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1.
Nitric Oxide ; 132: 15-26, 2023 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-36736618

RESUMEN

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.


Asunto(s)
Bradiquinina , Melanoma , Ratones , Animales , Bradiquinina/farmacología , Bradiquinina/metabolismo , Superóxidos , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Movimiento Celular
2.
Cell Biol Int ; 46(1): 158-169, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34719858

RESUMEN

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.


Asunto(s)
Adenocarcinoma/enzimología , Autofagia , Neoplasias del Colon/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Estrés Nitrosativo , Adenocarcinoma/genética , Adenocarcinoma/secundario , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Óxido Nítrico Sintasa de Tipo II/genética , Isoformas de Proteínas , Transducción de Señal
3.
Nitric Oxide ; 86: 1-11, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30772503

RESUMEN

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.


Asunto(s)
Proteínas Fúngicas/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Estrés Nitrosativo/fisiología , Paracoccidioides/fisiología , Proteínas ras/fisiología , Secuencia de Aminoácidos , Animales , Muerte Celular/fisiología , Proliferación Celular/fisiología , Expresión Génica/fisiología , Masculino , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/química , Óxido Nítrico/farmacología , Procesamiento Proteico-Postraduccional
4.
Nitric Oxide ; 93: 78-89, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31539562

RESUMEN

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4+T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2-36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4+T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4+T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4+T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , VIH-1/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Replicación Viral/fisiología , Infecciones por VIH , Humanos , Donantes de Óxido Nítrico/farmacología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacología , Familia-src Quinasas/metabolismo
5.
FEMS Yeast Res ; 18(2)2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29409063

RESUMEN

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a cause of disease in healthy and immunocompromised persons in Latin America. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. The development of the disease depends on factors associated with the host immune response and the infectious agent's characteristics, especially virulence. The oxidative stress response is an important virulence attribute in several fungi. In this study, we assessed the enzymatic repertoire of responses to oxidative stress in the Pb18 isolate with different degrees of virulence. The virulence of attenuated Pb18 (aPb18) strain was recovered after several animal passages. Virulent strain (vPb18) showed an effective fungal oxidative stress response and several genes involved in response to oxidative stress were up-regulated in this isolate. These genes expressed the same profile when we recovered the phenotypic virulence in attenuated strain aPb18. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18), and this process positively modulated the fungus's antioxidant repertoire.


Asunto(s)
Antioxidantes/metabolismo , Paracoccidioides/fisiología , Paracoccidioidomicosis/microbiología , Animales , Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Viabilidad Microbiana/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Paracoccidioides/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Virulencia
6.
FEMS Yeast Res ; 16(5)2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27268997

RESUMEN

Paracoccidioides brasiliensis and P. lutzii, thermally dimorphic fungi, are the causative agents of paracoccidioidomycosis (PCM). Paracoccidioides infection occurs when conidia or mycelium fragments are inhaled by the host, which causes the Paracoccidioides cells to transition to the yeast form. The development of disease requires conidia inside the host alveoli to differentiate into yeast cells in a temperature-dependent manner. We describe the presence of a two-component signal transduction system in P. brasiliensis, which we investigated by expression analysis of a hypothetical protein gene (PADG_07579) that showed high similarity with the dimorphism-regulating histidine kinase (DRK1) gene of Blastomyces dermatitidis and Histoplasma capsulatum This gene was sensitive to environmental redox changes, which was demonstrated by a dose-dependent decrease in transcript levels after peroxide stimulation and a subtler decrease in transcript levels after NO stimulation. Furthermore, the higher PbDRK1 levels after treatment with increasing NaCl concentrations suggest that this histidine kinase can play a role as osmosensing. In the mycelium-yeast (M→Y) transition, PbDRK1 mRNA expression increased 14-fold after 24 h incubation at 37°C, consistent with similar observations in other virulent fungi. These results demonstrate that the PbDRK1 gene is differentially expressed during the dimorphic M→Y transition. Finally, when P. brasiliensis mycelium cells were exposed to a histidine kinase inhibitor and incubated at 37°C, there was a delay in the dimorphic M→Y transition, suggesting that histidine kinases could be targets of interest for PCM therapy.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Histidina Quinasa/metabolismo , Paracoccidioides/citología , Paracoccidioides/genética , Oxidantes/metabolismo , Paracoccidioides/efectos de los fármacos , Paracoccidioides/enzimología , Transducción de Señal , Cloruro de Sodio/metabolismo , Temperatura
7.
Nitric Oxide ; 47: 40-51, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25819133

