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During a heart failure, higher amount of nucleoside diphosphate kinase (NDPK) enzyme in the sarcolemma membrane inhibits the synthesis of second messenger cyclic adenosine monophosphate (cAMP), which is required for the regulation of the calcium ion balance for normal functioning of the heart. In a dependent pathway, NDPK normally phosphorylates the stimulatory guanosine diphosphate, GDP(s), to a guanosine triphosphate, GTP(s), on the heterotrimeric (α, β and γ subunits) guanine nucleotide binding protein (G protein), resulting in the stimulation of the cAMP formation. In case of a heart failure, an increased quantity of NDPK also reacts with the inhibitory GDP(i), which is converted to a GTP(i), resulting in the inhibition of the cAMP formation. Typically, the βγ dimer of the G protein binds with hexameric NDPK-B/C complex and receives the phosphate at the residue His266 from residue His118 of NDPK-B. It is known that NDPK-C is required for NDPK-B to phosphorylate the G protein. In this work, the interactions between NDPK-B and NDPK-C are quantified in the presence and absence of graphene oxide (GO) as well as those between NDPK-B and GO through stability analysis involving hydrogen bonds, center of mass (COM), root mean square deviation (RMSD), and salt bridges, and energetics analysis involving van der Waals (VDW) and electrostatic energies. Furthermore, the role of water molecules at the interface of NDPK-B and NDPK-C as well as between NDPK-B and GO is investigated to understand the nature of interactions. It is found that the adsorption of NDPK-B on GO triggers a potential conformational change in the structure of NDPK-B, resulting in a diminished interaction with NDPK-C. This is confirmed through a reduced center of mass (COM) distance between NDPK-B and GO (from 40 A□ to 30 A□) and an increased COM distance between NDPK-B and NDPK-C (from 50 A□ to 60 A□). Furthermore, this is also supported by fewer salt bridges between NDPK-B and NDPK-C, and an increased number of hydrogen bonds formed by the interfacial water molecules. As NDPK-C is crucial to be complexed with NDPK-B for successful interaction of NDPK-B with the G protein, this finding shows that GO can suppress the interactions between NDPK-B/C and G proteins, thereby providing an additional insight into the role of GO in the heart failure mechanism.AUTHOR SUMMARYWe report a novel computational understanding of the interactions between the enzymes NDPK-B and NDPK-C with GO as a potential inhibitor to such interactions and its implications. These types of interactions can play influential roles in many biochemical processes including those that take place during heart failure. A second messenger called cAMP is needed for proper cardiac contraction through the actions of NDPK-B/NDPK-C. It is needed to study the interactions between NDPK-B and NDPK-C to control the synthesis of cAMP. Towards this end, GO is tested through molecular simulations to understand the interactions between NDPK-B and NDPK-C. Influencing or modifying such enzyme active sites has been very less explored and, in this work, the molecular simulations suggest that GO is able to interact with the active site of NDPK-B to provide a sustained cAMP synthesis for longer duration. We found that conformational changes within NDPK-B and NDPK-C influence the interactions between them and such conformational changes are found to be governed by their adsorption on GO. Finally, we found the role of interfacial water molecules between NDPK-B and GO to be crucial in maintaining the interface between them.

Background: The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. Objective: The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). Methods: The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. Results: NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. Conclusion: The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.

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.

Background: The aim of the present work was to evaluate the prognostic significance in patients with T1 breast cancer of tissue expression of the two oncosuppressors phosphatase and tensin homolog (PTEN) and non-metastatic clone 23 (NM23) as detected by immunohistochemistry. Materials and Methods: We prospectively analyzed 62 patients who underwent surgery for a T1 stage breast cancer. Expression of PTEN and NM23 was tested for correlation with clinical characteristics and clinical outcome. Results: Of the 62 patients considered for our study, 16 underwent mastectomy and 46 underwent conservative surgical treatment. The surgery was considered radical (R0) in all cases described. PTEN and NM23 expression was higher in patients with no lymph node metastases and no recurrent cancer at a mean follow-up of 36 months (range=6-48 months). This correlation was more evident when both PTNE and NM23 expression were highly expressed (p

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.

