NitFhit--Structure and Molecular Pharmacology

描述：(申请人描述)苍蝇和蠕虫中的FHIT同源物被编码为一种天然融合蛋白，带有一种氰化酶的同源物，该酶能分解吲哚-3-乙腈以产生植物生长和分化因子吲哚-3-乙酸。已经从人、小鼠和酵母中克隆了动物类型的腈酶同系物(NIT蛋白)。虽然在哺乳动物中不与FHIT多肽融合，但哺乳动物的Nit和FHIT具有几乎相同的表达模式，项目1表明Nit似乎将FHIT与其他相互作用的蛋白质联系起来。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功能的问题的一部分。项目3的目标如下。首先，我们将根据已经获得的天然的、硒的甲硫基和汞的I222晶体的衍射数据来确定NitFHit的3D结构。其次，我们将利用我们开发的生化分析方法获得NitFHit的底物和抑制剂，并表征生化相互作用的性质。第三，我们将产生NITS的活性位点突变体，并使用积累NIT底物的酵母细胞来鉴定和纯化这些酶的真正底物。NIT-FHIT的三维结构有望揭示NIT和FHIT之间通讯的本质，腈酶超家族酶的结构和化学特征，以及动物型腈酶同系物结合口袋的性质。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角色，并使破坏、放大或绕过它们的功能的新的化学手段成为可能。NITS底物捕获突变体的表达可能提供了另一种从基因上干扰信号的方法。NIT和FHIT的结构和药理学特征是一项长期计划的一部分，该计划旨在为具有失活的FHIT基因的肿瘤确定分子靶点，用于基因特异性化疗消除。

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.