Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers

BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. RESULTS: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs. We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction. CONCLUSIONS: Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.     

Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.     

Nit1 and Fhit tumor suppressor activities are additive

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit^?/?Nit1^?/? mice and observed that double knockout mice develop more spontaneous and carcinogen‐induced tumors than Fhit^?/? mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney‐derived double‐deficient cells appear not to activate the pChk2 pathway and when treated with H₂O₂, show little evidence of DNA damage, compared with wild type and Fhit^?/? cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas. J. Cell. Biochem. 107: 1097–1106, 2009. © 2009 Wiley‐Liss, Inc.     

Hint,Fhit, and GalT: function,structure, evolution,and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases

HIT (histidine triad) proteins, named for a motif related to the sequence HphiHphiHphiphi (phi, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the alpha-phosphate of ribonucleotides, and contain a approximately 30 kDa domain that is typically either a homodimer of approximately 15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5'-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in the development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding site. The potential roles of ASW and Hint in avian sexual development are discussed elsewhere. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases.     

β-catenin takes a HIT

Histidine triad (HIT) proteins represent a small family of nucleotide-binding and -hydrolyzing proteins, which attracted the attention of cancer biologists because their expression is lost in multiple human malignancies. To some of the family members including Fhit, Hint1 and Hint2, a tumor suppressive activity was assigned. Although highly similar in structure, their mode of action appears to be different as they are not able to compensate each other’s function. Surprisingly, in any reported assay system the enzymatic activity of the histidine triad proteins was not required for their tumor suppressor function. Until recently, little was known about the molecular mechanisms mediating the tumor suppressor activities of histidine triad proteins. The identification of new interaction partners started to shed light on the signaling pathways modulated by the HIT proteins. Here, we summarize these findings with special emphasis on the histidine triad proteins Hint1 and Fhit and their repressive activity on the β-catenin signaling function.     

Catalysis in the nitrilase superfamily

Recently, we defined the nitrilase superfamily as consisting of 12 families of amidases, N-acyltransferases and presumptive amidases, in addition to the family of plant and bacterial nitrilases for which the superfamily was named. A novel Glu-Lys-Cys catalytic triad, found at the crystallographically defined Nit active site of worm NitFhit, was postulated to constitute the catalytic residues for all members of the superfamily. Recent experimental results confirm the essentiality of the catalytic triad residues and specify the biochemical functions of additional branches and sub-branches of the nitrilase superfamily.     

A Fhit-ing Role in the DNA Damage Checkpoint Response

The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered.Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and neoplastic tissues, to provide rationales for effective replacement or reactivation of endogenous Fhit pathways in novel therapeutic or preventive approaches.     

Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit-/- cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit-/- cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression.     

Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.     

Desensitization of melanoma cells to autocrine TGF-beta isoforms

Histidine triad (HIT) proteins were until recently a superfamily of proteins that shared only sequence motifs. Crystal structures of nucleotide-bound forms of histidine triad nucleotide-binding protein (Hint) demonstrated that the conserved residues in HIT proteins are responsible for their distinctive, dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites. Hint-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Hint homologs are intracellular receptors for purine mononucleotides whose cellular function remains elusive. Fhit homologs bind and cleave diadenosine polyphosphates (Ap(n)A) such as ApppA and AppppA. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues. In invertebrates, Fhit homologs are encoded as fusion proteins with proteins related to plant and bacterial nitrilases that are candidate signaling partners in tumor suppression.     

The histidine triad protein Hint is not required for murine development or Cdk7 function

The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice. Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.     

乙酰基转移酶Tip60(KAT5)的功能研究进展

Tip60(KAT5)属于MYST乙酰基转移酶家族,同时它也是进化上非常保守的Nu A4蛋白质复合体的重要成员.过去十几年的研究证实,Tip60一方面可以作为转录调控因子结合核受体(如雄激素受体,AR)或c-MYC、AICD/Fe65、NCo R、E2F等转录因子来激活或抑制下游基因的表达,另一方面,KAT5可以乙酰化一系列蛋白来调控这些蛋白质的活性及稳定性,进而调控DNA损伤修复反应、细胞周期进程、细胞周期检查点的激活、凋亡、代谢及自噬等重要细胞功能.此外,Tip60在肿瘤的发生发展及转移、胚胎发育等过程中也发挥着至关重要的作用.本文将主要对Tip60近几年的研究进展做一个综述.     

