RESUMO
Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the "cross-talk" between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Citoplasma/metabolismo , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/química , Fosforilação , Serina/metabolismoRESUMO
In this work, surface magnetic molecularly imprinted polymers (SMMIPs) were synthesized and used as artificial receptors in the dispersive magnetic solid phase extraction (DMSPE) for capturing potential neuronal nitric oxide synthase-post synaptic density protein-95 (nNOS-PSD-95) uncouplers, which is known as neuroprotection against stroke. Factors that affected selective separation and adsorption of the artificial receptors, such as the amount of template, the types of functional monomer and porogen solvents, and the molar ratio of template/functional monomer/cross-linker were optimized. The artificial receptors were also characterized using fourier transformed infrared, scanning electron microscope, thermal gravimetric analysis and physical property measurement systems. Multiple interactions between template and SMMIPs led to larger binding capacities, faster binding kinetics, quicker separation abilities and more efficient selectivity than the surface magnetic nonimprinted polymers (SMNIPs). The SMMIPs were successfully applied to capture potential nNOS-PSD-95 uncouplers from complex samples, and eight compounds were seized and confirmed rapidly when combined with HPLC and MS. The detection of the new nNOS-PSD-95 uncouplers ranged from 0.001 to 1.500â¯mg/mL with correlation coefficients of 0.9990-0.9995. The LOD and LOQ were 0.10-0.68⯵g/mL and 0.47-2.11⯵g/mL, respectively. The neuroprotective effect and co-immunoprecipitation test in vitro revealed that Emodin-1-O-ß-d-glucoside, Rhaponticin, Gnetol and 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside have neuroprotective and uncoupling activities, and that they may be the new uncouplers of nNOS-PSD-95.
Assuntos
Proteína 4 Homóloga a Disks-Large/química , Óxido Nítrico Sintase Tipo I/química , Polímeros/química , Densidade Pós-Sináptica/química , Receptores Artificiais/química , Animais , Linhagem Celular Tumoral , Emodina/química , Glucosídeos/química , Magnetismo/métodos , Impressão Molecular/métodos , Fármacos Neuroprotetores/química , Células PC12 , Ratos , Extração em Fase Sólida/métodos , Solventes/química , Estilbenos/químicaRESUMO
Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) â Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) â Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) â Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.
Assuntos
Domínio Catalítico , Complexos de Coordenação/química , Cisteína/química , Ferro/química , Óxido Nítrico Sintase Tipo I/química , Animais , Sítios de Ligação/genética , Dicroísmo Circular/métodos , Complexos de Coordenação/metabolismo , Cisteína/genética , Cisteína/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/química , Heme/metabolismo , Ligação de Hidrogênio , Ferro/metabolismo , Ligantes , Modelos Moleculares , Mutação , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Oxirredução , Ratos , Espectrofotometria , Eletricidade Estática , TermodinâmicaRESUMO
UNLABELLED: Neuronal Nitric Oxide synthase (nNOS) is an attractive challenging target for the treatment of various neurodegenerative disorders. To date, several structure-based studies were conducted to search novel selective nNOS inhibitors. OBJECTIVE: Discovery of novel nNOS lead scaffolds through the integration of ligand-based threedimensional (3D) pharmacophore (s) with quantitative structure-activity relationship model. METHOD: The pharmacophoric space of ten structurally diverse sets acquired from 145 previously reported nNOS inhibitors was scrutinize to fabricate representative pharmacophores. Afterwards, genetic algorithm together with multiple linear regression analysis was applied to find out an optimal pharmacophoric models and 2D physicochemical descriptors able to produce optimal predictive QSAR equation (r(2) 116 =0.76, F = 353, r(2) LOO = 0.69, r(2) PRESS against 29 external test ligands =0.51). A minimum of three binding modes between ligands and nNOS binding pocket rationalized by the emergence of three pharmacophoric models in the QSAR equation were illustrated. The QSAR-selected pharmacophores were validated by receiver operating characteristic curves analysis and afterward invested as a tool for screening national cancer institute (NCI) database. RESULTS: Low micro molar novel nNOS inhibitors were revealed. CONCLUSION: Two structurally diverse compounds 148 and 153 demonstrated new scaffolds toward the discovery of potent nNOS inhibitors.
Assuntos
Simulação por Computador , Inibidores Enzimáticos/química , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/química , Animais , Ligantes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Quantitativa Estrutura-Atividade , RatosRESUMO
BACKGROUND: Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. METHODS AND RESULTS: Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker ß-tubulin in the proximal part of the ciliary axoneme. CONCLUSIONS: We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.
