RESUMO
BACKGROUND: It remains unknown whether the cutaneous microvascular responses are different between patients with scleroderma-associated pulmonary arterial hypertension (SSc-PAH) and SSc without pulmonary hypertension (PH). METHODS: We included 59 patients with SSc between March 2013 and September 2019. We divided patients into 4 groups: (a) no PH by right heart catheterization (RHC) (n = 8), (b) no PH by noninvasive screening tests (n = 16), (c) treatment naïve PAH (n = 16), and (d) PAH under treatment (n = 19). Microvascular studies using laser Doppler flowmetry (LDF) were done immediately after RHC or at the time of an outpatient clinic visit (group b). RESULTS: The median (IQR) age was 59 (54-68) years, and 90% were females. The responses to local thermal stimulation and postocclusive reactive hyperemia, acetylcholine, and sodium nitroprusside iontophoresis were similar among groups. The microvascular response to treprostinil was more pronounced in SSc patients without PH by screening tests (% change: 340 (214-781)) compared with SSc-PAH (naïve + treatment) (Perfusion Units (PU) % change: 153 (94-255) % [p = .01]). The response to A-350619 (a soluble guanylate cyclase (sGC) activator) was significantly higher in patients with SSc without PH by screening tests (PU % change: 168 (46-1,296)) than those with SSc-PAH (PU % change: 22 (15-57) % [p = .006]). The % change in PU with A350619 was directly associated with cardiac index and stroke volume index (R: 0.36, p = .03 and 0.39, p = .02, respectively). CONCLUSIONS: Patients with SSc-PAH have a lower cutaneous microvascular response to a prostacyclin analog treprostinil and the sGC activator A-350619 when compared with patients with SSc and no evidence of PH on screening tests, presumably due to a peripheral reduction in prostacyclin receptor expression and nitric oxide bioavailability.
RESUMO
Idiopathic pulmonary arterial hypertension (IPAH) is a rapidly progressive disease with several treatment options. Long-term mortality remains high with great heterogeneity in treatment response. Even though most of the pathology of IPAH is observed in the lung, there is systemic involvement. Platelets from patients with IPAH have characteristic metabolic shifts and defects in activation; therefore, we investigated whether they could be used to identify other disease-specific abnormalities. We used proteomics to investigate protein expression changes in platelets from patients with IPAH compared with healthy controls. Key abnormalities of nitric oxide pathway were tested in platelets from a larger cohort of unique patients with IPAH. Platelets showed abnormalities in the prostacyclin and nitric oxide pathways, which are dysregulated in IPAH and hence targets of therapy. We detected reduced expression of G protein αs and increased expression of the regulatory subunits of the cAMP-dependent protein kinase (PKA) type II isoforms, supporting an overall decrease in the activation of the prostacyclin pathway. We noted reduced levels of the soluble guanylate cyclase (sGC) subunits and increased expression of the phosphodiesterase type 5 A (PDE5A), conditions that affect the response to nitric oxide. Ensuing analysis of 38 unique patients with IPAH demonstrated considerable variation in the levels and specific activity of sGC, a finding with novel implications for personalized therapy. Platelets have some of the characteristic vasoactive signal abnormalities seen in IPAH and may provide comprehensive ex vivo mechanistic information to direct therapeutic decisions.
Assuntos
Plaquetas/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Proteoma/metabolismo , Guanilil Ciclase Solúvel/metabolismo , Adulto , Idoso , Plaquetas/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/análise , Adulto JovemRESUMO
Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and devastating disease characterized by vascular smooth muscle and endothelial cell proliferation leading to a narrowing of the vessels in the lung. The increased resistance in the lung and the higher pressures generated result in right heart failure. Nitric Oxide (NO) deficiency is considered a hallmark of IPAH and altered function of endothelial nitric oxide synthase (eNOS), decreases NO production. We recently demonstrated that glucose dysregulation results in augmented protein serine/threonine hydroxyl-linked N-Acetyl-glucosamine (O-GlcNAc) modification in IPAH. In diabetes, dysregulated glucose metabolism has been shown to regulate eNOS function through inhibition of Ser-1177 phosphorylation. However, the link between O-GlcNAc and eNOS function remains unknown. Here we show that increased protein O-GlcNAc occurs on eNOS in PAH and Ser-615 appears to be a novel site of O-GlcNAc modification resulting in reduced eNOS dimerization. Functional characterization of Ser-615 demonstrated the importance of this residue on the regulation of eNOS activity through control of Ser-1177 phosphorylation. Here we demonstrate a previously unidentified regulatory mechanism of eNOS whereby the O-GlcNAc modification of Ser-615 results in reduced eNOS activity and endothelial dysfunction under conditions of glucose dysregulation.
