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1.
J Immunol ; 202(5): 1549-1558, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30665935

RESUMEN

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.


Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Fagocitos/metabolismo , Superóxidos/metabolismo , Simportadores/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Aniones/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simportadores/genética
2.
Int Immunopharmacol ; 137: 112425, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-38851160

RESUMEN

The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , NADPH Oxidasas , Peptidilprolil Isomerasa de Interacción con NIMA , Neutrófilos , Humanos , Neutrófilos/inmunología , Neutrófilos/efectos de los fármacos , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Naftoquinonas/farmacología , Inflamación/inmunología , Células Cultivadas , Artritis Reumatoide/inmunología , Artritis Reumatoide/tratamiento farmacológico
3.
Blood ; 116(26): 5795-802, 2010 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-20956805

RESUMEN

Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.


Asunto(s)
NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Isomerasa de Peptidilprolil/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Western Blotting , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/química , Peptidilprolil Isomerasa de Interacción con NIMA , Naftoquinonas/farmacología , Neutrófilos/enzimología , Fosforilación , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo
4.
Free Radic Biol Med ; 160: 19-27, 2020 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-32758662

RESUMEN

Superoxide anion production by neutrophils is essential for host defense against microbes. Superoxide anion generates other reactive oxygen species (ROS) that are very toxic for microbes and host cells, therefore their excessive production could induce inflammatory reactions and tissue injury. Cyclic adenosine monophosphate (cAMP) elevating agents are considered to be physiological inhibitors of superoxide production by neutrophils but the mechanisms involved in this inhibitory effect are poorly understood. Superoxide is produced by the phagocyte NADPH oxidase, a complex enzyme composed of two membrane subunits, gp91phox or NOX2 and p22phox, and four cytosolic components p47phox, p67phox, p40phox, and Rac2. Except Rac2, these proteins are known to be phosphorylated upon neutrophil stimulation. Here we show that forskolin, an activator of the adenylate cyclase-cAMP-PKA pathway, induced phosphorylation of gp91phox/NOX2 and inhibited fMLF-induced NADPH oxidase activation in human neutrophils. H89, a PKA inhibitor prevented the forskolin-induced phosphorylation of gp91phox and restored NADPH oxidase activation. Furthermore, PKA phosphorylated the recombinant gp91phox/NOX2-cytosolic C-terminal region in vitro only on a few specific peptides containing serine residues, as compared to PKC. Interestingly, phosphorylation of NOX2-Cter by PKA alone did not induce interaction with the cytosolic components p47phox, p67phox and Rac2, however it induced inhibition of PKC-induced interaction. Furthermore, PKA alone did not induce NOX2 electron transfer activity, however it inhibited PKC-induced activation. These results suggest that PKA phosphorylates NOX2 in human neutrophils, a process essential to limit ROS production and inflammation under physiological conditions. Our data identify the cAMP-PKA-NOX2-axis as a critical gatekeeper of neutrophil ROS production.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Neutrófilos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Fagocitos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo
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