While extant organisms synthesize proteins using approximately 20 kinds of genetically coded amino acids, the earliest protein synthesis system is likely to have been much simpler, utilizing a reduced set of amino acids. However, which types of building blocks were involved in primordial protein synthesis remains unclear. Herein, we reconstructed three convergent sequences of an ancestral nucleoside diphosphate kinase, each comprising a 10 amino acid "alphabet," and found that two of these variants folded into soluble and stable tertiary structures. Therefore, an alphabet consisting of 10 amino acids contains sufficient information for creating stable proteins. Furthermore, re-incorporation of a few more amino acid types into the active site of the 10 amino acid variants improved the catalytic activity, although the specific activity was not as high as that of extant proteins. Collectively, our results provide experimental support for the idea that robust protein scaffolds can be built with a subset of the current 20 amino acids that might have existed abundantly in the prebiotic environment, while the other amino acids, especially those with functional sidechains, evolved to contribute to efficient enzyme catalysis.

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.

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.

The family of NME proteins represents a quite complex group of multifunctional enzymes [...]

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 Ni

Cancer stem cells (CSCs), which can self-renew and produce heterogeneous cancer cells, are the key factors during tumorigenesis. Transcription factors take essential effects on CSCs. However, the role of transcription factors in regulating the stemness of gastric cancer stem-like cells has not been well explored. In this investigation, it was found that transcription factor NME2 (NME/NM23 nucleoside diphosphate kinase 2) was upregulated in gastric cancer stem-like cells that sorted from the solid tumors of patients with gastric cancer and gastric cancer cell lines. NME2 could preserve the stemness of gastric cancer stem-like cells via suppressing their apoptosis. In vitro and in vivo data revealed that NME2 was crucial for maintaining the stemness of gastric cancer stem cells by enhancing the expression of anti-apoptosis genes. Consequently, our data contributed a new perspective to the relationship between transcription factor and the stemness maintenance of gastric cancer stem cells.

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.

NM23 (NME) is a metastasis suppressor that significantly reduces metastasis without affecting primary tumor size, however, the precise molecular mechanisms are not completely understood. We examined the role of dynamin (DNM2), a GTPase regulating membrane scission of vesicles in endocytosis, in NME1 and NME2 regulation of tumor cell motility and metastasis. Overexpression of NMEs in MDA-MB-231T and MDA-MB-435 cancer cell lines increased endocytosis of transferrin and EGF receptors (TfR and EGFR) concurrent with motility and migration suppression. The internalized vesicles, costained with Rab5, had AP2 depleted from the cell surface and exhibited increased Rab5-GTP levels, consistent with endocytosis. Dynamin inhibitors Iminodyn-22 and Dynole-34-2, or shRNA-mediated downregulation of DNM2, impaired NME's ability to augment endocytosis or suppress tumor cell motility. In a lung metastasis assay, NME1 overexpression failed to significantly suppress metastasis in the DNM2 knockdown MDA-MB-231T cells. Using the EGF–EGFR signaling axis as a model in MDA-MB-231T cells, NME1 decreased pEGFR and pAkt expression in a DNM2-dependent manner, indicating the relevance of this interaction for downstream signaling. NME–DNM2 interaction was confirmed in two-way coimmunoprecipitations. Transfection of a NME1 site–directed mutant lacking histidine protein kinase activity but retaining nucleoside diphosphate kinase (NDPK) activity showed that the NDPK activity of NME was insufficient to promote endocytosis or inhibit EGFR signaling. We show that addition of NME1 or NME2 to DNM2 facilitates DNM2 oligomerization and increases GTPase activity, both required for vesicle scission. NME–DNM2 interaction may contribute to metastasis suppression by altering tumor endocytic and motility phenotypes. Significance: NME1 suppresses metastasis via changes in tumor endocytosis and motility, mediated by dynamin (DNM2) GTPase activity.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium. In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium. However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15–30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

Precise control of stem cell (SC) proliferation ensures tissue homeostasis. In the Drosophila intestine, injury-induced regeneration involves initial activation of intestinal SC (ISC) proliferation and subsequent return to quiescence. These two phases of the regenerative response are controlled by differential availability of the BMP type I receptor Thickveins (Tkv), yet how its expression is dynamically regulated remains unclear. Here we show that during homeostasis, the E3 ubiquitin ligase Highwire and the ubiquitin-proteasome system maintain low Tkv protein expression. After ISC activation, Tkv is stabilized by proteasome inhibition and undergoes endocytosis due to the induction of the nucleoside diphosphate kinase Abnormal Wing Disc (AWD). Tkv internalization is required for the activation of the Smad protein Mad, and for the return to quiescence after a regenerative episode. Our data provide insight into the mechanisms ensuring tissue homeostasis by dynamic control of somatic stem cell activity., Regeneration after injury in the Drosophila intestine involves early activation of intestinal stem cells (ISCs) and subsequent return to quiescence. Here the authors show that return to quiescence by ISCs involves BMP Type I receptor Tkv protein stabilization along with AWD mediated internalization into endocytic vesicles.

