RESUMEN
Near-shore marine sediments deposited during the Paleocene-Eocene Thermal Maximum at Wilson Lake, NJ, contain abundant conventional and giant magnetofossils. We find that giant, needle-shaped magnetofossils from Wilson Lake produce distinct magnetic signatures in low-noise, high-resolution first-order reversal curve (FORC) measurements. These magnetic measurements on bulk sediment samples identify the presence of giant, needle-shaped magnetofossils. Our results are supported by micromagnetic simulations of giant needle morphologies measured from transmission electron micrographs of magnetic extracts from Wilson Lake sediments. These simulations underscore the single-domain characteristics and the large magnetic coercivity associated with the extreme crystal elongation of giant needles. Giant magnetofossils have so far only been identified in sediments deposited during global hyperthermal events and therefore may serve as magnetic biomarkers of environmental disturbances. Our results show that FORC measurements are a nondestructive method for identifying giant magnetofossil assemblages in bulk sediments, which will help test their ecology and significance with respect to environmental change.
Asunto(s)
Organismos Acuáticos/aislamiento & purificación , Sedimentos Geológicos/química , Magnetosomas/química , Organismos Acuáticos/química , Óxido Ferrosoférrico/química , Fósiles , Sedimentos Geológicos/análisis , Humanos , Fenómenos Magnéticos , Magnetismo , Fenómenos FísicosRESUMEN
Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria.
Asunto(s)
Alanina/química , Mitocondrias/metabolismo , Prolina/química , Liposomas Unilamelares/metabolismo , Proteína X Asociada a bcl-2/química , Alanina/metabolismo , Sustitución de Aminoácidos , Clonación Molecular , Citocromos c/metabolismo , Expresión Génica , Células HCT116 , Humanos , Liposomas/química , Liposomas/metabolismo , Mitocondrias/química , Mutación , Permeabilidad , Prolina/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Liposomas Unilamelares/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Disulfuros/química , Nucleósido-Difosfato Quinasa/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Disulfuros/efectos de la radiación , Estabilidad de Enzimas/efectos de la radiación , Calor , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Subunidades de Proteína/química , Alineación de Secuencia , Sulfatos , Rayos XRESUMEN
Despite the harsh environmental conditions in the world's oldest and driest desert, some salt flat or 'salar' environments in the Atacama Desert host standing bodies of water known as saline lakes. Evaporite minerals deposited within saline lakes result from the equilibrium of environmental, sedimentological, and biogeochemical processes that occur in the salar; consequently, these minerals are sensitive records of human activities and ecological, evolutionary, and geological changes. The objective of this study was to evaluate feedbacks between physical, chemical, and microbial processes that culminate in distinct trends in brine chemistry, saline lake morphology, and associated evaporite sediments. Using samples from the Puquios of the Salar de Llamara, Atacama Desert, northern Chile, an analysis of spatial gradients and vertical stratification of lake elemental chemistry and mineral saturation indices were integrated with a comprehensive analysis of lake morphology, including depth, slope gradient, substrate type, and mineralogy. Lake waters ranged from saline to hypersaline, and exhibited normal, well mixed and inverse stratification patterns, and results suggest a correlation with lake morphology in the Salar de Llamara. Saline to hypersaline lakes (>150 mS/cm) with stratified brines tended to have crystalline substrate and deep (>35 cm) and steep-sided lake morphologies, while unstratified lakes with lower electrical conductivity (<90 mS/cm and microbial substrates had gentle slopes and characteristically shallow depths (<30 cm). Differences in minor element chemistry (Mn and Sr) between saline lakes were observed on scales of meters to kilometers, and result in different accessory mineral assemblages. Quantification of the physical, chemical, and microbial feedbacks that produce the observed heterogeneity in these ecosystems provides key insight into the geochemical composition and lake morphology of saline lakes in extreme environments around the world.
Asunto(s)
Ecosistema , Lagos , Retroalimentación , Humanos , Sales (Química)RESUMEN
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.
