Functional and Structural Analysis of HIT Proteins

描述(由申请人提供)：组氨酸三联体(HIT)蛋白是由两个分支组成的核苷酸水解酶的超家族，我们已经从结构、生化和遗传学上对其进行了表征。哺乳动物的HINT和酵母的同系物HNT1可以降解天然产物AMPNH2，我们证明了酶的活性是发挥生物学功能所必需的。酵母hnt1突变体不能在高温下在半乳糖上生长，并且与TFIIK组分Kin28、Ccl1和Tfb3以及Cak1的突变体一起合成的活性较低。HIT超家族的FHIT分支包含水解二腺苷多磷酸(ApNA)的酶。人类FHIT缺失是肺等上皮性肿瘤中最早和最频繁的基因变化之一，这些肿瘤导致了美国每年10万人的死亡。虽然酵母遗传学实验证明FHIT同系物HNT2在体内控制ApNA水平，但特异性地阻断FHIT水解但不结合ApNA的能力并不阻止FHIT抑制肿瘤形成的能力。这些发现促成了一种类似RAS的FHIT功能模型，在该模型中，结晶学定义的FHIT底物类似物复合体被视为活性信号形式。这种结构导致了新的荧光底物、辅助抑制剂的合成和表征以及野生型和突变型酶的研究，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HNT2的细胞功能仍然是主要的悬而未决的问题，通过人工致死分析是容易解决的。这项建议的具体目标如下。1)我们将使用化学方法定义小分子和/或多肽HNT1底物，这些底物解释kin28和hnt1之间的遗传相互作用。2)我们将应用生化和遗传学方法来揭示hnt1缺乏的基因表达和碳源利用后果的机制。3)我们将完成与hNT2的合成致死和抑制筛选，并探索带有Apna结合的hnt2的蛋白质阵列，并利用这些基因来表征hNT2的功能。4)我们将使用我们纯化的一组FHIT突变体来严格检验FHIT底物复合体是FHIT的活性信号形式的假设。对HINT/HNT1的研究将阐明一种新发现的作用于TFIIK的保守调控机制，该机制影响碳源的利用和酵母细胞的活性，这似乎与导致共济失调并动眼失用的缺陷有关。对FHIT/HNT2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.