Assuntos
Óxido Nítrico Sintase Tipo III , Óxido Nítrico , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Serina/metabolismoAssuntos
Determinação da Pressão Arterial/métodos , Hipertensão Pulmonar/diagnóstico , Idoso , Cateterismo Cardíaco , Estudos Transversais , Feminino , Humanos , Hipertensão Pulmonar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Pressão Propulsora PulmonarRESUMO
Group 1 pulmonary hypertension (PH), i.e., pulmonary arterial hypertension (PAH), is associated with a metabolic shift favoring glycolysis in cells comprising the lung vasculature as well as skeletal muscle and right heart. We sought to determine whether this metabolic switch is also detectable in circulating platelets from PAH patients. We used Seahorse Extracellular Flux to measure bioenergetics in platelets isolated from group 1 PH (PAH), group 2 PH, patients with dyspnea and normal pulmonary artery pressures, and healthy controls. We show that platelets from group 1 PH patients exhibit enhanced basal glycolysis and lower glycolytic reserve compared with platelets from healthy controls but do not differ from platelets of group 2 PH or dyspnea patients without PH. Although we were unable to identify a glycolytic phenotype unique to platelets from PAH patients, we found that platelet glycolytic metabolism correlated with hemodynamic severity only in group 1 PH patients, supporting the known link between PAH pathology and altered glycolytic metabolism and extending this association to ex vivo platelets. Pulmonary artery pressure and pulmonary vascular resistance in patients with group 1 PH were directly associated with basal platelet glycolysis and inversely associated with maximal and reserve glycolysis, suggesting that PAH progression reduces the capacity for glycolysis even while demanding an increase in glycolytic metabolism. Therefore, platelets may provide an easy-to-harvest, real-time window into the metabolic shift occurring in the lung vasculature and represent a useful surrogate for interrogating the glycolytic shift central to PAH pathology.
Assuntos
Plaquetas/metabolismo , Glicólise , Hemodinâmica , Hipertensão Arterial Pulmonar/patologia , Idoso , Estudos de Casos e Controles , Metabolismo Energético , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/metabolismo , Índice de Gravidade de DoençaRESUMO
RATIONALE: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. OBJECTIVES: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response. FINDINGS: We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively). CONCLUSIONS: Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature.
Assuntos
Inalação , Metaboloma , Microvasos/metabolismo , Óxido Nítrico/análise , Biomarcadores , Voluntários Saudáveis , Humanos , Cinética , Microcirculação , Projetos Piloto , Pele/irrigação sanguíneaRESUMO
BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. METHODS AND RESULTS: Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked ß-N-acetylglucosamine modification, O-linked ß-N-acetylglucosamine transferase (OGT), and O-linked ß-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked ß-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. CONCLUSIONS: Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH.
Assuntos
Hipertensão Pulmonar Primária Familiar/enzimologia , N-Acetilglucosaminiltransferases/fisiologia , Adulto , Aloxano/farmacologia , Divisão Celular , Células Cultivadas , Progressão da Doença , Hipertensão Pulmonar Primária Familiar/mortalidade , Hipertensão Pulmonar Primária Familiar/patologia , Hipertensão Pulmonar Primária Familiar/cirurgia , Feminino , Glucose/metabolismo , Glicosilação , Hexosaminas/biossíntese , Hospitalização/estatística & dados numéricos , Fator C1 de Célula Hospedeira/fisiologia , Humanos , Transplante de Pulmão/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Artéria Pulmonar/patologia , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall. METHODS AND RESULTS: A monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichment of apoA1 compared with normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed that only a minority (<3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063≤d≤1.21). In contrast, the majority (>90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d>1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed ≈80% lower cholesterol efflux activity and ≈90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1. CONCLUSIONS: The function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired.
