Polyphosphate degradation by Nudt3-Zn2+ mediates oxidative stress response.

Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo development in zebrafish.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis.

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Nudt3 is an mRNA decapping enzyme that modulates cell migration.

Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin ß6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin ß6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin ß6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development.

The Arabidopsis protein AtTTM3 belongs to the CYTH superfamily named after its two founding members, the CyaB adenylate cyclase from Aeromonas hydrophila and the mammalian thiamine triphosphatase. In this study we report the three-dimensional structure of a plant CYTH domain protein, AtTTM3, determined at 1.9 Å resolution. The crystal structure revealed the characteristic tunnel architecture of CYTH proteins, which specialize in the binding of nucleotides and other organic phosphates and in phosphoryl transfer reactions. The ß barrel is composed of eight antiparallel ß strands with a cluster of conserved inwardly facing acidic and basic amino acid residues. Mutagenesis of these residues in the catalytic core led to an almost complete loss of enzymatic activity. We established that AtTTM3 is not an adenylate cyclase. Instead, the enzyme displayed weak NTP phosphatase as well as strong tripolyphosphatase activities similar to the triphosphate tunnel metalloenzyme proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM). AtTTM3 is most highly expressed in the proximal meristematic zone of the plant root. Furthermore, an AtTTM3 T-DNA insertion knockout line displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development.

[Capping strategies in RNA viruses]. / Stratégies de formation de la structure coiffe chez les virus à ARN.

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

Inactivation of both FHIT and p53 cooperate in deregulating proliferation-related pathways in lung cancer.

INTRODUCTION: FHIT and p53 are the two most commonly altered tumor suppressor genes in lung cancer, and their molecular status regulates sensitivity to anticancer drugs. Although their functions are independent, there is evidence that their pathways might be interconnected, but little is known at the molecular level. METHODS: Microarray profiling of FHIT-transduced lung cancer cells and modulation of FHIT levels by RNA interference in human bronchial cells were used to generate a signature of FHIT-regulated transcripts. Expression of these genes was evaluated by real-time polymerase chain reaction in 55 primary lung cancer samples characterized for FHIT and p53 expression by immunehistochemistry. RESULTS: A signature of FHIT-transcripts, particularly enriched in genes involved in cell cycle control, was identified. This signature showed overlap with p53-regulated genes, indicating possible crosstalk between these proteins. Consistently, transcriptional deregulation after FHIT modulation was higher in p53-negative cells. In primary lung cancers, inactivation of either gene was detected in 48 of 55 cases (87%) and both genes in 23 of 55 (42%) cases, confirming the central role of these pathways. Primary tumors with inactivation of both FHIT and p53 displayed the strongest deregulation of growth-related pathways with high levels of expression of CCNB1, BUB1, CDC6, TOP2A, MCM6, and CENPF. CONCLUSIONS: FHIT and p53 seem to rely on common mediators, and inactivation of both genes results in prominent deregulation of growth-related pathways in lung cancer cell lines and primary tumors. This reveals crosstalk between these proteins and suggests a possible distinctive phenotype for tumors with inactivation of both genes.

[Progress in polyphosphate and related metabolizing enzymes].

Inorganic polyphosphate (Poly P) is a polymer consisting of ten to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds, which is abundantly found in all organisms and nature. Here the basic facts of PolyP are summarized: genes regulated by polyP, role in DNA uptake, motility of microorganism, function in stress response, the virulence of pathogens, as well as the proliferation of mammary cancer cells, blood coagulation, cell calcification and the modulation of mitochondrial activity. Enzymes with activities requiring polyP, such as endopolyphosphatase, glucokinase, NAD kinase, AMP phosphotransferase are outlined too. The structure and activity of enzymes regulating polyP level such as polyphosphate kinase and exopolyphosphatase are noted. A thorough analysis of the mycobacterium tuberculosis PPX protein homologs and their biochemical activity is presented.

FHIT promotes the apoptosis of QBC939 by reducing the expression of cyclin D1.

BACKGROUND/AIMS: The fragile histidine triad (FHIT) gene is a candidate tumor suppressor gene. Recent studies have found that FHIT protein is lost in digestive system neoplasms. However, there is limited information on the effect of FHIT on apoptosis and the regulation functions on cyclin D1 in cholangiocarcinoma. METHODOLOGY: Eukaryotic expression plasmids with/without the FHIT gene were constructed and transfected into three groups in which cholangiocarcinoma cells were also divided. We observed the proliferation of cholangiocarcinoma by using the MTT method and flow cyclometry (FCM). Invasive ability was then determined via transwell chamber testing. In addition, to examine whether FHIT mRNA and protein expression were correlated with cyclin D1 expression, we used real-time RT-PCR and western blotting. RESULTS: The absorbance (A490) was decreased significantly (p<0.05) according to MTT, showing that proliferation had been inhibited and the apoptosis ratio after transfecting had increased significantly (p<0.05). The amount of cells which passed the filter in the transwell chamber was significantly less (p<0.05). The expression of cyclin D1 mRNA and the protein was decreased from the original cell line (p<0.05). CONCLUSIONS: The results suggest that increased FHIT gene activity can promote apoptosis of cholangiocarcinoma and reduce the expression level of cyclin D1.

