Functional and Structural Analysis of HIT Proteins

描述（由申请人提供）：组氨酸三联体（HIT）蛋白是核苷酸水解酶的一个超家族，由两个分支组成，我们已经在结构、生物化学和遗传学上进行了表征。哺乳动物Hint和酵母同源物Hnt 1水解天然产物AMPNH 2，我们表明酶活性是生物功能所必需的。酵母hnt 1突变体不能在半乳糖上在升高的温度下生长，并且与TFIIK组分Kin 28、Ccl 1和Tfb 3中的突变体以及与Cak 1的突变体在合成上不太可行。HIT超家族的Fhit分支含有水解二腺苷多磷酸（ApnA）的酶。人类FHIT基因的缺失是上皮性肿瘤（如肺）中最早和最常见的遗传变化之一，每年导致10万例美国死亡。虽然酵母遗传实验证明Fhit同源物Hnt 2控制体内ApnA水平，但特异性消除Fhit水解但不结合ApnA的能力不会阻断Fhit抑制肿瘤形成的能力。这些发现促成了Fhit功能的Ras样模型，其中晶体学定义的Fhit-底物类似物复合物被视为活性信号传导形式。该结构导致了新的荧光底物，辅助抑制剂合成和表征，以及野生型和突变体酶学，并开始使Fhit成像/诊断成为可能。尽管如此，Fhit作为肿瘤抑制因子的细胞机制和Hnt 2的细胞功能是通过合成致死分析可处理的主要未解决的问题。这项建议的具体目标如下。1)我们将使用化学方法来确定小分子和/或多肽Hnt 1底物，解释kin 28和hnt 1之间的遗传相互作用。2）我们将应用生物化学和遗传学方法来发现hnt 1缺陷的基因表达和碳源利用后果的机制。3)我们将完成一个合成的致死和抑制屏幕与hnt 2和探针与APnA结合Hnt 2的蛋白阵列，并使用这些基因来表征HNT 2的功能。4)我们将使用一系列我们已经纯化的Fhit突变体来严格测试Fhit-底物复合物是Fhit的活性信号形式的假设。Hint/Hnt 1的工作将阐明一个新发现的，保守的调节机制作用于TFIIK，影响酵母中的碳源使用和细胞活力，这似乎与共济失调与眼动性失用症的缺陷有关。Fhit/Hnt 2的研究旨在揭示最常丢失的癌症基因之一的途径，并可能导致识别预防和治疗肺癌和胃癌的新靶点。

DESCRIPTION (provided by applicant): Histidine triad (HIT) proteins are a superfamily of nucleotide hydrolases consisting of two branches we have characterized structurally, biochemically and genetically. Mammalian Hint and yeast homolog Hnt1 hydrolyze the natural product AMPNH2 and we showed that enzyme activity is required for biological function. Yeast hnt1 mutants fail to grow at elevated temperatures on galactose and are synthetically less viable with mutants in TFIIK components Kin28, Ccl1 and Tfb3, and with Cak1. The Fhit branch of the HIT superfamily contains enzymes that hydrolyze diadenosine polyphosphates (ApnA). Deletions in human FHIT are among the earliest and most frequent genetic changes in epithelial tumors such as lung, which are responsible for 100,000s of annual US deaths. While yeast genetic experiments prove that Fhit homolog, Hnt2, controls ApnA levels in vivo, specific ablation of the ability of Fhit to hydrolyze but not to bind ApnA does not block the ability of Fhit to suppress tumor formation. These discoveries contributed to a Ras-like model of Fhit function in which the crystallographically defined Fhit-substrate analog complex is seen as the active signaling form. The structure led to novel fluorescent substrates, aided inhibitor synthesis and characterization, and wild-type and mutant enzymology, and began to enable Fhit imaging/diagnostics. Nonetheless, the cellular mechanism of Fhit as tumor suppressor and cellular function of Hnt2 are major unsolved problems that are tractable via synthetic lethal analysis. Specific aims of this proposal are as follows. 1) We will use chemical methods to define the small molecule and/or polypeptide Hnt1 substrates that account for genetic interactions between kin28 and hnt1 2) We will apply biochemical and genetic methods to discover the mechanism for the gene expression and carbon source utilization consequences of hnt1-deficiency. 3) We will complete a synthetic lethal and suppression screen with hnt2 and probe a protein array with APnA-bound Hnt2 and use these genes to characterize HNT2 function. 4) We will use a collection of Fhit mutants we have purified to test rigorously the hypothesis that Fhit-substrate complexes are the active signaling form of Fhit. Work on Hint/Hnt1 will clarify a newly discovered, conserved regulatory mechanism acting on TFIIK that affects carbon source use and cell viability in yeast, which appears related to the defect responsible for ataxia with oculomotor apraxia. Work on Fhit/Hnt2 is designed to uncover the pathway of one of the most frequently lost cancer genes and may lead to identification of novel targets for prevention and treatment of lung and stomach cancer.