RESUMO
The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.
Assuntos
Mycobacterium tuberculosis/enzimologia , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Varredura Diferencial de Calorimetria , Cobre/análise , Cobre/metabolismo , Dimerização , Estabilidade de Medicamentos , Cinética , Oxirredução , Desnaturação Proteica , Dobramento de Proteína , TermodinâmicaRESUMO
In the melanoma M14 cell line, we found that the antimetastatic protein NM23/nucleoside diphosphate kinase binds to the promoters of the oncogene cMYC and of P53, a gene often mutated in human cancer (Cervoni et al. [2006] J. Cell. Biochem. 98:421-428). In a further study, we find now that IFI16, a transcriptional repressor, in both promoters binds to the G-rich fragment that also binds NM23/NDPK. These fragments possess non-B DNA structures. Moreover, by sequential chromatin immunoprecipitation (re-ChIP) we show that the two proteins (IFI16 and NM23/NDPK) are simultaneously bound in vivo to the same DNA fragments. Since P53 stimulates apoptosis and inhibits cellular growth, and cMYC promotes cell growth and, in several instances, also apoptosis, the presence of NM23 and IFI16 on the same DNA fragments suggests their common involvement in the reduced development of some tumors.
Assuntos
DNA/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Melanoma/patologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Proteínas Nucleares/fisiologia , Oligodesoxirribonucleotídeos/metabolismo , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.
Assuntos
Proteínas de Neoplasias/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fosfoproteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Catálise , Linhagem Celular Tumoral , Primers do DNA , Ativação Enzimática , Humanos , Cinética , Mutagênese Sítio-Dirigida , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma , Fosforilação , Dobramento de Proteína , Proteínas Recombinantes/metabolismoRESUMO
We isolated and analyzed by chromatin immunoprecipitation (ChIP) in viable M14 cells DNA sequences bound to the antimetastatic protein nucleoside diphosphate kinase (NM23/NDPK) to shed some light on the nuclear functions of this protein and on the mechanism by which it acts in development and cancer. We assessed the presence of selected sequences from promoters of platelet-derived growth factor A (PDGF-A), c-myc, myeloperoxidase (MPO), CD11b, p53, WT1, CCR5, ING1, and NM23-H1 genes in the cross-linked complexes. Quantitative PCR (Q-PCR) showed a substantial enrichment of the correlated oncosuppressor genes p53, WT1, ING1, and NM23-H1 in the immunoprecipitated (IP) DNA. This suggests that NM23/NDPK binding is involved in the transcription regulation of these genes. These results reveal new interactions that should help us to disclose the antimetastatic mechanism of NM23.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Melanoma/genética , Compostos Organometálicos/química , Fatores de Transcrição/metabolismo , Sequência de Bases/genética , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Cromatina/química , Cromatina/isolamento & purificação , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Genes myc/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Melanoma/secundário , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95 degrees C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.
Assuntos
Amidoidrolases/metabolismo , Proteínas Arqueais/metabolismo , Sulfolobus solfataricus/enzimologia , Amidas/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Alinhamento de Sequência , Especificidade por Substrato , Sulfolobus solfataricus/genética , TemperaturaRESUMO
Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.