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1.
Biochem Biophys Res Commun ; 516(3): 1039-1045, 2019 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-28698136

RESUMEN

Heterotrimeric G proteins composed of Gα, Gß and Gγ subunits are evolutionarily conserved signaling modules involved in diverse biological processes in plants and animals. The role and action of Gα remain largely enigmatic in plant innate immunity. We have recently demonstrated that Arabidopsis Gα (GPA1) is a key component of a new immune signaling pathway activated by bacteria-secreted proteases. Here we show that GPA1 is also involved in the signaling network of Arabidopsis in response to the bacterial flagellin epitope flg22. Specifically, GPA1 plays a pivotal role in an immune pathway involving the flg22 receptor FLS2, co-receptor BAK1, Regulator of G Signaling 1 (RGS1), and Arabidopsis Gß (AGB1), in which flg22 elicits GPA1/AGB1 dissociation from the FLS2/BAK1/RGS1 receptor complex. Consequently, we observed flg22-induced degradation of FLS2, BAK1 and RGS1 but not GPA1 or AGB1. We also found that GPA1 constitutively interacts with the NADPH oxidase RbohD to potentiate flg22-induced ROS burst independently of the central cytoplasmic kinase BIK1. Taken together, our work sheds multiple novel insights into the functions and regulatory mechanisms of GPA1 in Arabidopsis innate immunity.


Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Flagelina/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Epítopos/inmunología , Flagelina/química , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/inmunología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Inmunidad Innata/genética , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas RGS/genética , Proteínas RGS/inmunología , Proteínas RGS/metabolismo , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética
2.
J Exp Med ; 215(10): 2655-2672, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30194266

RESUMEN

The vascular endothelium is constantly exposed to mechanical forces, including fluid shear stress exerted by the flowing blood. Endothelial cells can sense different flow patterns and convert the mechanical signal of laminar flow into atheroprotective signals, including eNOS activation, whereas disturbed flow in atheroprone areas induces inflammatory signaling, including NF-κB activation. How endothelial cells distinguish different flow patterns is poorly understood. Here we show that both laminar and disturbed flow activate the same initial pathway involving the mechanosensitive cation channel Piezo1, the purinergic P2Y2 receptor, and Gq/G11-mediated signaling. However, only disturbed flow leads to Piezo1- and Gq/G11-mediated integrin activation resulting in focal adhesion kinase-dependent NF-κB activation. Mice with induced endothelium-specific deficiency of Piezo1 or Gαq/Gα11 show reduced integrin activation, inflammatory signaling, and progression of atherosclerosis in atheroprone areas. Our data identify critical steps in endothelial mechanotransduction, which distinguish flow pattern-dependent activation of atheroprotective and atherogenic endothelial signaling and suggest novel therapeutic strategies to treat inflammatory vascular disorders such as atherosclerosis.


Asunto(s)
Endotelio Vascular/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Integrinas/inmunología , Canales Iónicos/inmunología , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Endotelio Vascular/patología , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Integrinas/genética , Canales Iónicos/genética , Ratones , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología
3.
Bull Math Biol ; 76(6): 1352-75, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24809944

RESUMEN

Cyclic AMP is important for the resolution of inflammation, as it promotes anti-inflammatory signaling in several immune cell lines. In this paper, we present an immune cell specific model of the cAMP signaling cascade, paying close attention to the specific isoforms of adenylyl cyclase (AC) and phosphodiesterase that control cAMP production and degradation, respectively, in these cells. The model describes the role that G protein subunits, including Gαs, Gαi, and Gßγ, have in regulating cAMP production. Previously, Gαi activation has been shown to increase the level of cAMP in certain immune cell types. This increase in cAMP is thought to be mediated by ßγ subunits which are released upon Gα activation and can directly stimulate specific isoforms of AC. We conduct numerical experiments in order to explore the mechanisms through which Gαi activation can increase cAMP production. An important conclusion of our analysis is that the relative abundance of different G protein subunits is an essential determinant of the cAMP profile in immune cells. In particular, our model predicts that limited availability of ßγ subunits may both (i) enable immune cells to link inflammatory Gαi signaling to anti-inflammatory cAMP production thereby creating a balanced immune response to stimulation with low concentrations of PGE2, and (ii) prohibit robust anti-inflammatory cAMP signaling in response to stimulation with high concentrations of PGE2.