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.

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.

Trialkyltins and triaryltins function as nuclear retinoid X receptors (RXR) agonists due to their affinity to the ligand-binding domain of RXR subtypes and function as transcriptional activators. We present the data on combined effects of all-trans retinoic acid (ATRA), retinoic acid receptor (RAR) ligand and tributyltin chloride or triphenyltin chloride (RXR ligands) on protein pattern in MDA-MB-231 cells. Proteomic strategies based on bottom-up method were applied in this study. The total cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. By employing PDQuest™ software, we identified more than 30 proteins differently affected by the above compounds. For further studies, we selected specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or to cellular processes as apoptosis, regulation of gene transcription or epithelial-mesenchymal transition (annexin 5, nucleoside diphosphate kinase B and vimentin). We have found that treatment of MDA-MB-231 cells with triorganotins reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin and nucleoside diphosphate kinase B. These results demonstrate that RXR/RAR heterodimer may act under this experimental design as permissive heterodimer allowing activation of RXR by triorganotins.

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.

Metabolic reprogramming by oncogenic signaling is a hallmark of cancer. Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms inducing hyperactivation of Wnt/β-catenin signaling and strategies for targeting this pathway are incompletely understood. In this study, we find nucleoside diphosphate kinase 7 (NME7) to be a positive regulator of Wnt/β-catenin signaling. Upregulation of NME7 positively correlated with the clinical features of HCC. Knockdown of NME7 inhibited HCC growth in vitro and in vivo, whereas overexpression of NME7 cooperated with c-Myc to drive tumorigenesis in a mouse model and to promote the growth of tumor-derived organoids. Mechanistically, NME7 bound and phosphorylated serine 9 of GSK3β to promote β-catenin activation. Furthermore, MTHFD2, the key enzyme in one-carbon metabolism, was a target gene of β-catenin and mediated the effects of NME7. Tumor-derived organoids with NME7 overexpression exhibited increased sensitivity to MTHFD2 inhibition. In addition, expression levels of NME7, β-catenin, and MTHFD2 correlated with each other and with poor prognosis in patients with HCC. Collectively, this study emphasizes the crucial roles of NME7 protein kinase activity in promoting Wnt/β-catenin signaling and one-carbon metabolism, suggesting NME7 and MTHFD2 as potential therapeutic targets for HCC. Significance: The identification of NME7 as an activator of Wnt/β-catenin signaling and MTHFD2 expression in HCC reveals a mechanism regulating one-carbon metabolism and potential therapeutic strategies for treating this disease.

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.

Metastasis accounts for over 90% of cancer-related deaths, yet the mechanisms guiding this process remain unclear. Secreted nucleoside diphosphate kinase A and B (NDPK) support breast cancer metastasis. Proteomic evidence confirms their presence in breast cancer-derived extracellular vesicles (EVs). We investigated the role of EV-associated NDPK in modulating the host microenvironment in favor of pre-metastatic niche formation. We measured NDPK expression and activity in EVs isolated from triple-negative breast cancer (MDA-MB-231) and non-tumorigenic mammary epithelial (HME1) cells using flow cytometry, western blot, and ATP assay. We evaluated the effects of EV-associated NDPK on endothelial cell migration, vascular remodeling, and metastasis. We further assessed MDA-MB-231 EV-induced proteomic changes in support of pre-metastatic lung niche formation. NDPK-B expression and phosphotransferase activity were enriched in MDA-MB-231 EVs that promote vascular endothelial cell migration and disrupt monolayer integrity. MDA-MB-231 EV-treated mice demonstrate pulmonary vascular leakage and enhanced experimental lung metastasis, whereas treatment with an NDPK inhibitor or a P2Y1 purinoreceptor antagonist blunts these effects. We identified perturbations to the purinergic signaling pathway in experimental lungs, lending evidence to support a role for EV-associated NDPK-B in lung pre-metastatic niche formation and metastatic outgrowth. These studies prompt further evaluation of NDPK-mediated EV signaling using targeted genetic silencing approaches.

