Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

In-silico structural characterization and phylogenetic analysis of Nucleoside diphosphate kinase: A novel antiapoptotic protein of Porphyromonas gingivalis.

The Nucleoside diphosphate kinase (NDK) protein of Porphyromonas gingivalis (P. gingivalis) plays a crucial role in immune evasion and inhibition of apoptosis in host cells and has the potential to cause cancer. However, its structure has not yet been characterized. We used an in-silico approach to determine the 3D structure of the P. gingivalis NDK. Furthermore, structural characterization and functional annotation were performed using computational approaches. The 3D structure of NDK was predicted through homology modeling. The structural domains predicted for the model protein belong to the NDK family. Structural alignment of prokaryotic and eukaryotic NDKs with the model protein revealed the conservation of the domain region. Structure-based phylogenetic analysis depicted a significant evolutionary relationship between the model protein and the prokaryotic NDK. Functional annotation of the model confirmed structural homology, exhibiting similar enzymatic functions as NDK, including ATP binding and nucleoside diphosphate kinase activity. Furthermore, molecular dynamic (MD) simulation technique stabilized the model structure and provides a thermo-stable protein structure that can be used as a therapeutic target for further studies.

Molecular docking and simulation studies against nucleoside diphosphate kinase (NDK) of Pseudomonas aeruginosa with secondary metabolite identified by genome mining from paenibacillusehimensis.

Pseudomonas aeruginosa is one of the leading opportunistic pathogens that causes nosocomial pneumonia and mostly in people with cystic fibrosis. In the present study, an in-silicoapproach was adopted to identify the novel drug target against Pseudomonas aeruginosa by employing subtractive genomics and molecular docking studies. Each step in the subtractive genomics scrutinized the bacterial proteome and determined a potential drug target against Pseudomonas aeruginosa. 71 essential proteins were obtained from the subcellular localization method that resides in the extracellular region. Metabolic pathways were studied to elucidate the unique pathways where the involvement of proteins present in the pathogen was predicted and a total of 6 unique pathways were determined. By, Genome mining of the source organism Paenibacillusehimensis, 9 ligands were obtained. The molecular docking analysis between the binding site of target protein NDK and ligands was carried out by employing the AutoDock Vina tool. Based on the highest binding affinity, Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein with a lower binding energy of -7.5 kcal/mol, -7.4and -7.2 kcal/molrespectively were considered for the simulation studies. Molecular dynamics simulation studies showed the ligand in complex with protein was highly stable and rigid for a duration of 150 ns. For Paenibactin, AnabaenopeptinNZ857 and Nostamide Acomplex with protein, RMSD plot showed a deviation of ∼0.2-0.3 nm till ∼30ns/50 ns-110ns and further stabilized. The radius of the gyration plot clearly showed that the values stayed at ∼1.45 nm- 1.55 nm showing compactness and stability. The SASA stayed at the range ∼80nm2 and at least one total number of hydrogen bonds was shown throughout the 150 ns simulation for all three possible ligand-protein complexes. In the RMSF plot, the maximum fluctuation was ranged from ∼0.4-0.42 nm at the range between ∼57ns-60ns.The Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein showed a stable, rigid and compact interaction throughout the simulation of duration 150 ns.Communicated by Ramaswamy H. Sarma.

Magnesium and cell energetics: At the junction of metabolism of adenylate and non-adenylate nucleotides.

Free magnesium (Mg2+) represents a powerful signal arising from interconversions of adenylates (ATP, ADP and AMP). This is a consequence of the involvement of adenylate kinase (AK) which equilibrates adenylates and uses defined species of Mg-complexed and Mg-free adenylates in both directions of its reaction. However, cells contain also other reversible Mg2+-dependent enzymes that equilibrate non-adenylate nucleotides (uridylates, cytidylates and guanylates), i.e. nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinase (NDPK). Here, we propose that AK activity is tightly coupled to activities of NMPK and NDPK, linking adenylate equilibrium to equilibria of other nucleotides, and with [Mg2+] controlling the ratios of Mg-chelated and Mg-free nucleotides. This coupling establishes main hubs for adenylate-driven equilibration of non-adenylate nucleotides, with [Mg2+] acting as signal arising from all nucleotides rather than adenylates only. Further consequences involve an overall adenylate control of UTP-, GTP- and CTP-dependent pathways and the availability of substrates for RNA and DNA synthesis.

