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1.
Cell Death Differ ; 23(7): 1140-51, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26742431

RESUMO

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.


Assuntos
Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mitofagia , Nucleosídeo Difosfato Quinase D/metabolismo , Animais , Autofagia/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/toxicidade , Cardiolipinas/análise , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/patologia , Mitofagia/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Nucleosídeo Difosfato Quinase D/antagonistas & inibidores , Nucleosídeo Difosfato Quinase D/genética , Oxidopamina/farmacologia , Ligação Proteica , Interferência de RNA , Rotenona/farmacologia
2.
Gene ; 296(1-2): 87-97, 2002 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-12383506

RESUMO

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion, progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning, characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Núcleosídeo-Difosfato Quinase/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes/genética , Humanos , Hibridização In Situ , Íntrons , Isoenzimas/genética , Camundongos , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de Transcrição
3.
Eur J Biochem ; 268(7): 1972-81, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11277919

RESUMO

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.


Assuntos
Isoenzimas/genética , Isoenzimas/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Sequência de Aminoácidos , Western Blotting , Catálise , Estabilidade Enzimática , Temperatura Alta , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma/enzimologia , Desnaturação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Ureia/farmacologia
4.
J Biol Chem ; 275(19): 14264-72, 2000 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-10799505

RESUMO

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.


Assuntos
Mitocôndrias/enzimologia , Proteínas Monoméricas de Ligação ao GTP/genética , Núcleosídeo-Difosfato Quinase/genética , Fatores de Transcrição/genética , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , DNA Complementar , Humanos , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/isolamento & purificação , Nucleosídeo NM23 Difosfato Quinases , Nucleosídeo Difosfato Quinase D , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação
5.
J Cell Physiol ; 182(2): 144-9, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10623877

RESUMO

Tumor suppressor genes have a pivotal role in normal cells regulating cell cycle processes negatively. Furthermore, the inhibition of cell proliferation is a crucial step in the achievement of cell differentiation. Increasing evidence suggests that the nm23 genes, initially documented as suppressors of the invasive phenotype in some cancer types, are involved in the control of normal development and differentiation. In this review, we summarize some data concerning the involvement of the nm23 genes in development and differentiation, attempting to delineate an overall view of many facets of their biological role.


Assuntos
Diferenciação Celular/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/fisiologia , Envelhecimento/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Família Multigênica , Nucleosídeo NM23 Difosfato Quinases , Homologia de Sequência , Fatores de Transcrição/genética
6.
J Bioenerg Biomembr ; 32(3): 247-58, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11768308

RESUMO

Biochemical experiments over the past 40 years have shown that nucleoside diphosphate (NDP) kinase activity, which catalyzes phosphoryl transfer from a nucleoside triphosphate to a nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Over the past 10 years, eight human genes of the nm23/NDP kinase family have been discovered that can be separated into two groups based on analysis of their sequences. In addition to catalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has come recently from the discovery of the genes nm23 and awd, which encode NDP kinases and are involved in tumor metastasis and Drosophila development, respectively. Current work shows that the human NDP kinase genes are differentially expressed in tissues and that their products are targeted to different subcellular locations. This suggests that Nm23/NDP kinases possess different, but specific, functions within the cell, depending on their localization. The roles of NDP kinases in metabolic pathways and nucleic acid synthesis are discussed.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Núcleosídeo-Difosfato Quinase/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/classificação , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/classificação , Núcleosídeo-Difosfato Quinase/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo
7.
Biochemistry ; 38(22): 7265-72, 1999 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10353838

RESUMO

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.


Assuntos
Desoxirribonucleotídeos/química , Núcleosídeo-Difosfato Quinase/química , Animais , Catálise , Cristalografia por Raios X , Dictyostelium/enzimologia , Didesoxinucleosídeos/química , Cinética , Modelos Químicos , Relação Estrutura-Atividade , Nucleotídeos de Timina/química , Viscosidade , Zidovudina/química
8.
Exp Cell Res ; 246(2): 355-67, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9925751

RESUMO

The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.


Assuntos
Citoesqueleto/metabolismo , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Especificidade de Anticorpos , Neoplasias da Mama , Divisão Celular , Núcleo Celular , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Interfase , Microtúbulos/metabolismo , Mitose , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/imunologia , Coelhos , Fatores de Transcrição/imunologia , Células Tumorais Cultivadas , Vimentina/análise
9.
FEBS Lett ; 434(3): 289-94, 1998 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-9742940

RESUMO

We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.


Assuntos
Histonas/genética , Proteínas Monoméricas de Ligação ao GTP , Espermatozoides/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Clonagem Molecular , Primers do DNA , DNA Complementar , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Testículo/citologia
10.
J Biol Chem ; 272(25): 15599-602, 1997 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-9188446

RESUMO

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.


