NitFhit--Structure and Molecular Pharmacology

产品说明：蝇和蠕虫中的Fhit同系物被编码为与腈水解酶同系物的天然融合蛋白，腈水解酶是一种切割吲哚-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。虽然在哺乳动物中未融合到Fhit多肽，但哺乳动物Nit和Fhit具有几乎相同的表达模式，并且项目1已经显示Nit似乎将Fhit连接到其他相互作用蛋白。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，我们将确定的3D结构NitFhit从衍射数据的本地，硒甲硫酰和汞I222晶体，已经获得。 其次，我们将获得底物和NitFhit的抑制剂与我们已经开发的生化检测和生化相互作用的性质的特点。 第三，我们将产生活性位点突变体的NIT和使用酵母细胞积累NIT基板，以确定和纯化这些酶的真实基板。 Nit-Fhit的3D结构有望揭示Nit和Fhit之间的通信性质、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澄清这些酶的细胞作用，并使破坏，放大或绕过其功能的新化学手段成为可能。Nits的底物捕获突变体的表达可能提供了一种额外的方式来干扰遗传信号。 Nit和Fhit的结构和药理学表征是长期计划的一部分，以定义具有失活FHIT基因的肿瘤的分子靶点，用于基因型特异性化疗消除。

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.