Assuntos
Aorta/metabolismo , Apolipoproteína A-I/sangue , Apolipoproteína A-I/genética , Aterosclerose/metabolismo , Placa Aterosclerótica/metabolismo , Aciltransferases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Apolipoproteína A-I/imunologia , Linhagem Celular , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , OxirreduçãoRESUMO
AIM: Loss of superoxide dismutase (SOD) activity is a defining biochemical feature of asthma. However, mechanisms for the reduced activity are unknown. We hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation. RESULT: Activity assays of blood samples from asthmatics and healthy controls revealed impaired dismutase activity of copper-zinc SOD (CuZnSOD) in asthma. CuZnSOD purified from erythrocytes or airway epithelial cells from asthmatic was highly susceptible to oxidative inactivation. Proteomic analyses identified that inactivation was related to oxidation of cysteine 146 (C146), which is usually disulfide bonded to C57. The susceptibility of cysteines pointed to an alteration in protein structure, which is likely related to the loss of disulfide bond. We speculated that a shift to greater intracellular reducing potential might account for the change. Strikingly, measures of reduced and oxidized glutathione confirmed greater reducing intracellular state in asthma, compared with controls. Similarly, greater free thiol in CuZnSOD was confirmed by ~2-fold greater N-ethylmaleimide binding to C146 in asthma as compared with controls. INNOVATION: Greater reducing potential under a chronic inflammatory state of asthma, thus, leads to susceptibility of CuZnSOD to oxidative inactivation due to cleavage of C57-C146 disulfide bond and exposure of usually unavailable cysteines. CONCLUSION: Vulnerability of CuZnSOD influenced by redox likely amplifies injury and inflammation during acute asthma attacks when reactive oxygen species are explosively generated. Overall, this study identifies a new paradigm for understanding the chemical basis of inflammation, in which redox regulation of thiol availability dictates protein susceptibility to environmental and endogenously generated reactive species.
Assuntos
Asma/sangue , Cistina/química , Superóxido Dismutase/sangue , Adulto , Sequência de Aminoácidos , Asma/enzimologia , Plaquetas/enzimologia , Estudos de Casos e Controles , Ditiotreitol/química , Eritrócitos/enzimologia , Feminino , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/química , Masculino , Dados de Sequência Molecular , Oxidantes/química , Oxirredução , Estresse Oxidativo , Fragmentos de Peptídeos/química , Substâncias Redutoras/química , Superóxido Dismutase/químicaRESUMO
In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.
Assuntos
Calmodulina/química , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico/biossíntese , Animais , Calmodulina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , NADP/química , NADP/metabolismo , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo I/metabolismo , Oxirredução , Estrutura Terciária de Proteína , RatosRESUMO
Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease. Several processes are believed to lead to the fatal progressive pulmonary arterial narrowing seen in IPAH including vasoconstriction, cellular proliferation inflammation, vascular remodeling, abnormalities in the lung matrix, and in situ thrombosis. Nitric oxide (NO) produced by NO synthases (NOS) is a potent vasodilator and plays important roles in many other processes including platelet function. Reduced NO levels in patients with IPAH are known to contribute to the development of pulmonary hypertension and its complications. Platelet defects have been implied in IPAH, but original research supporting this hypothesis has been limited. Normal platelets are known to have NOS activity, but little is known about NOS expression and NO production by platelets in patients with IPAH. Here we characterized the phenotype of the platelets in IPAH and show a defect in their ability to be activated in vitro by thrombin receptor activating protein but not adenosine diphosphate. We also show that endothelial NOS (eNOS) levels in these platelets are reduced and demonstrate that NO is an important regulator of platelet function. Thus reduced levels of eNOS in platelets could impact their ability to regulate their own function appropriately.