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 µM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.

Effect of zinc supplementation on N-nitrosomethylbenzylamine-induced forestomach tumor development and progression in tumor suppressor-deficient mouse strains.

Zinc deficiency is associated with high incidences of esophageal and other cancers in humans and leads to a highly proliferative hyperplastic condition in the upper gastrointestinal tract in laboratory rodents. Zn replenishment reduces the incidence of lingual, esophageal and forestomach tumors in Zn-deficient rats and mice. While previous animal studies focused on Zn deficiency, we have investigated the effect of Zn supplementation on carcinogenesis in Zn-sufficient mice of wild-type and tumor suppressor-deficient mouse strains. All mice received N-nitrosomethylbenzylamine and half the mice of each strain then received Zn supplementation. At killing, mice without Zn supplementation had developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice developed an average of 7.0 versus 5.0 tumors for Zn supplemented (P < 0.05); Zn-supplemented Fhit-/- mice averaged 5.7 versus 8.0 for control mice (P < 0.01); Zn-supplemented Fhit-/-Nit1-/- mice averaged 5.4 versus 9.2 for control mice (P < 0.01) and Zn-supplemented Fhit-/-Rassf1a-/- (the murine gene) mice averaged 5.9 versus 9.1 for control mice (P < 0.01). Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit-/-Nit1-/-). Histological analysis of forestomach tissues also showed significant decreases in severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse strain. Thus, Zn supplementation significantly reduced tumor burdens in mice with multiple tumor suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final carcinogen dose, the reduction in tumor burden was the same as observed when supplementation began immediately after carcinogen dosing, suggesting that Zn supplementation may affect tumor progression rather than tumor initiation.

The role of the exopolyphosphatase PPX in avoidance by Neisseria meningitidis of complement-mediated killing.

The complement system is critical for immunity against the important human pathogen Neisseria meningitidis. We describe the isolation of a meningococcal mutant lacking PPX, an exopolyphosphatase responsible for cleaving cellular polyphosphate, a polymer of tens to hundreds of orthophosphate residues found in virtually all living cells. Bacteria lacking PPX exhibit increased resistance to complement-mediated killing. By site directed mutagenesis, we define amino acids necessary for the biochemical activity of meningococcal PPX, including a conserved glutamate (Glu(117)) and residues in the Walker B box predicted to be involved in binding to phosphate. We show that the biochemical activity of PPX is necessary for interactions with the complement. The relative resistance of the ppx mutant does not result from changes in structures (such as capsule, lipopolysaccharide, and factor H-binding protein), which are known to be required for evasion of this key aspect of host immunity. Instead, expression of PPX modifies the interaction of N. meningitidis with the alternative pathway of complement activation.

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Enzymology of RNA cap synthesis.

The 5' guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7-methyl guanosine linked through a 5'-5' inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co-transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway.

Fhit regulates invasion of lung tumor cells.

In many types of cancers, the fragile histidine triad (Fhit) gene is frequently targeted by genomic alterations leading to a decrease or loss of gene and protein expression. Fhit has been described as a tumor suppressor gene because of its ability to induce apoptosis and to inhibit proliferation of tumor cells. Moreover, several studies have shown a correlation between the lack of Fhit expression and tumor aggressiveness, thus suggesting that Fhit could be involved in tumor progression. In this study, we explored the potential role of Fhit during tumor cell invasion. We first showed that a low Fhit expression is associated with in vivo and in vitro invasiveness of tumor cells. Then, we showed that Fhit overexpression in Fhit-negative highly invasive NCI-H1299 cells by transfection of Fhit cDNA and Fhit inhibition in Fhit-positive poorly invasive HBE4-E6/E7 cells by transfection of Fhit small interfering RNA induce, respectively, a decrease and an increase in migratory/invasive capacities. These changes in cell behavior were associated with a reorganization of tight and adherens junction molecules and a regulation of matrix metalloproteinase and vimentin expression. These results show that Fhit controls the invasive phenotype of lung tumor cells by regulating the expression of genes associated with epithelial-mesenchymal transition.

Fragile histidine triad protein: structure, function, and its association with tumorogenesis.

BACKGROUND: The human fragile histidine triad (FHIT) gene is a putative tumor suppressor gene, which is located at chromosome region 3p14.2. It was suggested that the loss of heterozygosity (LOH), homozygous deletions, and abnormal expression of the FHIT gene were involved in several types of human malignancies. MATERIALS AND METHODS: To determine the role of FHIT in various cancers, we have performed structural and functional analysis of FHIT in detail. RESULTS AND DISCUSSION: The protein FHIT catalyzes the Mg(2+) dependent hydrolysis of P1-5 cent-O-adenosine-P3-5 cent-O-adenosine triphosphate, Ap3A, to AMP, and ADP. The reaction is thought to follow a two-step mechanism. Histidine triad proteins, named for a motif related to the sequence H-cent-H-cent-H-cent-cent- (cent, a hydrophobic amino acid), belong to superfamily of nucleotide hydrolases and transferases. This enzyme acts on the R-phosphate of ribonucleotides, and contain a approximately 30-kDa domain that is typically a homodimer of approximately 15 kDa polypeptides with catalytic site. CONCLUSION: Here we have gathered information is known about biological activities of FHIT, the structural and biochemical bases for their functions. Our approach may provide a comparative framework for further investigation of FHIT.

