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.

Modulation of small GTPase activity by NME proteins.

NME proteins are reported to influence signal transduction activity of small GTPases from the Ras superfamily by diverse mechanisms in addition to their generic NDP kinase activity, which replenishes the cytoplasmic pool of GTP. Comprehensive evidence shows that NME proteins modulate the activity of Ras GTPases, in particular members of the Rho family, via binding to their major activators GEFs. Direct interaction between several NMEs and Ras GTPases were also indicated in vitro and in vivo. These modes of regulation are mainly independent of the NME's kinase activity. NMEs also modulate the Ras-mediated signal transduction by interfering with the formation of a Ras signaling complex at the plasma membrane. In several examples, NMEs were proposed to perform the role of GAP proteins by promoting hydrolysis of the bound GTP, but this activity still requires additional verification. Early suggestions that NMEs can activate small GTPases by direct phosphorylation of the bound GDP, or by high-rate loading of GTP onto a closely apposed GTPase, were largely dismissed. In this review article, we survey and put into perspective published examples of identified and hypothetical mechanisms of Ras signaling modulation by NME proteins. We also point out involvement of NMEs in the transcriptional regulation of components of Ras GTPases-mediated signal transduction pathways, and reciprocal regulation of NME function by small GTPases, particularly related to NME's binding to membranes.

General and specific promotion of flagellar assembly by a flagellar nucleoside diphosphate kinase.

Nucleoside diphosphate kinases (NDKs) play a central role in diverse cellular processes using the canonical NDK activity or alternative mechanisms that remain poorly defined. Our study of dimeric NDK5 in a flagellar motility control complex, the radial spoke (RS), has revealed new modalities. The flagella in Chlamydomonas ndk5 mutant were paralyzed, albeit only deficient in three RS subunits. RS morphology appeared severely changed in averaged cryo-electron tomograms, suggesting that NDK5 is crucial for the intact spokehead formation as well as RS structural stability. Intriguingly, ndk5's flagella were also short, resembling those of an allelic spoke-less mutant. All ndk5's phenotypes were rescued by expressions of NDK5 or a mutated NDK5 lacking the canonical kinase activity. Importantly, the mutated NDK5 that appeared fully functional in ndk5 cells elicited a dominant-negative effect in wild-type cells, causing paralyzed short flagella with hypophosphorylated, less abundant, but intact RSs, and accumulated hypophosphorylated NDK5 in the cell body. We propose that NDK5 dimer is an RS structural subunit with an additional mechanism that uses cross-talk between the two NDK monomers to accelerate phosphorylation-related assembly of RSs and entire flagella.

Nucleoside diphosphate kinase (Ndk): A pleiotropic effector manipulating bacterial virulence and adaptive responses.

Nucleoside diphosphate kinase (Ndk) is a housekeeping enzyme that balances cellular nucleoside triphosphate (NTP) pools by catalyzing the reversible transfer of γ-phosphate from NTPs to nucleoside diphosphates (NDPs). In addition to its fundamental role in nucleotide metabolism, Ndk has roles in protein histidine phosphorylation, DNA cleavage/repair, and gene regulation. Recent studies have also revealed that Ndk secreted from bacteria is important in modulating virulence-associated phenotypes including quorum sensing regulation, type III secretion system activation, and virulence factor production. Moreover, after infection, Ndks released from bacteria are involved in regulating host defense activities, such as cell apoptosis, phagocytosis, and inflammatory responses. Given that Ndk exerts a pleiotropic effect on bacterial virulence and bacteria-host interactions, the biological significance of the bacterial Ndks during infection is intriguing. This review will provide a synopsis of the current knowledge regarding the biological properties and roles of Ndks in regulating bacterial virulence and adaptation and will discuss in depth the biological significance of Ndk during bacteria-host interactions.

NME7 is a functional component of the γ-tubulin ring complex.

As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.

Valproyl-CoA inhibits the activity of ATP- and GTP-dependent succinate:CoA ligases.

BACKGROUND: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences. OBJECTIVE: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL). METHODS: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure. RESULTS: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes. DISCUSSION: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.

Cellular function and molecular structure of ecto-nucleotidases.

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

Novel inhibitors of the Pseudomonas aeruginosa virulence factor LasB: a potential therapeutic approach for the attenuation of virulence mechanisms in pseudomonal infection.

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed "second-generation" antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (K(i) = 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

NME genes in epithelial morphogenesis.