Asunto(s)
ADN/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Sitios de Unión , Línea Celular Tumoral , Humanos , Melanoma/patología , Nucleósido Difosfato Quinasas NM23/fisiología , Proteínas Nucleares/fisiología , Oligodesoxirribonucleótidos/metabolismo , Fosfoproteínas/fisiología , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismoRESUMEN
Nucleoside diphosphate kinases (NDPK) are encoded by the NME genes, also called NM23. They catalyze the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high energy phospho-histidine intermediate [1, 2]. Besides their known functions in the control of intracellular nucleotide homeostasis, they are involved in multiple physiological and pathological cellular processes such as differentiation, development, metastastic dissemination or cilia functions. Over the past 15 years, ten human genes have been discovered encoding partial, full length, and/or tandemly repeated Nm23/NDPK domains, with or without N-or C-terminal extensions and/or additional domains. These genes encode proteins exhibiting different functions at various tissular and subcellular localizations. Most of these genes appear late in evolution with the emergence of the vertebrate lineage. This review summarizes the present knowledge on these multitalented proteins.
Asunto(s)
Cilios/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Neoplasias/patología , Nucleósido-Difosfato Quinasa/metabolismo , Animales , Cilios/genética , Humanos , Isoenzimas/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Mitocondrias/enzimología , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia/genética , Nucleósido-Difosfato Quinasa/genética , Filogenia , Unión Proteica , Transducción de SeñalRESUMEN
Amyloid fibrils are ordered beta-sheet protein or peptide polymers. The benzothiazole dye Thioflavin-T (ThT) shows a strong increase in fluorescence upon binding to amyloid fibrils and has hence become the most commonly used amyloid-specific dye. In spite of this widespread use, the mechanism underlying specific binding and fluorescence enhancement upon interaction with amyloid fibrils remains largely unknown. Recent contradictory reports have proposed radically different modes of binding. We have studied the interaction of ThT with fibrils of the prion forming domain of the fungal HET-s prion protein assembled at pH 2 in order to try to gain some insight into the general mechanism of ThT-binding and fluorescence. We found that ThT does not bind to HET-s(218-289) fibrils as a micelle as previously proposed in the case of insulin fibrils. We have measured binding kinetics, affinity and stoichiometry at pH values above and below the pI of the HET-s(218-289) fibrils and found that binding is dramatically affected by pH and ionic strength. Binding is poor at acidic pH, presumably as a result of repulsive electrostatic interaction between the positively charged ThT molecule and the fibril surface. Finally, we found that ThT acquires chiral properties when it is fibril-bound. These results are discussed in relation to the different ThT-binding modes that have been proposed.
Asunto(s)
Amiloide/química , Proteínas Fúngicas/química , Tiazoles/química , Benzotiazoles , Colorantes Fluorescentes/farmacología , Humanos , Concentración de Iones de Hidrógeno , Insulina/química , Cinética , Podospora/metabolismo , Polímeros/química , Priones , Unión Proteica , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
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.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismo , Fosfoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Catálisis , Línea Celular Tumoral , Cartilla de ADN , Activación Enzimática , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Nucleósido Difosfato Quinasas NM23 , Neuroblastoma , Fosforilación , Pliegue de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Nucleósido-Difosfato Quinasa/química , Secuencia de Aminoácidos , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismo , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Citosol/metabolismo , Células Epiteliales/citología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23/química , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Sistema Respiratorio/citología , Adulto JovenRESUMEN
Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.
Asunto(s)
Membrana Celular/metabolismo , Dinaminas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Fusión de Membrana , Mitocondrias/metabolismo , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasa D/metabolismoRESUMEN
Amyloid beta (Aß) peptides produced by APP cleavage are central to the pathology of Alzheimer's disease. Despite widespread interest in this issue, the relationship between the auto-assembly and toxicity of these peptides remains controversial. One intriguing feature stems from their capacity to form anti-parallel ß-sheet oligomeric intermediates that can be converted into a parallel topology to allow the formation of protofibrillar and fibrillar Aß. Here, we present a novel approach to determining the molecular aspects of Aß assembly that is responsible for its in vivo toxicity. We selected Aß mutants with varying intracellular toxicities. In vitro, only toxic Aß (including wild-type Aß42) formed urea-resistant oligomers. These oligomers were able to assemble into fibrils that are rich in anti-parallel ß-sheet structures. Our results support the existence of a new pathway that depends on the folding capacity of Aß .