Assuntos
Cílios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Mucosa Respiratória/enzimologia , Brônquios/química , Brônquios/enzimologia , Células Cultivadas , Cílios/química , Cílios/metabolismo , Humanos , Imuno-Histoquímica , Pólipos Nasais/química , Pólipos Nasais/enzimologia , Óxido Nítrico Sintase Tipo I/química , Mucosa Respiratória/química , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
The binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) enables formation of the output state of nNOS for nitric oxide production. Essential to NOS function is the geometry and dynamics of CaM docking to the NOS oxygenase domain, but little is known about these details. In the present work, the domain docking in a CaM-bound oxygenase/FMN (oxyFMN) construct of nNOS was investigated using the relaxation-induced dipolar modulation enhancement (RIDME) technique, which is a pulsed electron paramagnetic resonance technique sensitive to the magnetic dipole interaction between the electron spins. A cysteine was introduced at position 110 of CaM, after which a nitroxide spin label was attached at the position. The RIDME study of the magnetic dipole interaction between the spin label and the ferric heme centers in the oxygenase domain of nNOS revealed that, with increasing [Ca(2+)], the concentration of nNOS·CaM complexes increases and reaches a maximum at [Ca(2+)]/[CaM] ≥ 4. The RIDME kinetics of CaM-bound nNOS represented monotonous decays without well-defined oscillations. The analysis of these kinetics based on the structural models for the open and docked states has shown that only about 15 ± 3% of the CaM-bound nNOS is in the docked state at any given time, while the remaining 85 ± 3% of the protein is in the open conformations characterized by a wide distribution of distances between the bound CaM and the oxygenase domain. The results of this investigation are consistent with a model that the Ca(2+)-CaM interaction causes CaM docking with the oxygenase domain. The low population of the docked state indicates that the CaM-controlled docking between the FMN and heme domains is highly dynamic.
Assuntos
Calmodulina/química , Óxido Nítrico Sintase Tipo I/química , Animais , Cálcio/química , Calmodulina/genética , Simulação por Computador , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli , Heme/química , Cinética , Fenômenos Magnéticos , Modelos Moleculares , Óxido Nítrico Sintase Tipo I/genética , Ratos , Marcadores de Spin , TransfecçãoRESUMO
Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.
Assuntos
Córtex Cerebral/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Córtex Cerebral/citologia , Chlorocebus aethiops , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Células PC12 , Fosforilação , Cultura Primária de Células , Ligação Proteica , Proteína Quinase C/química , Proteína Quinase C/genética , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND: Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. METHODOLOGY: In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, ß-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and ß-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01). CONCLUSIONS: These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.
Assuntos
Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/imunologia , Pectinidae/enzimologia , Pectinidae/imunologia , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Western Blotting , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemócitos/enzimologia , Humanos , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Funções Verossimilhança , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Pectinidae/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
π-π Interactions play an important role in the stability of protein structures. In the present study, we have analyzed the influence of π-π interactions in eNOS and nNOS proteins. The contribution of these π-π interacting residues in sequential separation, secondary structure involvement, solvent accessibility and stabilization centers has been evaluated. π-π interactions stabilize the core regions within eNOS and nNOS proteins. π-π interacting residues are evolutionary conserved. There is a significant number of π-π interactions in spite of the lesser natural occurrences of π-residues in eNOS and nNOS proteins. In addition to π-π interactions, π residues also form π-π networks in both eNOS and nNOS proteins which might play an important role in the structural stability of these protein structures.
Assuntos
Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo I/química , Modelos Moleculares , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Conformação ProteicaRESUMO
We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser(847) in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser(1412)in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser(1412) after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24h, after 15min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser(1412), PKA, and p-PKA at Thr(197) were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser(1412) significantly increased between 1 and 6h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser(1412) and PKA at Thr(197) occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr(197), which then phosphorylates nNOS at Ser(1412) in the subgranular layer of the dentate gyrus.