Leishmania donovani secretory protein nucleoside diphosphate kinase b localizes in its nucleus and prevents ATP mediated cytolysis of macrophages.

Leishmania donovani pathogenicity is closely linked to its ability to live and replicate in the hostile environment of macrophages. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. For some pathogens, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor mediated, ATP-induced death of infected macrophages. Here, Leishmanaia donovani nucleoside diphosphate kinase (LdNDKb) was cloned, expressed and purified by Ni2+-NTA affinity chromatography to elucidate its biological significance. The presence of secreted form of LdNDKb in the medium was confirmed by Western blot analysis. Interestingly, cellular localization by confocal microscopy showed that this protein was localized in the nucleus, inner leaflet of membrane and on the flagella of this parasite which indicates its multiple role in the life cycle of Leishmania donovani. Its possibility to bind with DNA was confirmed by gel retardation assay and electrophoretic mobility shift assay (EMSA) which show the binding with linear and supercoiled is not sequence specific. Further, treatment of J774 macrophages with recombinant LdNdKb and periodate oxidized ATP - a P2X7 receptor antagonist, inhibited ATP-induced cytolysis in vitro, as determined by lactate dehydrogenise release from J774 macrophages. Thus, LdNDKb prevents ATP-mediated host-cell plasma membrane permeabilization by hydrolyzing extracellular ATP, thereby, preserving the integrity of the host cells for the benefit of the parasite. This study indicates that LdNDKb could be explored for its potentiality as a drug/vaccine target against visceral leishmaniasis.

NDK/NME proteins: a host-pathogen interface perspective towards therapeutics.

No effective vaccine is available for any parasitic disease. The treatment to those is solely dependent on chemotherapy, which is always threatened due to development of drug resistance in bugs. This warrants identification of new drug targets. Here, we discuss Nucleoside diphosphate kinases (NDKs) of pathogens that alter host's intra and extracellular environment, as novel drug targets to simultaneously tackle multiple pathogens. NDKs having diverse functions, are highly conserved among prokaryotes and eukaryotes (the mammal NDKs are called NMEs [non-metastatic enzymes]). However, NDKs and NMEs have been separately analysed in the past for their structure and functions. The role of NDKs of pathogen in modulation of inflammation, phagocytosis, apoptosis, and ROS generation in host is known. Conversely, its combined contribution in host-pathogen interaction has not been studied yet. Through the sequence and domain analysis, we found that NDKs can be classified in two groups. One group comprised NMEs 1-4 and few NDKs of select essential protozoan parasites and the bacterium Mycobacterium tuberculosis. The other group included NME7 and the other NDKs of those parasites, posing challenges in the development of drugs specifically targeting pathogen NDKs, without affecting NME7. However, common drugs targeting group 2 NDKs of pathogens can be designed, as NME7 of group 2 is expressed only in ciliated host cells. This review thus analyses comparatively for the first time the structures and functions of human NMEs and pathogen NDKs and predicts the possibilities of NDKs as drug targets. In addition, pathogen NDKs have been now provided a nomenclature in alignment with the NMEs of humans.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

Heterozygous Nme7 Mutation Affects Glucose Tolerance in Male Rats.

Complex metabolic conditions such as type 2 diabetes and obesity result from the interaction of numerous genetic and environmental factors. While the family of Nme proteins has been connected so far mostly to development, proliferation, or ciliary functions, several lines of evidence from human and experimental studies point to the potential involvement of one of its members, NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) in carbohydrate and lipid metabolism. As a complete lack of Nme7 is semilethal in rats, we compared morphometric, metabolic, and transcriptomic profiles of standard diet-fed heterozygous Nme7+/- on male rats vs. their wild-type Nme7+/+ controls. Nme7+/- animals showed increased body weight, adiposity, higher insulin levels together with decreased glucose tolerance. Moreover, they displayed pancreatic islet fibrosis and kidney tubular damage. Despite no signs of overt liver steatosis or dyslipidemia, we found significant changes in the hepatic transcriptome of Nme7+/- male rats with a concerted increase of expression of lipogenic enzymes including Scd1, Fads1, Dhcr7 and a decrease of Cyp7b1 and Nme7. Network analyses suggested possible links between Nme7 and the activation of Srebf1 and Srebf2 upstream regulators. These results further support the implication of NME7 in the pathogenesis of glucose intolerance and adiposity.

