Function Of Nucleoside Diphosphate Kinase Research Articles

NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

A Comparative Transcriptome Analysis Reveals Physiological Maturation Properties of Mycelia in Pleurotus tuoliensis.

Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30–60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes—including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases—were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.

Molecular and structural basis of nucleoside diphosphate kinase–mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus

The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encoding gene copies as assessed by qPCR. Using gene-knockout and complementation experiments, we found that AfNDK regulates spore and sclerotia development and is involved in plant virulence as assessed in corn and peanut seed-based assays. An antifungal test with the inhibitor azidothymidine suppressed AfNDK activity in vitro and prevented spore production and sclerotia formation in A. flavus, confirming AfNDK's regulatory functions. Crystallographic analysis of AfNDK, coupled with site-directed mutagenesis experiments, revealed three residues (Arg-104, His-117, and Asp-120) as key sites that contribute to spore and sclerotia development. These results not only enrich our knowledge of the regulatory role of this important protein in A. flavus, but also provide insights into the prevention of A. flavus infection in plants and seeds, as well as into the structural features relevant for future antifungal drug development.

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis.

The primary structure and function of nucleoside diphosphate kinase (NDK), a substrate non-specific enzyme involved in the maintenance of nucleotide pools is also implicated to play pivotal roles in many other cellular processes. NDK is conserved from bacteria to human and forms a homotetramer or hexamer to exhibit its biological activity. However, the nature of the functional oligomeric form of the enzyme differs among different organisms. The functional form of NDKs from many bacterial systems, including that of the human pathogen, Mycobacterium tuberculosis (MtuNDK), is a hexamer, although some bacterial NDKs are tetrameric in nature. The present study addresses the oligomeric property of MsmNDK and how a dimer, the basic subunit of a functional hexamer, is stabilized by hydrogen bonds and hydrophobic interactions. Homology modeling was generated using the three-dimensional structure of MtuNDK as a template; the residues interacting at the monomer-monomer interface of MsmNDK were mapped. Using recombinant enzymes of wild type, catalytically inactive mutant, and monomer-monomer interactive mutants of MsmNDK, the stability of the dimer was verified under heat, SDS, low pH, and methanol. The predicted residues (Gln17, Ser24 and Glu27) were engaged in dimer formation, however the mutated proteins retained the ATPase and GTPase activity even after introducing single (MsmNDK- Q17A, MsmNDK-E27A, and MsmNDK-E27Q) and double (MsmNDK-E27A/Q17A) mutation. However, the monomer-monomer interaction could be abolished using methanol, indicating the stabilization of the monomer-monomer interaction by hydrophobic interaction.

Down-regulation of expression and function of nucleoside diphosphate kinase in insulin-secreting β-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.

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.

The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates

Mollicutes are wall-less bacteria and cause various diseases in humans, animals and plants. They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis. Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays a critical role in the synthesis of nucleic acids precursors, i.e. NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date. Therefore, it would be of interest to know how Mollicutes synthesize dNTPs/NTPs without NDK. To answer this question, nucleoside monophosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs/dNTPs. In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli. The recombinant enzymes were purified and characterized. These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs. The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP synthesis, in general, were much higher (up to 100-fold) than that of (d)NTP. Equilibrium studies with adenylate kinase suggested that the rates of NTPs/dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs. These results strongly indicate that NMPKs could substitute the NDK function in vivo.

Amino Acid Residues Involved in Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase from Mycobacterium tuberculosis

Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of approximately 16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5'-adenosine 3'-phosphate, and adenosine 3'-phosphate 5'-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.

Putative functions of nucleoside diphosphate kinase in plants and fungi.

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. 1) By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red-far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. 2) NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. 3) In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. 4) In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1Pro72His) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as "light-induced polarity of perithecia." In wild-type in darkness the beak was formed at random places on perithecia, and in ndkPro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.

Regulation of dynamin by nucleoside diphosphate kinase.

Nucleoside diphosphate (NDP) kinase is required for multiple cellular functions, including cell growth, motility, and differentiation, and its loss is associated with pathologies including tumor metastasis. A recent study has revealed a previously unknown function for NDP kinase as positive regulator of dynamin, a GTPase essential for endocytosis. In this review we describe the evidence that NDP kinase function is essential for endocytosis and also elaborate on a mechanism for NDP kinase regulation of dynamin. Recently documented interactions between endocytosis and cell signaling have revealed new insights into potential mechanisms of cancer. In this context, we discuss the possible relevance of NDP kinase and dynamin interaction for tumor suppression.

Interactions between Escherichia coliNucleoside-diphosphate Kinase and DNA

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.

Analysis of light signal transduction affecting morphogenesis of perithecia in Neurospora crassa

The development of perithecia in Neurospora crassa is under the control of light, nutritional condition such as nitrogen starvation and temperature. Blue light can stimulate(i)the formation of protoperithecia on nitrogen deficient media, (ii)positive bending of perithecial beak, and(iii)the ejection of ascospores from asci in the perithecia. We found that the position of the formation of perithecial beak was dependent on light.(iv)Light stimulated the positioning of perithecial beak on the upperward of it, which we defined as“ light induced polarity of perithecia”. We developed in vitro systems to analyse molecular mechanism of light signal transduction that used the membrane and soluble fractions of mycelial extract. UV-A light induced the binding of ATP/GTP to 58kDA, 77kDa, 83kDa, and 129kDa proteins in the membrane fraction. Blue light stimulated the ADP-ribosylation of 38kDa and 56kDa proteins in the soluble fraction. Blue light also stimulated the phosphorylation of 15kDa protein in the membrane fraction. We isolated a mutant missing completely the phosphorylation of 15kDa protein in the membrane fraction. The mutation designated as psp(phosphorylation of small protein)was mapped on LGVR between al-3and his-6. The psp mutats showed blindness in the light induced polarity of perithecia. The 15kDa protein was purified and the cDNA was cloned. It was identified as nucleoside diphosphate(NDP)kinase. The cDNA for NDP kinase prepared from psp mutants included a mutation of amino acid replacement from Pro 72to His. The putative function of NDP kinase in the light signal transduction to perithecial morphogenesis was discussed.

Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.

nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.