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Animales , Dicroismo Circular , Microscopía Electrónica de Transmisión , Células PC12 , RatasRESUMEN
Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Nucleósido-Difosfato Quinasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Sales (Química) , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The human nucleoside diphosphate (NDP) kinase A, product of the NME1 gene also named NM23-H1, is known as a metastasis suppressor protein. A naturally occurring variant, S120G, identified in neuroblastomas, possesses native three-dimensional structure and enzymatic activity but displays reduced conformational stability and a folding defect with the accumulation of a "molten globule" folding intermediate during refolding in vitro. As such intermediate has been postulated to be involved in amyloid formation, NDP kinase A may serve as a model protein for studying the relationship between folding intermediates and amyloid fibrils. The NDP kinase A S120G was heated in phosphate buffer (pH 7.0). The protein precipitated as amyloid fibrils, as demonstrated by electron microscopy, Congo red, and thioflavin T binding and FTIR spectroscopy. The NDP kinase A S120G, at neutral pH and at moderate temperature experiences a transition towards amyloid fibrils. The aggregation process was faster if seeded by preformed fibrils. The fibrils presented a large proteinase K-resistant core not including residue Gly 120, as shown by mass spectrometry. This suggests that the aggregation process is triggered by the reduced stability of the S120G variant and not by a specific increase in the kinase domain intrinsic aggregation propensity at the place of mutation. This constitutes one of the few reports on a protein involved in cancer biology able to aggregate into amyloid structures under mild conditions.
Asunto(s)
Amiloide/química , Amiloide/genética , Mutación , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Neuroblastoma/genética , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Electrónica de Transmisión , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TemperaturaRESUMEN
The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7M urea and of 2M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.
Asunto(s)
Metilaminas/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/genética , Mutación Puntual , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Nucleósido Difosfato Quinasas NM23/genética , Proteínas de Neoplasias/genética , Pliegue de Proteína/efectos de los fármacos , Urea/farmacologíaRESUMEN
The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.
Asunto(s)
Mutación Missense/genética , Neuroblastoma/genética , Nucleósido-Difosfato Quinasa/genética , Pliegue de Proteína , Proteínas Supresoras de Tumor/genética , Cromatografía en Gel , Humanos , Nucleósido Difosfato Quinasas NM23 , TermodinámicaRESUMEN
Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.
Asunto(s)
Adenosina Difosfato/química , Modelos Moleculares , Nucleósido-Difosfato Quinasa/química , Cristalización , Humanos , Nucleósido Difosfato Quinasas NM23 , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Genes Supresores de Tumor/fisiología , Melanoma/genética , Compuestos Organometálicos/química , Factores de Transcripción/metabolismo , Secuencia de Bases/genética , Sitios de Unión , Biomarcadores de Tumor/metabolismo , Cromatina/química , Cromatina/aislamiento & purificación , Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Genes myc/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melanoma/metabolismo , Melanoma/secundario , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23 , Nucleósido-Difosfato Quinasa/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: The metastasis-suppressing role of the NM23 gene in the metastatic spread of solid tumors is still debated. We examined the role of NM23 in tumor development and metastatic dissemination by using transgenic mice that lack mouse NM23 (NM23-M1) in two mouse models of hepatocellular carcinoma (HCC) that recapitulate all steps of tumor progression. METHODS: We induced HCC in mice that contained (NM23-M1(+/+)) or lacked (NM23-M1(-/-)) NM23-M1 by diethylnitrosamine injection or by a crossing scheme that transferred a transgene that leads to liver expression of simian virus 40 large T antigen (ASV mice). We used microscopic examination and immunohistochemistry to analyze tumor progression. Expression of Nm23 protein isoforms (Nm23-M1 and Nm23-M2) and several tumor markers was analyzed in the primary tumor and in metastases by Western blotting. The statistical significance of differences in the incidence of Nm23-M2 overexpression in null mice relative to that in wild-type mice was tested by a one-sided Fisher's exact test. The statistical significance of differences in the incidence of metastases was examined using one-sided chi-square tests. All other statistical tests were two-sided. RESULTS: In both models, Nm23-M1 and/or Nm23-M2 were overexpressed in the primary liver tumors compared with nontumor liver tissue; however, the lack of the NM23-M1 gene had no effect on primary tumor formation in either model. ASV mice developed pulmonary metastases that were positive for the Hep-Par 1 antibody, which recognizes a specific hepatocyte antigen, whereas the few pulmonary nodules that developed in diethylnitrosamine-injected mice were negative for this antigen. Statistically significantly more ASV/NM23-M1(-/-) mice than ASV/NM23-M1(+/+) mice developed lung metastases (69.2% versus 37.5%; difference = 31.7%, 95% confidence interval = 13.1% to 50.3%; P<.001). In ASV/NM23-M1(+/+) mice, immunohistochemical staining for Nm23-M1 was highly heterogeneous among the primary liver tumors, but weak or negative among lung metastases. CONCLUSIONS: The lack of NM23-M1 expression promotes metastasis in the SV40 animal model of liver carcinogenesis.