c-MYC is deregulated in triple negative breast cancer (TNBC) pointing to be a promising biomarker for breast cancer treatment. Precise level of MYC expression is important in the control of cellular growth and proliferation. Designing of c-MYC-targeted antidotes to restore its basal level of cellular expression holds an optimistic approach towards anti-cancer treatment. MYC transcription is dominantly controlled by Nuclear Hypersensitive Element III-1 (NHEIII1) upstream of the promoter region possessing G-Quadruplex silencer element (Pu-27). We have investigated the selective binding-interaction profile of a natural phytophenolic compound Curcumin with native MYC G-quadruplex by conducting an array of biophysical experiments and in silico based Molecular Docking and Molecular Dynamic (MDs) simulation studies. Curcumin possesses immense anti-cancerous properties. We have observed significantly increased stability of MYC-G Quadruplex and thermodynamic spontaneity of Curcumin-MYC GQ binding with negative ΔG value. Transcription of MYC is tightly regulated by a complex mechanism involving promoters, enhancers and multiple transcription factors. We have used Curcumin as a model drug to understand the innate mechanism of controlling deregulated MYC back to its basal expression level. We have checked MYC-expression at transcriptional and translational level and proceeded for Chromatin Immuno-Precipitation assay (ChIP) to study the occupancy level of SP1, Heterogeneous nuclear ribonucleoprotein K (hnRNPK), Nucleoside Diphosphate Kinase 2 (NM23-H2) and Nucleolin at NHEIII1 upon Curcumin treatment of MDA-MB-231 cells. We have concluded that Curcumin binding tends to drive the equilibrium towards stable G-quadruplex formation repressing MYC back to its threshold-level. On retrospection of the synergistic effect of upregulated c-MYC and BCL-2 in cancer, we have also reported a new pathway [MYC-E2F-1-BCL-2-axis] through which Curcumin trigger apoptosis in cancer cells. Communicated by Ramaswamy H. Sarma

The fission and fusion processes determine mitochondrial morphology and function, in which signaling lipids play pivotal roles. Phosphatidic acid (PA) generated by PLD6, a mitochondrial phospholipase D, is essential for mitochondrial fusion; however, the underlying mechanism is still unclear. We previously discovered that a nucleoside diphosphate kinase, NME3, is critical for mitochondrial fusion yet through a kinase-independent activity. Here we demonstrate that NME3 functions as a mitochondrial tethering factor through binding to PA with its N-terminal amphipathic helix, enriching at the tip of mitochondria, and clustering membranes via its oligomeric structure. Optogenetic induction of PLD activity enhances NME3 recruitment to mitochondria in a catalytic-dependent manner, and NME3 is required for PLD6-mediated mitochondrial fusion. Together, PLD6-generated PA on mitochondria promotes NME3 binding, thereby increasing mitochondrial tethering to facilitate membrane fusion.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles.Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention.The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets.Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets.NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

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.

To date, a variety of Brucella effector proteins have been found to mediate host cell secretion, autophagy, inflammation, and other signal pathways, but nuclear effector proteins have not yet been reported. We identified the first Brucella nucleomodulin, BspJ, and we screened out the BspJ interaction host proteins NME/NM23 nucleoside diphosphate kinase 2 (NME2) and creatine kinase B (CKB) through yeast two-hybrid and co-immunoprecipitation assays. These proteins are related to the host cell energy synthesis, metabolism, and apoptosis pathways. Brucella nucleomodulin BspJ will decrease the expression level of NME2 and CKB. In addition, BspJ gene deletion strains promoted the apoptosis of macrophages and reduced the intracellular survival of Brucella in host cells. In short, we found nucleomodulin BspJ may directly or indirectly regulate host cell apoptosis through the interaction with NME2 and CKB by mediating energy metabolism pathways in response to the intracellular circulation of Brucella infection, but the mechanism needs further study.

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 A, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Our present study aimed to identify host cell proteins that may interact with avian reovirus (ARV) σA protein and their potential effect on ARV replication. The ARV structural protein σA has been demonstrated to suppress interferon production and confirmed to activate the PI3K/Akt pathway. However, host cell factors interacting with σA to affect ARV replication remain unknown. In current study, a cDNA library of chicken embryo fibroblasts (CEFs) was constructed, and host cell proteins interacting with σA were screened by a yeast two-hybrid system. We identified four candidate cellular proteins that interact with ARV σA protein. Among them, Gallus NME/NM23 nucleoside diphosphate kinase 2 (NME2) was further validated as a σA-binding protein through co-immunoprecipitation. The key interaction domain was identified at amino acids (aa) 121-416 in NME2 and at aa 71-139 in σA, respectively. We demonstrated that overexpression of NME2 substantially inhibited ARV replication. In addition silencing NME2 by small interfering RNAs (siRNAs) resulted in marked enhancement of ARV replication. Our work has demonstrated that NME2 is a σA-binding protein that may affect ARV replication in CEF cells.

Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s-1 ), specificity constant (626 ± 110.0 mm-1 ·s-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s-1 ) and lowest specificity constant (40.2 ± 4.4 mm-1 ·s-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).