Asunto(s)
Adenilil Ciclasas/inmunología , AMP Cíclico/inmunología , Modelos Inmunológicos , Transducción de Señal/inmunología , Línea Celular , Simulación por Computador , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Subunidades beta de la Proteína de Unión al GTP/inmunología , Subunidades gamma de la Proteína de Unión al GTP/inmunología , Cinética , Receptor de Anafilatoxina C5a/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/inmunología
4.
J Immunol ; 192(10): 4774-82, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24733850

RESUMEN

Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαißγ protein-dependent and ß-arrestin-related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gßγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. ß-arrestin2 recruitment was studied in OXE-R-overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks ß-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE-triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE-induced Ca(2+) flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gßγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi-biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.


Asunto(s)
Bencenoacetamidas/farmacología , Benzotiazoles/farmacología , Eosinófilos/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Monocitos/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Receptores Eicosanoides/antagonistas & inhibidores , Ácidos Araquidónicos/inmunología , Arrestinas/inmunología , Antígeno CD11b/inmunología , Antígenos CD18/inmunología , Calcio/inmunología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Eosinófilos/citología , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Células HEK293 , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Monocitos/citología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/citología , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Receptores Eicosanoides/inmunología , beta-Arrestinas
5.
J Immunol ; 192(5): 2186-94, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24510965

RESUMEN

The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.


Asunto(s)
Quinasas de Receptores Acoplados a Proteína-G/inmunología , Inhibidores de Disociación de Guanina Nucleótido/inmunología , Complejos Multiproteicos/inmunología , Receptores de Interleucina-8B/inmunología , Animales , Calcio/inmunología , Señalización del Calcio/genética , Señalización del Calcio/inmunología , Línea Celular , Membrana Celular/genética , Membrana Celular/inmunología , Quinasas de Receptores Acoplados a Proteína-G/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación de la Expresión Génica/fisiología , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Fosforilación/genética , Fosforilación/inmunología , Transporte de Proteínas/fisiología , Receptores de Interleucina-8B/genética
6.
J Histochem Cytochem ; 62(5): 347-54, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24423937

RESUMEN

G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Anticuerpos/inmunología , Western Blotting , Femenino , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Humanos , Embarazo
7.
J Immunol ; 192(5): 2349-56, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24477912

RESUMEN

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (LT) B4 (LTB4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that LTB4 control of MyD88 expression involves the generation of microRNAs. Our data show that LTB4, via its receptor B leukotriene receptor 1 (BLT1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. LTB4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the LTB4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and LT-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR BLT1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.


Asunto(s)
Regulación de la Expresión Génica/inmunología , Leucotrieno B4/inmunología , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , MicroARNs/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Animales , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación de la Expresión Génica/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Leucotrieno B4/genética , Macrófagos Peritoneales/patología , Ratones , Ratones Noqueados , MicroARNs/genética , Factor 88 de Diferenciación Mieloide/genética , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/inmunología
8.
Indian J Biochem Biophys ; 51(6): 467-75, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25823218

RESUMEN

Guanine nucleotide regulatory proteins (G proteins) play a key role in the regulation of various signal transduction systems, including adenylyl cyclase/cAMP and phospholipase C (PLC)/phosphatidyl inositol (PI) turnover, which are implicated in the modulation of a variety of physiological functions, such as platelet functions, including platelet aggregation, secretion, and clot formation and cardiovascular functions, including arterial tone and reactivity. Several abnormalities in adenylyl cyclase activity, cAMP levels and G proteins have been shown to be responsible for the altered cardiac performance and vascular functions observed in cardiovascular disease states. The enhanced or unaltered levels of inhibitory G proteins (Giα) and mRNA have been reported in different models of hypertension, whereas Gsα levels are shown to be unaltered. The enhanced levels of Giα proteins precede the development of blood pressure and suggest that overexpression of Gi proteins may be one of the contributing factors for the pathogenesis of hypertension. The levels of vasoactive peptides including ET-1 and Ang II and growth factors are augmented in hypertension and contribute to the enhanced expression of Giα proteins in hypertension. In addition, oxidative stress due to enhanced levels of Ang II and ET-1 is enhanced in hypertension and may also be responsible for the enhanced expression of Giα proteins observed in hypertension. Furthermore, Ang II- and ET-1-induced transactivation of growth factor receptor through the activation of MAP kinase signaling is also shown to contribute to the augmented levels of Giα in hypertension. Thus, it appears that the enhanced levels of vasoactive peptides by increasing oxidative stress and transactivation growth factor receptors enhance MAP kinase activity that contribute to the enhanced expression of Giα proteins responsible for the pathogenesis of hypertension. In this review, we describe the role of vasoactive peptides and the signaling mechanisms responsible for the enhanced expression of Giα proteins in hypertension.