Enlightened protein: Fhit tumor suppressor protein structure and function and its role in the toxicity of protoporphyrin IX-mediated photodynamic reaction.

The Fhit tumor suppressor protein possesses Ap(3)A (diadenosine triphosphate - ApppA) hydrolytic activity in vitro and its gene is found inactive in many pre-malignant states due to gene inactivation. For several years Fhit has been a widely investigated protein as its cellular function still remains largely unsolved. Fhit was shown to act as a molecular 'switch' of cell death via cascade operating on the influence of ATR-Chk1 pathway but also through the mitochondrial apoptotic pathway. Notably, Fhit was reported by our group to enhance the overall eradication effect of porphyrin-mediated photodynamic treatment (PDT). In this review the up-to-date findings on Fhit protein as a tumor suppressor and its role in PDT are presented.

Intramitochondrial calcium regulation by the FHIT gene product sensitizes to apoptosis.

Despite the growing interest in the Fhit tumor suppressor protein, frequently deleted in human cancers, the mechanism of its powerful proapoptotic activity has remained elusive. We here demonstrate that Fhit sensitizes the low-affinity Ca(2+) transporters of mitochondria, enhancing Ca(2+) uptake into the organelle both in intact and in permabilized cells, and potentiating the effect of apoptotic agents. This effect can be attributed to the fraction of Fhit sorted to mitochondria, as a fully mitochondrial Fhit (a chimeric protein including a mitochondrial targeting sequence) retains the Ca(2+) signaling properties of Fhit and the proapoptotic activity of the native protein (whereas the effects on the cell cycle are lost). Thus, the partial sorting of Fhit to mitochondria allows to finely tune the sensitivity of the organelle to the highly pleiomorphic Ca(2+) signals, synergizing with apoptotic challenges. This concept, and the identification of the molecular machinery, may provide ways to act on apoptotic cell death and its derangement in cancer.

Fragile gene product, Fhit, in oxidative and replicative stress responses.

Though the fragile histidine triad gene product, Fhit, was discovered and characterized as a tumor suppressor 13 years ago, its sequence, structure, and cellular location did not provide clues to aid discovery of its mechanisms of suppression. Recently, using chemical cross-linkers and immunoprecipitation, a Fhit protein complex was identified that includes Hsp60 and Hsp10 which may mediate Fhit stability and mitochondrial localization, where Fhit binds and stabilizes ferredoxin reductase (Fdxr); when Fdxr is overexpressed, it can lead to production of reactive oxygen species (ROS) that induce apoptosis. Cancer cells expressing endogenous or exogenous Fhit, when exposed to H(2)O(2), an oxidative stress, produce higher levels of apoptosis-inducing ROS than matched, Fhit-negative cells; the Fhit-negative cancer cells survive, carrying DNA damage. In addition to this mitochondrial function, Fhit-overexpression in cancer cells exposed to replicative stress-inducing agents leads to enhanced caspase 3 activation and apoptosis, due to defective Chk1 activation. Thus, damage to the fragile FHIT locus leads to reduced expression of Fhit protein, and makes a two-pronged contribution to development of preneoplastic clonal expansion: (1) absence or reduction of Fhit leads to reduced expression of Fdxr and reduced ROS-induced apoptosis; (2) cells that escape ROS- or replicative stress-induced apoptosis can carry misrepaired DNA damage. The aberrant DNA damage response checkpoint in Fhit-deficient preneoplasias and cancers may make these lesions targets for inhibitors of proteins such as Parp1 and Chk1 with important roles in checkpoint responses, as observed for BRCA1-deficient cancer cells that also exhibit DNA damage repair deficiencies.

[FHIT--tumor suppressor protein involved in induction of apoptosis and cell cycle regulation]. / FHIT--bialko supresorowe zaangazowane w indukcje apoptozy i regulacje cyklu komórkowego.

The fragile histidine triad (Fhit) protein is a diadenosine triphosphate hydrolase belonging to the histidine triad family of nucleotide-binding proteins. Fhit is a tumor suppressor protein which plays an important role in pro-apoptotic signalling, cell cycle control and sensitivity to DNA damaging agents. The Ap3A--hydrolase activity of Fhit is not required for exertion of its tumor suppressor function. The FHIT gene is located in FRA3B region--the most common fragile site in the human genome. Loss of FHIT is observed in a variety of tumors and in premalignant states. Re-expression of FHIT in human cancer cells leads to inhibition of tumorogenecity and restoration of caspase-dependent apoptosis. Viral-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. Therefore, treatment with the FHIT gene may constitute a novel clinical approach for gene therapy of human cancers.