NitFhit--Structure and Molecular Pharmacology

NitFhit--结构和分子药理学

• 批准号: 6313442
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6313442
• 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，腈水解酶是一种裂解吲哚 3 乙腈以产生植物生长和分化因子吲哚 3 乙酸的酶。 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。尽管未与哺乳动物中的 Fhit 多肽融合，但哺乳动物 Nit 和 Fhit 具有几乎相同的表达模式，并且项目 1 表明 Nit 似乎将 Fhit 与其他相互作用蛋白连接起来。鉴于 Nit 和 Fhit 之间的这些联系，理解 Nit 的结构和活性被视为理解 Fhit 功能问题的一部分。项目3的目标如下。 首先，我们将根据已获得的天然 I222 晶体、硒代甲硫氨酰晶体和汞化 I222 晶体的衍射数据确定 NitFhit 的 3D 结构。 其次，我们将通过我们开发的生化测定获得 NitFhit 的底物和抑制剂，并表征生化相互作用的性质。 第三，我们将生成 Nits 活性位点突变体，并使用积累 Nit 底物的酵母细胞来识别和纯化这些酶的真实底物。 Nit-Fhit 的 3D 结构有望揭示 Nit 和 Fhit 之间通讯的性质、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。与 Nit 和 Fhit 相互作用的小分子的发现有望阐明这些酶的细胞作用，并使破坏、放大或绕过其功能的新化学方法成为可能。 Nits 底物捕获突变体的表达可能提供另一种扰乱遗传信号传导的方法。 Nit 和 Fhit 的结构和药理学表征是长期计划的一部分，该计划旨在确定具有失活 FHIT 基因的肿瘤的分子靶点，以实现基因型特异性化疗消除。