Semi-Lethal Primary Ciliary Dyskinesia in Rats Lacking the Nme7 Gene.

NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) is a member of a gene family with a profound effect on health/disease status. NME7 is an established member of the ciliome and contributes to the regulation of the microtubule-organizing center. We aimed to create a rat model to further investigate the phenotypic consequences of Nme7 gene deletion. The CRISPR/Cas9 nuclease system was used for the generation of Sprague Dawley Nme7 knock-out rats targeting the exon 4 of the Nme7 gene. We found the homozygous Nme7 gene deletion to be semi-lethal, as the majority of SDNme7-/- pups died prior to weaning. The most prominent phenotypes in surviving SDNme7-/- animals were hydrocephalus, situs inversus totalis, postnatal growth retardation, and sterility of both sexes. Thinning of the neocortex was histologically evident at 13.5 day of gestation, dilation of all ventricles was detected at birth, and an external sign of hydrocephalus, i.e., doming of the skull, was usually apparent at 2 weeks of age. Heterozygous SDNme7+/- rats developed normally; we did not detect any symptoms of primary ciliary dyskinesia. The transcriptomic profile of liver and lungs corroborated the histological findings, revealing defects in cell function and viability. In summary, the knock-out of the rat Nme7 gene resulted in a range of conditions consistent with the presentation of primary ciliary dyskinesia, supporting the previously implicated role of the centrosomally located Nme7 gene in ciliogenesis and control of ciliary transport.

The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Diverse roles of nucleoside diphosphate kinase in genome stability and growth fitness.

Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, catalyses reversible transfer of the γ phosphate from nucleoside triphosphates to nucleoside diphosphates and functions to maintain the pools of ribonucleotides and deoxyribonucleotides in the cell. As even a minor imbalance in the nucleotide pools can be mutagenic, NDK plays an antimutator role in maintaining genome integrity. However, the mechanism of the antimutator roles of NDK is not completely understood. In addition, NDKs play important roles in the host-pathogen interactions, metastasis, gene regulation, and various cellular metabolic processes. To add to these diverse roles of NDK in cells, a recent study now reveals that NDK may even confer mutator phenotypes to the cell by acting on the damaged deoxyribonucleoside diphosphates that may be formed during the oxidative stress. In this review, we discuss the roles of NDK in homeostasis of the nucleotide pools and genome integrity, and its possible implications in conferring growth/survival fitness to the organisms in the changing environmental niches.

Optimization of nucleotide sugar supply for polysaccharide formation via thermodynamic buffering.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

The Function of NM23-H1/NME1 and Its Homologs in Major Processes Linked to Metastasis.

Metastasis suppressor genes (MSGs) inhibit different biological processes during metastatic progression without globally influencing development of the primary tumor. The first MSG, NM23 (non-metastatic clone 23, isoform H1) or now called NME1 (stands for non-metastatic) was identified some decades ago. Since then, ten human NM23 paralogs forming two groups have been discovered. Group I NM23 genes encode enzymes with evolutionarily highly conserved nucleoside diphosphate kinase (NDPK) activity. In this review we summarize how results from NDPKs in model organisms converged on human NM23 studies. Next, we examine the role of NM23-H1 and its homologs within the metastatic cascade, e.g. cell migration and invasion, proliferation and apoptosis. NM23-H1 homologs are well known inhibitors of cell migration. Drosophila studies revealed that AWD, the fly counterpart of NM23-H1 is a negative regulator of cell motility by modulating endocytosis of chemotactic receptors on the surface of migrating cells in cooperation with Shibire/Dynamin; this mechanism has been recently confirmed by human studies. NM23-H1 inhibits proliferation of tumor cells by phosphorylating the MAPK scaffold, kinase suppressor of Ras (KSR), resulting in suppression of MAPK signalling. This mechanism was also observed with the C. elegans homolog, NDK-1, albeit with an inverse effect on MAPK activation. Both NM23-H1 and NDK-1 promote apoptotic cell death. In addition, NDK-1, NM23-H1 and their mouse counterpart NM23-M1 were shown to promote phagocytosis in an evolutionarily conserved manner. In summary, inhibition of cell migration and proliferation, alongside actions in apoptosis and phagocytosis are all mechanisms through which NM23-H1 acts against metastatic progression.