Asunto(s)
Angiotensina II/inmunología , Vasos Sanguíneos/inmunología , Endotelina-1/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Hipertensión/inmunología , Transducción de Señal/inmunología , Sistema Vasomotor/inmunología , Animales , Presión Sanguínea/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Modelos Cardiovasculares , Modelos Inmunológicos
9.
Glycoconj J ; 30(9): 819-23, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23877649

RESUMEN

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/inmunología , Oligodendroglía/inmunología , Proteínas Proto-Oncogénicas c-fyn/inmunología , Sulfoglicoesfingolípidos/inmunología , Familia-src Quinasas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Oligodendroglía/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Ratas , Sulfoglicoesfingolípidos/metabolismo , Familia-src Quinasas/metabolismo
10.
PLoS Pathog ; 8(11): e1003040, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23166501

RESUMEN

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.


Asunto(s)
Entamoeba histolytica/inmunología , Entamebiasis/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Proteínas Protozoarias/inmunología , Factores de Virulencia/inmunología , Animales , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Entamoeba histolytica/enzimología , Entamoeba histolytica/genética , Entamebiasis/enzimología , Entamebiasis/genética , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/genética , Regulación de la Expresión Génica/inmunología , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Células Jurkat , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Factores de Intercambio de Guanina Nucleótido Rho , Transcripción Genética/inmunología , Factores de Virulencia/biosíntesis , Factores de Virulencia/química
11.
J Immunol ; 189(10): 4797-805, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23041570

RESUMEN

The complement anaphylatoxins C3a, C5a, and desarginated C5a (C5a(desArg)) play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3a receptor and C5a receptor (C5aR). The role of C5a(desArg) in inducing cell activation has been often neglected, because the affinity of C5a(desArg) for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5a(desArg) to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5a(desArg) induce significant levels of cell activation that are even higher than those achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, because it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of G(α) proteins upon C5aR engagement by C5a or C5a(desArg). Further, mass spectrometric characterization of plasma-derived C5a and C5a(desArg) provided important insight into the posttranslational modification pattern of these anaphylatoxins, which includes glycosylation at Asn(64) and partial cysteinylation at Cys(27). Although the context-specific physiological contribution of C5a(desArg) has to be further explored, our data suggest that C5a(desArg) acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.


Asunto(s)
Bioensayo/métodos , Complemento C3/inmunología , Complemento C5a/inmunología , Receptores de Complemento/inmunología , Animales , Línea Celular Tumoral , Complemento C3/análisis , Complemento C3/genética , Complemento C5a/análisis , Complemento C5a/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Humanos , Péptidos Cíclicos/farmacología , Ratas , Receptor de Anafilatoxina C5a , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/genética
12.
Protein Pept Lett ; 19(3): 277-81, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21933130

RESUMEN

Like those in mammals, heterotrimeric G protein complexes have been implicated in signal transduction pathways in plants; however, the subunits themselves have not been isolated. In this study, the rice heterotrimeric G protein subunits α (Gα) and ß (Gß) were purified by affinity chromatography using anti-Gα and -Gß antibodies and SDS-PAGE. Six and seven peptides, respectively, were identified by mass spectrometry and identified as the translation products of the Gα gene RGA1 and Gß gene RGB1. During purification, the subunits dissociated easily from the G protein complex.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/análisis , Subunidades alfa de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Oryza/química , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Anticuerpos/inmunología , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/inmunología , Espectrometría de Masas , Datos de Secuencia Molecular , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Resinas Sintéticas/química
13.
Neurosci Lett ; 499(2): 127-31, 2011 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-21651958

RESUMEN

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Bulbo Olfatorio/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Secuencia de Aminoácidos , Animales , Peces , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Inmunohistoquímica , Datos de Secuencia Molecular , Bulbo Olfatorio/química , Bulbo Olfatorio/inmunología , Mucosa Olfatoria/química , Mucosa Olfatoria/inmunología , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/inmunología
14.
J Biol Chem ; 286(12): 10466-75, 2011 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-21233205

