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The transcription-inhibitory G-Quadruplex(Pu27-GQ) at c-MYC promoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increase c-MYC transcription. Here, we used Inosine 5'-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties.
The mechanisms that structure the mammalian endoplasmic reticulum (ER) network are not fully understood. Here we show that salt extraction of semi-intact normal rat kidney (NRK) fibroblasts and subsequent incubation of the extracted cells with ATP resulted in dramatic ER network retraction. Under these conditions, addition of a single protein, Nucleoside Diphosphate Kinase B (NDKB), was sufficient to reverse the retraction and to promote ER network extension. The underlying mechanism of membrane extension involved direct lipid binding, as NDKB bound phosphatidylinositol (PtdIns)(4)P, PtdIns(4,5)P(2) and phosphatidic acid (PA); binding to these anionic lipids required clusters of basic residues on the surface of the NDKB hexamer; and amino acid changes in NDKB that blocked lipid binding also blocked ER network extension. Remarkably, purified NDKB transformed a uniform population of synthetic lipid vesicles into extensive membrane networks, and this also required its phospholipid-binding activity. Altogether these results identify a protein sufficient to scaffold extended membrane networks, and suggest a possible role for NDKB-like proteins, as well as phosphoinositides and/or acidic phospholipids, in modulating ER network morphogenesis.
Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.
Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme found in all organisms and cell types, which catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The gene encoding for NDPK from Drosophila melanogaster was amplified from the genomic DNA. The recombinant NDPK (rNDPK) was overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA agarose affinity chromatography, HiTrap SP HP cation exchange chromatography and HiLoad 16/60 Superdex 200 gel filtration chromatography. The gel filtration chromatography and analytical ultracentrifugation showed that rNDPK was a trimer in solution. The binding affinity of NDPs with rNDPK, measured by isothermal titration calorimetry, indicated that the purines nucleotides show higher binding affinity compared with pyrimidines. The rNDPK had a definite nuclease activity in vitro, which could cleave supercoiled plasmid DNA, but had no effect on dsDNA and ssDNA. Furthermore, the structure for NDPK was determined by using the sitting drop vapor diffusion method. In the final model, the asymmetric unit is made of three molecules, each of which consists of a four-stranded anti-parallel β-sheets and seven α-helices. Sequence alignment and structure comparison illustrated that the simulated nucleotide-binding active site are conserved.
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.
MDA-MB-435S human breast cancer cells (435S) secrete nucleoside diphosphate kinase (NDPK) that supports metastases and is inhibited by epigallocatechin gallate (EGCG) and ellagic acid (EA). We hypothesise that 435S cell-secreted NDPK-B supports tumour formation by modulating ATP levels locally to activate endothelial cell (EC) P2Y receptor-mediated angiogenesis. Epigallocatechin gallate (IC50=8-10 microM) and EA (IC50=2-3 microM) suppressed 435S cell growth, but had less effect on human CD31+ EC growth. Epigallocatechin gallate (IC50=11 microM) and EA (IC50=1 microM) also prevented CD31+ EC tubulogenesis on Matrigeltrade mark. 435S cell-conditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 microM), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y 1 receptor activation by NDPK-generated nucleotide (100 microM ATP) or by 10 microM 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y 1 antagonist MRS2179 (10 microM). Physiological amounts of purified as well as 435S cell-secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 microM MRS2179, indicating a P2Y 1 receptor-mediated pathway. These results support the notion that secreted NDPK mediates angiogenesis via P2Y receptor signalling and suggests that novel inhibitors of NDPK may be useful as therapeutics.
Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.
The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.
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.
We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk.
Aspergillus fumigatus is a major pathogen of invasive pulmonary aspergillosis with high mortality rate. The nucleoside diphosphate kinase of A. fumigatus, AfNDK (also called SwoH) is essential for its viability, however, its structural characteristic was unknown. In this study, we solved the crystal structure of AfNDK and found that it exists predominantly in form of tetramer in solution. Oligomeric form rather than dimeric form was essential for its kinase activity. The Arg30 and the C terminal amino acids were crucial for dimer-dimer interaction and the viability of A. fumigatus. Mutation V83F might make the secondary structure α5 helix protrude outward so that the whole protein structure became unstable at higher temperature, which might subsequently result to the inviability of A. fumigatus under 44 °C. In conclusion, the crystal structure of AfNDK was for the first time analyzed and the stability of the tetrameric form with dimer-dimer interaction were crucial for its function in A. fumigatus.
Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. It is also secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions.
Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.
Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.
In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.
Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.
The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.
Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.
Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.
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