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses

BACKGROUND NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.

Characterization of Nme5-Like Gene/Protein from the Red Alga Chondrus Crispus.

The Nme gene/protein family of nucleoside diphosphate kinases (NDPK) was originally named after its member Nm23-H1/Nme1, the first identified metastasis suppressor. Human Nme proteins are divided in two groups. They all possess nucleoside diphosphate kinase domain (NDK). Group I (Nme1-Nme4) display a single type NDK domain, whereas Group II (Nme5-Nme9) display a single or several different NDK domains, associated or not associated with extra-domains. Data strongly suggest that, unlike Group I, none of the members of Group II display measurable NDPK activity, although some of them autophosphorylate. The multimeric form is required for the NDPK activity. Group I proteins are known to multimerize, while there are no data on the multimerization of Group II proteins. The Group II ancestral type protein was shown to be conserved in several species from three eukaryotic supergroups. Here, we analysed the Nme protein from an early branching eukaryotic lineage, the red alga Chondrus crispus. We show that the ancestral type protein, unlike its human homologue, was fully functional multimeric NDPK with high affinity to various types of DNA and dispersed localization throughout the eukaryotic cell. Its overexpression inhibits both cell proliferation and the anchorage-independent growth of cells in soft agar but fails to deregulate cell apoptosis. We conclude that the ancestral gene has changed during eukaryotic evolution, possibly in correlation with the protein function.

Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli.

The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

Expression profiles of p53/p73, NME and GLI families in metastatic melanoma tissue and cell lines.

Unlike other tumours, TP53 is rarely mutated in melanoma; however, it fails to function as a tumour suppressor. We assume that its functions might be altered through interactions with several families of proteins, including p53/p73, NME and GLI. To elucidate the potential interplay among these families we analysed the expression profiles of aforementioned genes and proteins in a panel of melanoma cell lines, metastatic melanoma specimens and healthy corresponding tissue. Using qPCR a higher level of NME1 gene expression and lower levels of Δ40p53ß, ΔNp73, GLI1, GLI2 and PTCH1 were observed in tumour samples compared to healthy tissue. Protein expression of Δ133p53α, Δ160p53α and ΔNp73α isoforms, NME1 and NME2, and N'ΔGLI1, GLI1FL, GLI2ΔN isoforms was elevated in tumour tissue, whereas ∆Np73ß was downregulated. The results in melanoma cell lines, in general, support these findings. In addition, we correlated expression profiles with clinical features and outcome. Higher Δ133p53ß and p53α mRNA and both GLI1 mRNA and GLI3R protein expression had a negative impact on the overall survival. Shorter overall survival was also connected with lower p53ß and NME1 gene expression levels. In conclusion, all examined genes may have implications in melanoma development and functional inactivity of TP53.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

MiR-35 buffers apoptosis thresholds in the C. elegans germline by antagonizing both MAPK and core apoptosis pathways.

Apoptosis is a genetically programmed cell death process with profound roles in development and disease. MicroRNAs modulate the expression of many proteins and are often deregulated in human diseases, such as cancer. C. elegans germ cells undergo apoptosis in response to genotoxic stress by the combined activities of the core apoptosis and MAPK pathways, but how their signalling thresholds are buffered is an open question. Here we show mir-35-42 miRNA family play a dual role in antagonizing both NDK-1, a positive regulator of MAPK signalling, and the BH3-only pro-apoptotic protein EGL-1 to regulate the magnitude of DNA damage-induced apoptosis in the C. elegans germline. We show that while miR-35 represses EGL-1 by promoting transcript degradation, repression of NDK-1 may be through sequestration of the transcript to inhibit translation. Importantly, dramatic increase in NDK-1 expression was observed in cells about to die. In the absence of miR-35, increased NDK-1 activity enhanced MAPK signalling that lead to significant increases in germ cell death. Our findings demonstrate that NDK-1 acts upstream of (or in parallel to) EGL-1, and that miR-35 targets both egl-1 and ndk-1 to fine-tune cell killing in response to genotoxic stress.