Assuntos
Antígenos de Neoplasias/genética , Proteínas Monoméricas de Ligação ao GTP , Neuroblastoma/enzimologia , Núcleosídeo-Difosfato Quinase/genética , Mutação Puntual , Dobramento de Proteína , Fatores de Transcrição/genética , Antígenos de Neoplasias/química , Cromatografia em Gel , Dicroísmo Circular , Glicina , Humanos , Cinética , Peso Molecular , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma/genética , Núcleosídeo-Difosfato Quinase/química , Serina , Fatores de Transcrição/química
11.
Hum Genet ; 99(4): 550-7, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9099850

RESUMO

A novel human nm23/nucleoside diphosphate (NDP) kinase gene, called nm23-H4, was identified by screening a human stomach cDNA library with a probe generated by amplification by reverse transcription-polymerase chain reaction. The primers were designed from publicly available database cDNA sequences selected according to their homology to the human nn23-H1 putative metastasis suppressor gene. The full-length cDNA sequence predicts a 187 amino acid protein possessing the region homologous to NDP kinases with all residues crucial for nucleotide binding and catalysis, strongly suggesting that Nm23-H4 possesses NDP kinase activity. It shares 56, 55 and 60% identity with Nm23-H1, Nm23-H2 and DR-Nm23, respectively, the other human Nm23 proteins isolated so far. Compared with these proteins, Nm23-H4 contains an additional NH2-terminal region that is rich in positively charged residues and could indicate routing to mitochondria. The nm23-H4 gene has been localised to human chromosomal band 16p13.3. The corresponding 1.2 kb mRNA is widely distributed and expressed in a tissue-dependent manner, being found at very high levels in prostate, heart, liver, small intestine and skeletal muscle tissues and in low amounts in the brain and in blood leucocytes. Nm23-H4 naturally possesses the Pro-Ser substitution equivalent to the K-pn mutation (P97S) of Drosophila.


Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 16 , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Humanos , Dados de Sequência Molecular , Família Multigênica , Nucleosídeo NM23 Difosfato Quinases , Nucleosídeo Difosfato Quinase D , Filogenia , Homologia de Sequência de Aminoácidos
12.
Structure ; 3(12): 1307-14, 1995 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-8747457

RESUMO

BACKGROUND: Nucleoside diphosphate (NDP) kinases provide precursors for DNA and RNA synthesis. In mammals, these enzymes are also involved in cell regulations. Human NDP kinase B, product of the human nm23-H2 gene, is both an enzyme and a transcription factor. It activates transcription of the c-myc oncogene independently of its catalytic function, by binding to its promoter DNA. How do the two functions coexist? RESULTS: Recombinant human NDP kinase B was co-crystallized with GDP. The X-ray structure was solved at 2.0 A resolution by molecular replacement from the homologous Drosophila Awd protein. Both enzymes are homo-hexamers with a characteristic beta alpha beta beta alpha beta fold. GDP binds near the active site His118. The guanine base is in a surface cleft and interacts with the C terminus of another subunit. CONCLUSIONS: The beta alpha beta beta alpha beta fold, also present in the 'palm' domain of Escherichia coli DNA polymerase I and HIV reverse transcriptase, is both a mononucleotide- and a polynucleotide-binding fold. If NDP kinase B binds DNA in the same way as the polymerases, the enzyme must undergo a conformation change in order to carry out gene activation.


Assuntos
Proteínas de Drosophila , Guanosina Difosfato/química , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/química , Conformação Proteica , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA Polimerase I/química , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica , Guanosina Difosfato/metabolismo , Humanos , Hormônios de Inseto/química , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , Ligação Proteica , Multimerização Proteica , DNA Polimerase Dirigida por RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Ativação Transcricional
13.
Eur J Biochem ; 234(1): 200-7, 1995 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-8529641

RESUMO

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/metabolismo , Fosfotransferases/metabolismo , Serina/metabolismo , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Aminoácidos/análise , Sítios de Ligação , Humanos , Cinética , Nucleosídeo NM23 Difosfato Quinases , Fosforilação , Células Tumorais Cultivadas , Ureia/química
14.
Gut ; 37(5): 712-20, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8549951

RESUMO

Although a reduced expression of nm23 has been shown to correlate with a high metastatic potential in some human cancers, in colorectal cancers, conflicting data have been reported. As there are two homologous genes, nm23-H1 and nm23-H2, which encode the A and B subunits of nucleoside diphosphate kinase, efficient and simplified techniques were designed to selectively study nm23-H1 and -H2 expression in 35 colorectal cancers at both the protein and mRNA levels by immunoblotting, immunohistochemistry, and reverse transcription polymerase chain reaction (RT PCR) using specific antibodies and primers. Nm23-H1 and Nm23-H2 proteins were overexpressed in tumours compared with adjacent mucosa. This overexpression was lost, however, in some advanced cases: 89% and 81% of TNM (tumour, node, metastases) stages 0-II showed Nm23-H1 and -H2 overexpression, respectively, which significantly differed from 47% and 38% of stage III-IV tumours. Similar results were seen with nm23-H1 mRNA. Heterogenous labelling of tumoral cells was seen by immunohistological staining. This suggests a dichotomy: an overexpression of nm23-H1 and -H2 linked to early stages of cancer and a loss of nm23-H1 overexpression seen in more advanced stages. Therefore specific nm23-H1 determination should be evaluated as a prognostic factor in human colorectal carcinoma.


Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP , Fatores de Transcrição/análise , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Neoplasias Colorretais/patologia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Estadiamento de Neoplasias , Núcleosídeo-Difosfato Quinase/análise
15.
Nucleic Acids Res ; 23(19): 3858-64, 1995 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-7479028

RESUMO

Recently, a DNA binding protein 'PUF' was purified that binds to a poly-pyrimidine rich element in the human c-myc promoter. Cloning of the corresponding gene surprisingly identified this putative transcription factor as isoform B of the enzyme nucleoside diphosphate kinase (NDPK-B) [Postel et al. (1993) Science, 261, 478-480], the product of the potential metastasis suppressor gene nm23-H2. Using different recombinant NDP kinases, we demonstrate by electrophoretic mobility shift analysis (EMSA) that the NDP kinase DNA binding properties are predominantly observed with human isoform B. Unlike typical DNA binding proteins that are involved in transcriptional regulation, binding occurs to single-stranded DNA rather than to a double-stranded oligonucleotide. As a consequence, complexes of single-stranded DNA and NDPK-B are generated from double-stranded oligonucleotide hybrids in an ATP independent manner. In addition to the c-myc element, NDPK-B is binding in vitro to a variety of poly-pyrimidine rich sequences including dC or dT homo-oligomers, (CT)n dinucleotide repeats, the initiator region of the Adenovirus major late promoter and even poly-pyrimidine rich RNAs. The possible consequences of these findings in understanding the multiple roles of NDP kinase are discussed.


Assuntos
Isoenzimas/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Pirimidinas/metabolismo , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Genes myc , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA/metabolismo , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas
16.
J Mol Biol ; 243(5): 873-90, 1994 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-7966307

RESUMO

The X-ray structure of the nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been refined at 1.8 A resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 A. The Dictyostelium structure is described in detail and compared to those of Drosophila and Myxococcus xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular alpha/beta domain. The four-stranded beta-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve alpha-helices and a 20-residue segment called the Kpn loop. The beta-sheet is regular except for a beta-bulge in edge strand beta 2 and a gamma-turn at residue Ile120 just preceding strand beta 4. The latter may induce strain in the main chain near the active site His122. The alpha 1 beta 2 motif participates in forming dimers within the hexamer, helices alpha 1 and alpha 3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyostelium are conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococcus xanthus enzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.


Assuntos
Dictyostelium/enzimologia , Núcleosídeo-Difosfato Quinase/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Ligação de Hidrogênio , Dados de Sequência Molecular , Myxococcus/enzimologia , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
18.
Bull Cancer ; 80(8): 717-22, 1993 Aug.
Artigo em Francês | MEDLINE | ID: mdl-8204954

RESUMO

The nm23-H1 gene encoding the nucleoside diphosphate (NDP) kinase A has been proposed as a tumor metastasis suppressor. Two important features emerge from published data: 1) an inverse correlation between the metastatic invasion and the level of NDP kinase/Nm23 was observed in melanomas, hepatocellular carcinomas and, in some studies, on breast carcinomas; 2) an overexpression of NDP kinase/Nm23 was observed in several solid tumors as compared to normal surrounding tissues, positively correlated with aggressiveness in the case of neuroblastomas. The level of NDP kinase/Nm23 in tumors appears to be altered in different ways, related or not to the metastatic potential, depending on the tissue of origin. Its evaluation as a prognostic or diagnostic marker of tumor invasion and aggressiveness deserves further study.


Assuntos
Regulação Enzimológica da Expressão Gênica , Metástase Neoplásica/genética , Neoplasias/genética , Núcleosídeo-Difosfato Quinase/genética , Humanos , Neoplasias/enzimologia , Núcleosídeo-Difosfato Quinase/metabolismo , Prognóstico
19.
Anal Biochem ; 209(1): 6-8, 1993 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-8385421

RESUMO

A sensitive assay for nucleoside diphosphate kinase which utilizes microtiter plates is described. ATP, formed in the reaction between dGTP and ADP, is trapped by the glycerol kinase reaction. A colored compound is generated by glycerol-3-phosphate oxidase and peroxidase. This assay is useful for testing a large number of samples generated by chromatographic techniques or for screening purposes.


Assuntos
Núcleosídeo-Difosfato Quinase/análise , Peroxidases/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Fosfatos de Dinucleosídeos/farmacologia , Glicerol Quinase/metabolismo , Cinética , Microquímica/métodos , Núcleosídeo-Difosfato Quinase/metabolismo , Oxirredução
20.
EMBO J ; 11(9): 3203-8, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1324167

RESUMO

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.


Assuntos
Dictyostelium/enzimologia , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/química , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Nucleosídeo NM23 Difosfato Quinases , Conformação Proteica , Proteínas/química , Homologia de Sequência do Ácido Nucleico
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