定义肿瘤分子靶点的长期计划的一部分 具有失活的 FHIT 基因，用于基因型特异性化疗 消除。