The p53 family: same response, different signals?

TP53, the gene that encodes p53, is a well-defined tumor suppressor gene that is frequently mutated in human cancers. Recently, two proteins homologous to p53, termed p73 and p63, were identified. Current data indicate that both p73 and p63, like p53, can induce cell-cycle arrest and apoptosis, suggesting that they might also be tumor suppressors. However, the physiological signals that can regulate p53, for example, DNA damage, have no effect on p73, as tested in several cell lines. Furthermore, the signaling pathways by which p73 (and possibly p63) induces cell-cycle arrest and apoptosis appear to be similar to those of p53, but also have important differences. Thus, the p53 family proteins are closely related but might have distinct physiological functions.     

Protein expression profiling identifies cyclophilin A as a molecular target in Fhit-mediated tumor suppression

Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.     

Actions of aprataxin in multiple DNA repair pathways

Mutations in the Aptx gene lead to a neurological disorder known as ataxia oculomotor apraxia-1. The product of Aptx is Aprataxin (Aptx), a DNA-binding protein that resolves abortive DNA ligation intermediates. Aprataxin catalyzes the nucleophilic release of adenylate groups covalently linked to 5' phosphate termini, resulting in termini that can again serve as substrates for DNA ligases. Here we show that Aprataxin acts preferentially on adenylated nicks and double-strand breaks rather than on single-stranded DNA. Moreover, we show that whereas the catalytic activity of Aptx resides within the HIT domain, the C-terminal zinc finger domain provides stabilizing contacts that lock the enzyme onto its high affinity AMP-DNA target site. Both domains are therefore required for efficient AMP-DNA hydrolase activity. Additionally, we find a role for Aprataxin in base excision repair, specifically in the removal of adenylates that arise from abortive ligation reactions that take place at incised abasic sites in DNA. We suggest that Aprataxin may have a general proofreading function in DNA repair, removing DNA adenylates as they arise during single-strand break repair, double-strand break repair, and in base excision repair.     

Who's on first in the cellular response to DNA damage?

Cellular DNA-repair pathways involve proteins that have roles in other DNA-metabolic processes, as well as those that are dedicated to damage removal. Several proteins, which have diverse functions and are not known to have roles in DNA repair, also associate with damaged DNA. These newly discovered interactions could either facilitate or hinder the recognition of DNA damage, and so they could have important effects on DNA repair and genetic integrity. The outcome for the cell, and ultimately for the organism, might depend on which proteins arrive first at sites of DNA damage.     

The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4

Ataxia-oculomotor apraxia 1 (AOA1) is an autosomal recessive neurodegenerative disease that is reminiscent of ataxia-telangiectasia (A-T). AOA1 is caused by mutations in the gene encoding aprataxin, a protein whose physiological function is currently unknown. We report here that, in contrast to A-T, AOA1 cell lines exhibit neither radioresistant DNA synthesis nor a reduced ability to phosphorylate downstream targets of ATM following DNA damage, suggesting that AOA1 lacks the cell cycle checkpoint defects that are characteristic of A-T. In addition, AOA1 primary fibroblasts exhibit only mild sensitivity to ionising radiation, hydrogen peroxide, and methyl methanesulphonate (MMS). Strikingly, however, aprataxin physically interacts in vitro and in vivo with the DNA strand break repair proteins XRCC1 and XRCC4. Aprataxin possesses a divergent forkhead associated (FHA) domain that closely resembles the FHA domain present in polynucleotide kinase, and appears to mediate the interactions with CK2-phosphorylated XRCC1 and XRCC4 through this domain. Aprataxin is therefore physically associated with both the DNA single-strand and double-strand break repair machinery, raising the possibility that AOA1 is a novel DNA damage response-defective disease.     

生长抑制因子(ING)抑癌基因家族的研究进展

生长抑制因子(inhibitor of growth,ING)家族成员是候选的抑癌基因.ING蛋白参与磷脂酰肌醇介导的脂类信号转导通路及激素介导的通路,能够与组蛋白乙酰转移酶、去乙酰化酶等结合参与染色质的重构,调节基因的转录,与p53协同作用,抑制细胞生长,诱导细胞凋亡和DNA损伤修复.ING家族成员通过对基因表达的表观遗传学调控将细胞周期、细胞凋亡和衰老等生物学过程有机联系起来.