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 患有肾癌，并且在一个区域内，在许多 人类肿瘤。Hint和FHIT是两个分支的原型成员 新发现的低分子量二聚体HIT超家族 核苷酸结合蛋白。提示蛋白质在进化过程中是保守的 自从细菌、真核细菌和古生菌分离以来。FHIT蛋白是一种 在真核植物中发现分解二腺苷的不同分支 聚磷酸盐，如ApppA，尽管它们在整个进化过程中保持保守 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。私家侦探已经 确定了HINT中无核苷酸和无核苷酸的原子结构 核苷酸结合的形式，生长的FHIT晶体，并已开始生成 突变酵母菌株对S同源基因功能的了解 酿酒。具体地说，建议1)确定精确度 同源基因的细微和粗大变化的生理后果 HNT1和HNT2蛋白在酵母中的表达以及哺乳动物HINT和FHIT在酵母中的表达 酵母菌，探索突变、抑制剂和相互作用的影响 蛋白质；2)测定嘌呤单核苷酸结合活性和 纯化的野生型和野生型二腺苷多磷酸水解酶活性 突变的HNT1和HNT2蛋白与哺乳动物HIT的行为比较 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 S活性的分子基础 和功能。结构信息(目标3)已在 突变体的设计将提供额外的生理(目标1)和 生化(目标2)信息。这一综合战略预计将 详细了解中的提示和FHIT的功能 真核细胞及其理解FHIT的细胞和结构基础 肿瘤中的损伤。

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.