FUNCTIONAL AND STRUCTURAL ANALYSIS OF HIT PROTEINS

描述：位于3p14.2的FHIT基因处于共分离的易位中， 肾癌和在一个区域内，这是同源破坏，在许多 人类肿瘤 HINT和FHIT是两个分支的典型成员， 新认识到的HIT超家族的低分子量，二聚 核苷酸结合蛋白。 HINT蛋白在进化中是保守的 自细菌、真核生物和古生菌分离以来， FHIT蛋白是一种 在真核生物中发现的水解二腺苷的独特分支 多磷酸盐，如AppPA，尽管它们在整个进化过程中是保守的， 以及FHIT在癌症病因学或并发症中的意义， HIT蛋白的功能在任何细胞中都不清楚。 私家侦探具有 确定了HINT的原子结构， 核苷酸结合的形式，生长晶体的FHIT，并已开始产生 突变酵母菌株，以了解S 啤酒。 具体而言，建议1）确定精确的 生理后果的微妙和总的变化，在同源 酵母中的Hnt 1和Hnt 2蛋白以及在酵母中表达的哺乳动物HINT和FHIT蛋白， 酵母，探索突变的影响，抑制剂和相互作用 2）测量嘌呤单核苷酸肽结合活性，和 纯化的野生型二腺苷多磷酸水解酶活性和 突变的Hnt 1和Hnt 2蛋白，比较它们与哺乳动物HIT的行为 蛋白质，并测试细胞功能对这些蛋白质的依赖性， 参数;以及3）确定FHIT晶体的X射线结构， 已经生长，阐明了FHIT活性的分子基础 和功能 结构信息（目标3）已经在 设计突变体，提供额外的生理（目标1）和 生物化学（目标2）信息。 这一综合战略预计将 详细了解HINT和FHIT的功能， 真核细胞和理解FHIT的细胞和结构基础 肿瘤中的病变。

DESCRIPTION:The FHIT gene at 3p14.2 is at a translocation that cosegregates with renal cancer and within a region that is homozygously disrupted in many human tumors. HINT and FHIT are the prototypical members of two branches of the newly recognized HIT superfamily of low molecular weight, dimeric nucleotide-binding proteins. HINT proteins have been conserved in evolution since the separation of bacteria, eukarya and archaea. FHIT proteins are a distinct branch found within eukarya that hydrolyze diadenosine polyphosphates such as ApppA Despite their conservation throughout evolution and the implication of FHIT in the etiology or complications of cancer, the function of HIT proteins is not understood in any cell. The P.I. has determined the atomic structure of HINT in its nucleotide-free and nucleotide-bound forms, grown crystals of FHIT, and has begun to generate mutant yeast strains to understand the function of the homologous genes in S cerevisiae. Specifically, it is proposed 1) to determin precise physiological consequences of subtle and gross alterations in the homologous Hnt1 and Hnt2 proteins in yeast and of mammalian HINT and FHIT expressed in yeast, exploring the effects of mutations, inhibitors and interacting proteins; 2) to measure purine mononucleotide binding activity and diadenosine polyphosphate hydrolase activity of purified wild-type and mutant Hnt1 and Hnt2 proteins, comparing their behaviors with mammalian HIT proteins and testing the dependence of cellular functions on these parameters; and 3) to determine the X-ray structure of FHIT crystals that have already been grown, elucidating the moleular basis of FHIT s activity and function. Structural information (aim 3) is already in use in the design of mutants which will provide additional physiological (aim 1) and biochemical (aim 2) information. This integrated strategy is expected to provide a detailed understanding of the functions of HINT and FHIT in eukaryotic cells and a cellular and structural basis for understanding FHIT lesions in tumors.