Assuntos
Plaquetas/patologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Óxido Nítrico/metabolismo , Agregação Plaquetária/fisiologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Difosfato de Adenosina/metabolismo , Adulto , Plaquetas/metabolismo , Plaquetas/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Hipertensão Pulmonar/genética , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregação Plaquetária/genética , Contagem de Plaquetas/métodos , Vasoconstrição/genética , Vasoconstrição/fisiologiaRESUMO
Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target. Surprisingly, we found that GAPDH plays a key role. GAPDH was associated with iNOS in cells. Pure GAPDH bound tightly to heme or to iNOS in an NO-sensitive manner. GAPDH knockdown inhibited heme insertion into iNOS and a GAPDH mutant with defective heme binding acted as a dominant negative inhibitor of iNOS heme insertion. Exposing cells to NO either from a chemical donor or by iNOS induction caused GAPDH to become S-nitrosylated at Cys152. Expressing a GAPDH C152S mutant in cells or providing a drug to selectively block GAPDH S-nitrosylation both made heme insertion into iNOS resistant to the NO inhibition. We propose that GAPDH delivers heme to iNOS through a process that is regulated by its S-nitrosylation. Our findings may uncover a fundamental step in intracellular heme trafficking, and reveal a mechanism whereby NO can govern the process.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Heme/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Camundongos , Mutação , Ligação ProteicaRESUMO
Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified function in protein folding, energy metabolism, antioxidant capacity, and membrane permeability. Nitration of heat shock protein 60 in vitro was found to decrease its ATP hydrolysis and interaction with proinsulin, suggesting a mechanism by which protein nitration could diminish insulin secretion. This was supported by our finding of a decrease in stimulated insulin secretion following glycolytic stress in cultured cells. Our results reveal that protein tyrosine nitration may be a previously unrecognized factor in beta-cell dysfunction and the pathogenesis of diabetes.
Assuntos
Diabetes Mellitus/fisiopatologia , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Nitratos/metabolismo , Estado Pré-Diabético/fisiopatologia , Tirosina/metabolismo , Animais , Chaperonina 60/metabolismo , Diabetes Mellitus/patologia , Humanos , Hiperglicemia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulinoma/patologia , Insulinoma/fisiopatologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Estado Pré-Diabético/patologiaRESUMO
Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.
Assuntos
Mononucleotídeo de Flavina/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Mononucleotídeo de Flavina/química , Óxido Nítrico Sintase Tipo I/química , Oxirredução , Reação em Cadeia da Polimerase , Potenciometria , Conformação Proteica , RatosRESUMO
Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes.
Assuntos
Adipócitos/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Glucose/metabolismo , Nitratos/metabolismo , Células 3T3-L1 , Transporte Ativo do Núcleo Celular , Adipócitos/patologia , Animais , Proteínas de Ligação a Ácido Graxo/química , Glucose/química , Camundongos , Nitratos/química , Oxirredução , Estresse Oxidativo , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Tirosina/química , Tirosina/metabolismoRESUMO
The C-terminal tail (CT) of neuronal nitric oxide synthase (nNOS) is a regulatory element that suppresses nNOS activities in the absence of bound calmodulin (CaM). A crystal structure of the nNOS reductase domain (nNOSr) (Garcin, E. D., Bruns, C. M., Lloyd, S. J., Hosfield, D. J., Tiso, M., Gachhui, R., Stuehr, D. J., Tainer, J. A., and Getzoff, E. D. (2004) J. Biol. Chem. 279, 37918-37927) revealed how the first half of the CT interacts with nNOSr and thus provided a template for detailed studies. We generated truncation mutants in nNOS and nNOSr to test the importance of 3 different regions of the CT. Eliminating the terminal half of the CT (all residues from Ile1413 to Ser1429), which is invisible in the crystal structure, had almost no impact on NADP+ release, flavin reduction, flavin autoxidation, heme reduction, reductase activity, or NO synthesis activity, but did prevent an increase in FMN shielding that normally occurs in response to NADPH binding. Additional removal of the CT alpha-helix (residues 1401 to 1412) significantly increased the NADP+ release rate, flavin autoxidation, and NADPH oxidase activity, and caused hyper-deshielding of the FMN cofactor. These effects were associated with increased reductase activity and slightly diminished heme reduction and NO synthesis. Further removal of residues downstream from Gly1396 (a full CT truncation) amplified the aforementioned effects and in addition altered NADP+ interaction with FAD, relieved the kinetic suppression on flavin reduction, and further diminished heme reduction and NO synthesis. Our results reveal that the CT exerts both multifaceted and regiospecific effects on catalytic activities and related behaviors, and thus provide new insights into mechanisms that regulate nNOS catalysis.