Assuntos
Isquemia Encefálica/metabolismo , Giro Denteado/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Prosencéfalo/patologia , Serina/metabolismo , Animais , Western Blotting , Isquemia Encefálica/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Óxido Nítrico Sintase Tipo I/química , Fosforilação , RatosRESUMO
Research into neonatal ischemic brain damage is impeded by the lack of a complete understanding of the initial hemodynamic mechanisms resulting in a lesion, particularly that of NO-mediated vascular mechanisms. In a neonatal stroke rat model, we recently show that collateral recruitment contributes to infarct size variability. Non-specific and selective NO synthase (NOS) inhibition was evaluated on cerebral blood-flow changes and outcome in a P7 rat model of arterial occlusion (left middle cerebral artery electrocoagulation with 50 min occlusion of both common carotid arteries). Blood-flow changes were measured by using ultrasound imaging with sequential Doppler recordings in both internal carotid arteries and basilar trunk. Cortical perfusion was measured by using laser Doppler flowmetry. We showed that global NOS inhibition significantly reduced collateral support and cortical perfusion (collateral failure), and worsened the ischemic injury in both gender. Conversely, endothelial NOS inhibition increased blood-flows and aggravated volume lesion in males, whereas in females blood-flows did not change and infarct lesion was significantly reduced. These changes were associated with decreased phosphorylation of neuronal NOS at Ser(847) in males and increased phosphorylation in females at 24h, respectively. Neuronal NOS inhibition also increased blood-flows in males but not in females, and did not significantly change infarct volumes compared to their respective PBS-treated controls. In conclusion, both nNOS and eNOS appear to play a key role in modulating arterial blood flow during ischemia mainly in male pups with subsequent modifications in infarct lesion.
Assuntos
Infarto Encefálico/enzimologia , Circulação Cerebrovascular/fisiologia , Hipóxia-Isquemia Encefálica/enzimologia , Leucomalácia Periventricular/enzimologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Animais , Animais Recém-Nascidos , Infarto Encefálico/patologia , Infarto Encefálico/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Recém-Nascido , Leucomalácia Periventricular/patologia , Leucomalácia Periventricular/fisiopatologia , Masculino , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/química , Ratos , Ratos WistarRESUMO
Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys(331), one of the zinc-tetrathiolate cysteines, was identified as the key site of nNOS S-nitrosylation. In addition, we also found that nNOS is highly S-nitrosylated in resting rat hippocampal neurons and the enzyme undergos denitrosylation during the process of rat brain ischemia/reperfusion. Intrestingly, the process of nNOS denitrosylation is coupling with the decrease of nNOS phosphorylation at Ser(847), a site associated with nNOS activation. Further more, we document that nNOS denitrosylation could be suppressed by pretreatment of neurons with MK801, an antagonist of NMDAR, GSNO, EGTA, BAPTA, W-7, an inhibitor of calmodulin as well as TrxR1 antisense oligonucleotide (AS-ODN) respectively. Taken together, our data demonstrate that the denitrosylation of nNOS induced by calcium ion influx is a NMDAR-dependent process during the early stage of ischemia/reperfusion, which is majorly mediated by thioredoxin-1 (Trx1) system. nNOS dephosphorylation may be induced by the enzyme denitrosylation, which suggest that S-nitrosylation/denitrosylation of nNOS may be an important mechanism in regulating the enzyme activity.
Assuntos
Óxido Nítrico Sintase Tipo I/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Apoptose , Isquemia Encefálica/metabolismo , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Cálcio/metabolismo , Cistina/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Masculino , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/química , Fosforilação , Ratos , Traumatismo por Reperfusão/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k(on) constants at 4.5°C: 6.6×10(8)M(-1)s(-1) for nNOS(726-749), 2.9×10(8)M(-1)s(-1) for eNOS(492-511) and 6.1×10(8)M(-1)s(-1) for iNOS(507-531), 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca(2+) chelation with ETDA: 3.7s(-1) for nNOS(726-749), 4.5s(-1) for eNOS(492-511), and 0.063s(-1) for iNOS(507-531). Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS(507-531) was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.
Assuntos
Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/química , Bovinos , Clonagem Molecular , Transporte de Elétrons , Escherichia coli , Citometria de Fluxo , Corantes Fluorescentes/análise , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/genética , Oxirredução , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transformação BacterianaAssuntos
Luz , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/química , Peptídeos/farmacologia , Animais , Ligantes , Camundongos , Conformação Molecular , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
The thermodynamics of cofactor binding to the isolated reductase domain (Red) of nNOS and its mutants have been studied by isothermal titration calorimetry. The NADP(+) and 2',5'-ADP binding stoichiometry to Red were both 1:1, consistent with a one-site kinetic model instead of a two-site model. The binding constant (K(D) = 71 nM) and the large heat capacity change (ΔC(p) = -440 cal mol(-1) K(-1)) for 2',5'-ADP were remarkably different from those for NADP(+) (1.7 µM and -140 cal mol(-1) K(-1), respectively). These results indicate that the nicotinamide moiety as well as the adenosine moiety has an important role in binding to nNOS. They also suggest that the thermodynamics of the conformational change in Red caused by cofactor binding are significantly different from the conformational changes that occur in cytochrome c reductase, in which the nicotinamide moiety of the cofactor is not essential for binding. Analysis of the deletion mutant of the autoinhibitory helix (RedΔ40) revealed that the deletion resulted in a decrease in the binding affinity of 2',5'-ADP with more unfavorable enthalpy gain. In the case of RedCaM, which contains a calmodulin (CaM) binding site, the presence of Ca(2+)/CaM caused a 6.7-fold increase in the binding affinity for 2',5'-ADP that was mostly due to the favorable entropy change. These results are consistent with a model in which Ca(2+)/CaM induces a conformational change in NOS to a flexible "open" form from a "closed" form that locked by cofactor binding, and this change facilitates the electron transfer required for catalysis.