RESUMEN

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gßγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gα(o) compared with rat Gα(i1). In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación Viral de la Expresión Génica/inmunología , Tolerancia Inmunológica/inmunología , Proteínas de Insectos/inmunología , Polydnaviridae/inmunología , Proteínas Virales/inmunología , Avispas/inmunología , Animales , Drosophila melanogaster , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Polydnaviridae/genética , Polydnaviridae/metabolismo , Ratas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Avispas/genética , Avispas/metabolismo , Avispas/virología
15.
Aging Cell ; 10(2): 208-19, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21108728

RESUMEN

Innate immune responses to pathogens are governed by the nervous system. Here, we investigated the molecular mechanism underlying innate immunity in Caenorhabditis elegans against Escherichia coli OP50, a standard laboratory C. elegans food. Longevity was compared in worms fed live or UV-killed OP50 at low or high density food condition (HDF). Expression of the antimicrobial gene lys-8 was approximately 5-fold higher in worms fed live OP50, suggesting activation of innate immunity upon recognition of OP50 metabolites. Lifespan was extended and SOD-3 mRNA levels were increased in gpa-9-overexpressing gpa-9XS worms under HDF in association with robust induction of insulin/IGF-1 signaling (IIS). Expression of ins-7 and daf-28 that control lys-8 expression was reduced in gpa-9XS, indicating that GPA-9-mediated immunity is due in part to ins-7 and daf-28 downregulation. Our results suggest that OP50 metabolites in amphid neurons elicit innate immunity through the IIS pathway, and identify GPA-9 as a novel regulator of both the immune system and aging in C. elegans.


Asunto(s)
Proteínas de Caenorhabditis elegans/inmunología , Caenorhabditis elegans/inmunología , Caenorhabditis elegans/fisiología , Dieta , Escherichia coli/inmunología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Inmunidad Innata , Envejecimiento , Animales , Proteínas de Caenorhabditis elegans/genética , Escherichia coli/patogenicidad , Subunidades alfa de la Proteína de Unión al GTP/genética , Insulina/inmunología , Factor I del Crecimiento Similar a la Insulina/inmunología , Transducción de Señal/inmunología
16.
Proc Natl Acad Sci U S A ; 107(31): 13788-93, 2010 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-20647387

RESUMEN

Signal transduction pathways that regulate longevity, immunity, and stress resistance can profoundly affect organismal survival. We show that a signaling module formed by the G protein alpha subunit, Gqalpha, and one of its downstream signal transducer phospholipase C beta (PLCbeta) can differentially affect these processes. Loss of Gqalpha and PLCbeta functions result in increased sensitivity to pathogens and oxidative stress but confer life span extension. Gqalpha and PLCbeta modulate life span and immunity noncell autonomously by affecting the activity of insulin/IGF1 signaling (IIS). In addition, Gqalpha and PLCbeta function cell autonomously within the intestine to affect the activity of the p38 MAPK pathway, an important component of Caenorhabditis elegans immune and oxidative stress response. p38 MAPK activity in the intestine is regulated by diacylglycerol levels, a product of PLCbeta's hydrolytic activity. We provide genetic evidence that life span is largely determined by IIS, whereas p38 MAPK signaling is the primary regulator of oxidative stress in PLCbeta mutants. Pathogen sensitivity of Gqalpha and PLCbeta mutants is a summation of the beneficial effects of decreased IIS through reduced neuronal secretion and the detrimental effects of reduced activity of intestinal p38 MAPK. We propose a model whereby Gqalpha signaling differentially regulates pathogen sensitivity, oxidative stress, and longevity through cell autonomous and noncell autonomous effects on p38 MAPK and insulin/IGF1 signaling, respectively.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Inmunidad Innata , Longevidad , Estrés Oxidativo , Transducción de Señal , Animales , Caenorhabditis elegans/inmunología , Proteínas de Caenorhabditis elegans/inmunología , Diglicéridos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación de la Expresión Génica , Fosfolipasa C beta/metabolismo
17.
Immunology ; 129(2): 257-67, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19824922