The NME family of genes encodes highly conserved multifunctional proteins that have been shown to participate in nucleic acid metabolism, energy homeostasis, cell signaling, and cancer progression. Some family members, particularly isoforms 1 and 2, have attracted extensive interests because of their potential anti-metastasis activity. Unfortunately, there have been few consensus mechanistic explanations for this critical function because of the numerous molecular functions ascribed to these proteins, including nucleoside diphosphate kinase, protein kinase, nuclease, transcription factor, growth factor, among others. In addition, different studies showed contradictory prognostic correlations between NME expression levels and tumor progression in clinical samples. Thus, analyses using pliable in vivo systems have become critical for unraveling at least some aspects of the complex functions of this family of genes. Recent works using the Drosophila genetic system have suggested a role for NME in regulating epithelial cell motility and morphogenesis, which has also been demonstrated in mammalian epithelial cell culture. This function is mediated by promoting internalization of growth factor receptors in motile epithelial cells, and the adherens junction components such as E-cadherin and ß-catenin in epithelia that form the tissue linings. Interestingly, NME genes in epithelial cells appear to function in a defined range of expression levels. Either down-regulation or over-expression can perturb epithelial integrity, resulting in different aspects of epithelial abnormality. Such biphasic functions provide a plausible explanation for the documented anti-metastatic activity and the suspected oncogenic function. This review summarizes these recent findings and discusses their implications.

The NM23 family in development.

The NM23 (non-metastatic 23) family is almost universally conserved across all three domains of life: eubacteria, archaea and eucaryotes. Unicellular organisms possess one NM23 ortholog, whilst vertebrates possess several. Gene multiplication through evolution has been accompanied by structural and functional diversification. Many NM23 orthologs are nucleoside diphosphate kinases (NDP kinases), but some more recently evolved members lack NDP kinase activity and/or display other functions, for instance, acting as protein kinases or transcription factors. These members display overlapping but distinct expression patterns during vertebrate development. In this review, we describe the functional differences and similarities among various NM23 family members. Moreover, we establish orthologous relationships through a phylogenetic analysis of NM23 members across vertebrate species, including Xenopus laevis and zebrafish, primitive chordates and several phyla of invertebrates. Finally, we summarize the involvement of NM23 proteins in development, in particular neural development. Carcinogenesis is a process of misregulated development, and NM23 was initially implicated as a metastasis suppressor. A more detailed understanding of the evolution of the family and its role in vertebrate development will facilitate elucidation of the mechanism of NM23 involvement in human cancer.

Down-regulation of expression and function of nucleoside diphosphate kinase in insulin-secreting beta-cells under in vitro conditions of glucolipotoxicity.

Previously, we reported a significant reduction in expression and the activity of nucleoside diphosphate kinase (NDP kinase) in islets derived from the Goto-Kakizaki rat (GK rat), an animal model for type 2 diabetes. Herein, we examined the effects of chronic exposure of insulin-secreting beta-(INS 832/13) cells to high glucose (a model for glucotoxicity), palmitate (a model for lipotoxicity), or glucose plus palmitate (a model for glucolipotoxicity) on the expression and activity of nm23-H1 (NDP kinase A) and nm23-H2 (NDP kinase B). Our findings indicate a marked reduction in the expression of both nm23-H1 and nm23-H2 and the associated NDP kinase activity under each of these conditions. A cell-permeable analog of ceramide (CER) also mimicked the effects of palmitate in significantly reducing the expression of nm23-H1 and nm23-H2 and NDP kinase activity in these cells. These findings suggest that de novo generation of intracellular CER from palmitate might represent at least one of the signaling steps involved in lipid-induced effects on NDP kinase expression and function in beta-cells. Based on these data, we conclude that glucolipotoxic conditions significantly impair expression and function of NDP kinase in pancreatic beta-cells. Potential significance of these findings, specifically at the level of abnormal G-protein activation and impaired insulin secretion under glucolipotoxic conditions is discussed.

YNK1, the yeast homolog of human metastasis suppressor NM23, is required for repair of UV radiation- and etoposide-induced DNA damage.

In humans, NM23-H1 is a metastasis suppressor whose expression is reduced in metastatic melanoma and breast carcinoma cells, and which possesses the ability to inhibit metastatic growth without significant impact on the transformed phenotype. NM23-H1 exhibits three enzymatic activities in vitro, each with potential to maintain genomic stability, a 3'-5' exonuclease and two kinases, nucleoside diphosphate kinase (NDPK), and protein histidine kinase. Herein we have investigated the potential contributions of NM23 proteins to DNA repair in the yeast, Saccharomyces cerevisiae, which contains a single NM23 homolog, YNK1. Ablation of YNK1 delayed repair of UV- and etoposide-induced nuclear DNA damage by 3-6h. However, YNK1 had no impact upon the kinetics of MMS-induced DNA repair. Furthermore, YNK1 was not required for repair of mitochondrial DNA damage. To determine whether the nuclear DNA repair deficit manifested as an increase in mutation frequency, the CAN1 forward assay was employed. An YNK1 deletion was associated with increased mutation rates following treatment with either UV (2.6x) or MMS (1.6 x). Mutation spectral analysis further revealed significantly increased rates of base substitution and frameshift mutations following UV treatment in the ynk1Delta strain. This study indicates a novel role for YNK1 in DNA repair in yeast, and suggests an anti-mutator function that may contribute to the metastasis suppressor function of NM23-H1 in humans.