Assuntos
Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/metabolismo , Sequência de Aminoácidos , Animais , Simulação por Computador , Redutases do Citocromo/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Heme/metabolismo , Cinética , Dados de Sequência Molecular , Mutação , NADP/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Oxirredução , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K(m)O(2) for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.
Assuntos
Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Sequência de Aminoácidos , Animais , Redutases do Citocromo/metabolismo , Flavinas/metabolismo , Heme/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Óxido Nítrico Sintase Tipo III/genética , Oxirredução , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
RATIONALE: A cellular prooxidant state promotes cells to neoplastic growth, in part because of modification of proteins and their functions. Reactive nitrogen species formed from nitric oxide (NO) or its metabolites, can lead to protein tyrosine nitration, which is elevated in lung cancer. OBJECTIVE: To determine the alteration in these NO derivatives and the role they may play in contributing to lung carcinogenesis. METHODS: We analyzed levels of NO, nitrite (NO2-), nitrate (NO3-), and the location of the protein nitration and identified the proteins that are modified. MEASUREMENTS AND MAIN RESULTS: Although exhaled NO and NO2- were increased, endothelial NO synthase or inducible NO synthase expression was similar in the tumor and tumor-free regions. However, immunohistochemistry showed that nitrotyrosine was increased in the tumor relative to non-tumor-bearing sections. We used proteomics to identify the modified proteins (two-dimensional polyacrylamide gel electrophoresis; mass spectrometry). Both the degree of nitration and the protein nitration profile were altered. We identified more than 25 nitrated proteins, including metabolic enzymes, structural proteins, and proteins involved in prevention of oxidative damage. Alterations of the biology of NO metabolites and nitration of proteins may contribute to the mutagenic processes and promote carcinogenesis. CONCLUSIONS: This study provides evidence in favor of a role for reactive nitrogen and oxygen species in lung cancer.
Assuntos
Neoplasias Pulmonares/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Idoso , Testes Respiratórios , Feminino , Humanos , Neoplasias Pulmonares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/análise , Proteínas , Proteômica , Espécies Reativas de Nitrogênio/análise , Espécies Reativas de Oxigênio/análise , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies. The other halves remained in storage media for 7 additional days and were also processed for immunohistochemistry. Mouse monoclonal antibody to nitrotyrosine adducts was used to define the localisation of these epitopes. The density of antibody staining was observed and quantified on a digital camera system and statistically analysed. Immunostaining in the epithelium was greater in tissues recovered after 10 days in storage compared to the intensity of staining after 3 days of storage (p<0.0001). No staining was evident in the epithelium in sections exposed to non-immune mouse IgG. Western blot analysis was performed on epithelial cells scraped from corneal surfaces of one-half of four donor corneas in storage for 3 days and from the other half at 10 days of storage. Nitrated BSA was used as a positive control. After extraction and homogenisation, identical protein concentrations of each sample were loaded per lane on 10% gels and subjected to SDS-PAGE. Proteins were blotted and probed with the anti-nitrotyrosine antibody. Western blot immunoreactivity was detected in epithelial samples at the 3 and 10 day recovery times with the latter samples showing greater staining intensity. Nitrated protein, thought to indicate toxic peroxynitrite formation, accumulates in the human corneal epithelium with time of storage. Our study shows that there is an association between increased nitrated protein and storage time.
Assuntos
Epitélio Corneano/química , Proteínas do Olho/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , Adulto , Western Blotting/métodos , Células Epiteliais/química , Proteínas do Olho/análise , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Oxidantes/análise , Oxidantes/metabolismo , Ácido Peroxinitroso/análise , Técnicas de Cultura de Tecidos/métodos , Tirosina/imunologiaRESUMO
Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.