RESUMEN

Infection of mice with the gastrointestinal nematode Trichuris muris represents a valuable tool to investigate and dissect intestinal immune responses. Resistant mouse strains respond to T. muris infection by mounting a T helper type 2 response. Previous results have shown that CD4(+) T cells play a critical role in protective immunity, and that CD4(+) T cells localize to the infected large intestinal mucosa to confer protection. Further, transfer of CD4(+) T cells from immune mice to immunodeficient SCID mice can prevent the development of a chronic infection. In the current study, we characterize the protective CD4(+) T cells, describe their chemokine receptor expression and explore the functional significance of these receptors in recruitment to the large intestinal mucosa post-T. muris infection. We show that the ability to mediate expulsion resides within a subpopulation of CD4(+) T cells marked by down-regulation of CD62L. These cells can be isolated from intestine-draining mesenteric lymph nodes (MLN) from day 14 post-infection, but are rare or absent in MLN before this and in spleen at all times post-infection. Among CD4(+) CD62L(low) MLN cells, the two most abundantly expressed chemokine receptors were CCR6 and CXCR3. We demonstrate for the first time that CD4(+) CD62L(low) T-cell migration to the large intestinal mucosa is dependent on the family of G alpha(i)-coupled receptors, to which chemokine receptors belong. CCR6 and CXCR3 were however dispensable for this process because neutralization of CCR6 and CXCR3 did not prevent CD4(+) CD62L(low) cell migration to the large intestinal mucosa during T. muris infection.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Inmunidad Mucosa , Mucosa Intestinal/metabolismo , Tricuriasis/inmunología , Trichuris/inmunología , Traslado Adoptivo , Animales , Anticuerpos Bloqueadores/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD4-Positivos/patología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Inmunidad Mucosa/efectos de los fármacos , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Toxina del Pertussis/farmacología , Receptores CCR6/metabolismo , Receptores CXCR3/metabolismo , Tricuriasis/patología , Trichuris/patogenicidad
18.
J Neurophysiol ; 100(3): 1420-32, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18562551

RESUMEN

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Opioides/metabolismo , Ganglio Estrellado/citología , Animales , Anticuerpos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Norepinefrina/farmacología , Péptidos Opioides/farmacología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Transfección/métodos , omega-Conotoxina GVIA/farmacología , Receptor de Nociceptina , Nociceptina
19.
Neuroreport ; 18(11): 1141-5, 2007 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-17589315

RESUMEN

The cholinergic system in the central nervous system plays an important role in higher brain functions, through muscarinic receptors. The nucleus tractus solitarius is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of acetylcholine (Ach) on VDCC currents (I(Ca)) in the nucleus tractus solitarius using patch-clamp recording methods. In 68 out of 99 neurons, an application of ACh caused inhibition of N-type and P/Q-type I(Ba) in a concentration-dependent manner. Pretreatments with AF-DX116 (muscarinic M2 receptor antagonist) attenuated the ACh-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the ACh-induced inhibition of I(Ba). These results indicate that ACh inhibits N-type and P/Q-type VDCCs via Gi-protein betagamma subunits mediated by M2 receptors in nucleus tractus solitarius.


Asunto(s)
Calcio/metabolismo , Inhibición Neural/fisiología , Neuronas/fisiología , Receptor Muscarínico M2/fisiología , Núcleo Solitario/citología , Acetilcolina/farmacología , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Pirenzepina/análogos & derivados , Pirenzepina/farmacología , Ratas , Ratas Wistar , Receptor Muscarínico M2/antagonistas & inhibidores
20.
Immunology ; 122(1): 116-23, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17484771

RESUMEN

Heterotrimeric G(i) proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of G(i) proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS induced production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 and interferon-gamma-inducible protein-10 (IP-10); SA induced TNF-alpha, and IL-1beta production; and GBS induced TNF-alpha, IL-6, IL-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. In contrast to the role of G(i) proteins as a positive regulator of mediators, LPS-induced production of MIP-1alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Galpha(i1/3) (-/-) mice, and SA-induced MIP-1alpha production was increased in both groups of Galpha(i) protein-depleted mice. LPS-induced production of KC and IL-1beta, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. These data suggest that G(i2) and G(i1/3) proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.


Asunto(s)
Quimiocinas/biosíntesis , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Staphylococcus aureus/inmunología , Streptococcus agalactiae/inmunología , Animales , Células Cultivadas , Citocinas/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Genotipo , Ratones , Ratones Noqueados
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