Measurement of human breast tumor cell-secreted shNDPK-B in a murine breast cancer model suggests its role in metastatic progression.

Human breast cancers metastasize early in tumorigenesis and distant lesions, though dormant are very likely extant at the time of diagnosis and treatment in the majority of cases. Removal of primary tumors by surgeons as an imperative of the current treatment approach, also removes inhibitory factors secreted by the primary tumor that had maintained the dormancy of the metastases. We have identified a factor secreted by human breast cancer cells that supports the formation of blood vessels and may be a principal early factor supporting the growth and development of metastases in human disease. Here we demonstrate for the first time that this factor, secreted (s) human (h) nucleoside diphosphate kinase type B (shNDPK-B), product of the nm23-h2 gene, can be detected specifically with high sensitivity (50 pg/ml; 2.5 pM) in an ELISA assay of our own design. We further demonstrate that shNDPK-B is released into the circulation in immunocompromized mice carrying the human breast carcinoma cell MDA-MB-231. These data support the hypothesis that shNDPK-B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development.

Molecular cloning and analysis of function of nucleoside diphosphate kinase (NDPK) from the scallop Chlamys farreri.

Nucleoside diphosphate kinase (NDPK) is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP. NDPK regulates a variety of eukaryotic cellular activities including cell proliferation, development, and differentiation. The ndpk cDNA was cloned from the hemocytes of the scallop Chlamys farreri and designated Cf-ndpk. The full-length sequence of Cf-ndpk consists of 715 bp encoding a polypeptide of 153 amino acids with a calculated molecular mass of 16927.52 daltons and pI of 7.64. The mRNA expression and distribution of Cf-ndpk in both bacterially challenged and unchallenged scallops were studied by Northern blotting and in situ hybridization. The results showed that Cf-ndpk transcripts were present in hemocytes, gill, adductor muscle, mantle, digestive gland, foot, and gonad, and the expression level increased in hemocytes after bacterial challenge. Recombinant Cf-NDPK expressed in Escherichia coli could transfer the terminal phosphate between UDP and ATP. The Cf-NDPK protein was present in all tested tissues including foot, adductor muscle, digestive gland, gonad, mantle, gill, and hemolymph. It was up-regulated in hemolymph after bacterial challenge. Taken together, these results suggest that NDPK may play roles in the innate immune response of scallop.

Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet beta-cell.

Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet beta-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the beta-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prokaryotic and eukaryotic cellular signal transduction. Most notably, phoshohistidine accounts for 6% of total protein phosphorylation in eukaryotes, which makes it nearly 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine and phosphothreonine. However, very little is known about the number of proteins with phosphohistidines, since they are highly labile and are rapidly lost during phosphoamino acid identification under standard experimental conditions. The overall objectives of this review are to: (i) summarize the existing evidence indicating the subcellular distribution and characterization of various histidine kinases in the islet beta-cell, (ii) describe evidence for functional regulation of these kinases by agonists of insulin secretion, (iii) present a working model to implicate novel regulatory roles for histidine kinases in the receptor-independent activation, by glucose, of G-proteins endogenous to the beta-cell, (iv) summarize evidence supporting the localization of protein histidine phosphatases in the islet beta-cell and (v) highlight experimental evidence suggesting potential defects in the histidine kinase signalling cascade in islets derived from the Goto-Kakizaki (GK) rat, a model for type 2 diabetes. Potential avenues for future research to further decipher regulatory roles for protein histidine phosphorylation in physiological insulin secretion are also discussed.

Awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion.

Border cell migration during Drosophila melanogaster oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. Here we show that the metastasis suppressor gene homolog Nm23/awd is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion.

[Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling].

The results of numerous investigations during the last 20 years have shown that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its beta-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to the GDP in the active center of G protein alpha-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.

Crystallographic and biochemical analysis of rotavirus NSP2 with nucleotides reveals a nucleoside diphosphate kinase-like activity.

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.