Assuntos
Calmodulina/química , Óxido Nítrico Sintase Tipo I/química , Termodinâmica , Animais , Sítios de Ligação , Biocatálise , Calmodulina/metabolismo , Bovinos , Modelos Moleculares , Óxido Nítrico Sintase Tipo I/metabolismo , Oxirredução , Ligação Proteica , Estrutura Quaternária de Proteína , RatosRESUMO
The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in recent years. However, the lack of sequence information and clones of shrimp NOS cDNA limits further study on its characterization and function in this species. In this report, the cDNA of NOS contained full-length ORF was cloned from the Pacific white shrimp, Litopenaeus vannamei. It was of 4680 bp, including a 5'-terminal untranslated region (UTR) of 278 bp, a 3'-terminal UTR of 862 bp, which contained 5 ATTTA repeats, and an open reading frame (ORF) of 3540 bp encoding a polypeptide of 1179 amino acids. It contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time reverse transcription PCR analysis revealed L. vannamei NOS (LvNOS) to be expressed in most shrimp tissues, with highest expression in the hepatopancreas and weakest expression in skin. The expression of LvNOS after challenge with LPS and poly I:C was tested in hemocytes, hepatopancreas and nerve. The results indicated that the NOS transcript level could be induced in hemocytes by injection with LPS. The highest expression was in the hemocyte, with 8.8 times (at 3 h) as much as that in the control (p < 0.05). However, sharp down-regulation of NOS was found in hepatopancreas and nerve after LPS and poly I:C injection (p < 0.05). These results suggested that NOS might play an important role in shrimp's defense against pathogenic infection.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Penaeidae/enzimologia , Penaeidae/genética , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo I/química , Penaeidae/classificação , Penaeidae/imunologia , Filogenia , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether the loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that the expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK-specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.
Assuntos
Distrofias Musculares/enzimologia , Distrofias Musculares/fisiopatologia , Óxido Nítrico Sintase Tipo I/metabolismo , Fosfofrutoquinases/metabolismo , Regulação Alostérica , Animais , Flavina-Adenina Dinucleotídeo/metabolismo , Glicogênio/metabolismo , Glicólise , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Modelos Moleculares , Distrofias Musculares/genética , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Condicionamento Físico Animal , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.
Assuntos
Estradiol/fisiologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/biossíntese , Receptores de Estrogênio/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo I/química , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/química , Receptores de N-Metil-D-Aspartato/químicaRESUMO
The tight regulation of intracellular calcium levels is essential for the normal function of a great many cellular processes, and disruption of this regulation, resulting in sustained increases in intracellular-free calcium, has been associated with numerous diseases. One of the several transporters involved in calcium homeostasis is a P-type ATPase known as the plasma membrane calcium/calmodulin-dependent ATPase (PMCA) which is involved in calcium extrusion from the cytosol to the extracellular compartment. It has long been established that in many cell types, in particular non-excitable cells, the primary role of PMCA is in the bulk transport of intracellular calcium; however, its role in excitable cells is less clear. In the heart, for example, calcium is essential for contractile function as well as being a key messenger in signal transduction pathways; however, the mechanisms by which the cardiomyocyte distinguishes between these roles of calcium remain unclear. It is perhaps the transporters not involved in the contractile cycle (such as PMCA) that are able to carry non-contractile signals. This review will highlight the role of PMCA as a modulator of signal transduction pathways and in particular the role of isoform 4 in the regulation of the nitric oxide signalling pathway.
Assuntos
Óxido Nítrico Sintase Tipo I/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Cálcio/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Transdução de Sinais/fisiologiaRESUMO
Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.