RESUMEN

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Óxido Nítrico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismo , Bradiquinina/farmacología , Células Endoteliales/metabolismo , Humanos , Óxido Nítrico/biosíntesis
8.
J Proteome Res ; 13(10): 4259-71, 2014 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-25145636

RESUMEN

Few virulence factors have been identified for Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in P. brasiliensis. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how Paracoccidioides modulates the host-pathogen interaction to its advantage.


Asunto(s)
Proteínas Fúngicas/metabolismo , Paracoccidioides/patogenicidad , Proteómica , Espectrometría de Masas , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/metabolismo , Reacción en Cadena de la Polimerasa , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Virulencia
9.
Arch Biochem Biophys ; 558: 14-27, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-24960080

RESUMEN

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Bradiquinina/farmacología , Receptores ErbB/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Óxido Nítrico/biosíntesis , Fosforilación/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Conejos , S-Nitrosotioles/metabolismo , Tirosina/metabolismo
10.
Med Mycol ; 52(2): 187-95, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24577000

RESUMEN

Paracoccidioides brasiliensis and P. lutzii are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis (PCM). Previously, we characterized the PbMDJ1 gene. This gene encodes P. brasiliensis chaperone Mdj1, which in yeast is a mitochondrial member of the J-domain family, whose main function is to regulate cognate Hsp70 activities. We produced rabbit polyclonal antibody antirecombinant PbMdj1 (rPbMdj1), which labeled the protein not only in mitochondria but also at the cell wall of P. brasiliensis yeasts of isolate Pb18. Here we used anti-rPbMdj1 in confocal microscopy to localize Mdj1 in Pb18 and other fungal isolates grown at different temperatures. Dual intracellular and cell surface pattern were initially seen in yeast-phase P. brasiliensis Pb3, Pb18 (control), P. lutzii Pb01, and Histoplasma capsulatum. Pb18 and Aspergillus fumigatus hyphae as well as Pb3 pseudo hyphae formed at 36°C were labeled predominantly along the cell surface. Preferential surface localization was observed by 72 h of yeast-mycelium thermotransition. It was interesting to observe that anti-rPbMdj1 concentrated at the surface tip and branching points of A. fumigatus hyphae grown at 36°C, suggesting a role in growth, whereas at 23°C, anti-rPbMdj1 was distributed along the hyphal surface. In Pb3, Pb18, and Pb01 mitochondrial extracts, the antibodies revealed a specific 55-kDa band, which corresponds to the processed Mdj1 size. The presence of Mdj1 on the fungal cell wall suggests that this protein could also play a role in the interaction with the host.


Asunto(s)
Aspergillus fumigatus/química , Pared Celular/química , Histoplasma/química , Mitocondrias/química , Paracoccidioides/química , Factores de Transcripción/análisis , Animales , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/efectos de la radiación , Histoplasma/crecimiento & desarrollo , Histoplasma/efectos de la radiación , Hifa/química , Microscopía Confocal , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/efectos de la radiación , Conejos , Temperatura
11.
Fungal Genet Biol ; 47(2): 179-89, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19825426

RESUMEN

Glycoprotein gp70 is an important intracellular antigen from Paracoccidioides brasiliensis that elicits both humoral and cellular immune responses. Herein, the PbGP70 gene cloning from isolate Pb18 using internal peptide sequence information is reported. The deduced protein sequence bears two N-glycosylation sites, antigenic sites and two mouse T-cell epitopes. Anti-recombinant gp70 (rPbgp70) polyclonal antibodies reacted with a 70-kDa component in total cell extract of P. brasiliensis, while MAbC5F11 and paracoccidioidomycosis patients' sera recognized rPbgp70. Confocal microscopy with anti-rPbgp70 and MAbC5F11 showed intense staining and cytoplasmatic co-localization. The protein sequence belongs to the flavoprotein monooxygenase family which groups important anti-oxidative bioactive compounds. We found increased PbGP70 transcript accumulation under oxidative stress induced by H(2)O(2), during fungal growth and in macrophage phagocyted/bound yeasts. Therefore, gp70 might play a dual role in P. brasiliensis by both eliciting immune cellular and humoral responses in the host and protecting the fungus from oxidative stress generated by phagocytic cells.


Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Paracoccidioides/enzimología , Paracoccidioides/genética , Secuencia de Aminoácidos , Animales , Southern Blotting , Células Cultivadas , Flavoproteínas/metabolismo , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peróxido de Hidrógeno/farmacología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Paracoccidioides/efectos de los fármacos , Paracoccidioides/crecimiento & desarrollo , Homología de Secuencia de Aminoácido
12.
Front Microbiol ; 11: 1184, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32582109

RESUMEN

The fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the causative agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. This fungus is considered a facultative intracellular pathogen that is able to survive and replicate inside macrophages. The survival of the fungus during infection depends on its adaptability to various conditions, such as nitrosative/oxidative stress produced by the host immune cells, particularly alveolar macrophages. Currently, there is little knowledge about the Paracoccidioides spp. signaling pathways involved in the fungus evasion mechanism of the host defense response. However, it is known that some of these pathways are triggered by reactive oxygen species and reactive nitrogen species (ROS/RNS) produced by host cells. Considering that the effects of NO (nitric oxide) on pathogens are concentration dependent, such effects could alter the redox state of cysteine residues by influencing (activating or inhibiting) a variety of protein functions, notably S-nitrosylation, a highly important NO-dependent posttranslational modification that regulates cellular functions and signaling pathways. It has been demonstrated by our group that P. brasiliensis yeast cells proliferate when exposed to low NO concentrations. Thus, this work investigated the modulation profile of S-nitrosylated proteins of P. brasiliensis, as well as identifying S-nitrosylation sites after treatment with RNS. Through mass spectrometry analysis (LC-MS/MS) and label-free quantification, it was possible to identify 474 proteins in the S-nitrosylated proteome study. With this approach, we observed that proteins treated with NO at low concentrations presented a proliferative response pattern, with several proteins involved in cellular cycle regulation and growth being activated. These proteins appear to play important roles in fungal virulence. On the other hand, fungus stimulated by high NO concentrations exhibited a survival response pattern. Among these S-nitrosylated proteins we identified several potential molecular targets for fungal disease therapy, including cell wall integrity (CWI) pathway, amino acid and folic acid metabolisms. In addition, we detected that the transnitrosylation/denitrosylation redox signaling are preserved in this fungus. Finally, this work may help to uncover the beneficial and antifungal properties of NO in the P. brasiliensis and point to useful targets for the development of antifungal drugs.

13.
Biochem Biophys Res Commun ; 369(4): 1001-6, 2008 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-18325324

RESUMEN

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.


Asunto(s)
Apoptosis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Flavonoides/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , S-Nitrosoglutatión/farmacología
14.
Toxicol Appl Pharmacol ; 233(2): 227-37, 2008 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-18786557

RESUMEN

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.


Asunto(s)
S-Nitrosoglutatión/farmacología , S-Nitrosotioles/metabolismo , Tiorredoxinas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitritos/metabolismo , Fosforilación/efectos de los fármacos , S-Nitrosoglutatión/administración & dosificación , Transducción de Señal/efectos de los fármacos , Tirosina/análogos & derivados , Tirosina/metabolismo
15.
Free Radic Res ; 52(5): 592-604, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29651879

RESUMEN

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.


Asunto(s)
Anoicis/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Familia-src Quinasas/genética , Animales , Anoicis/genética , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Melanoma Experimental/enzimología , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Óxido Nítrico/química , Donantes de Óxido Nítrico/química , Nitroprusiato/química , Compuestos Nitrosos/química , Transducción de Señal , Células Tumorales Cultivadas , Familia-src Quinasas/metabolismo
16.
Microbes Infect ; 19(1): 34-46, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27590702

RESUMEN

Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress, which are regulated by phosphatases and kinases activities. Furthermore, we present additional evidence for a functional two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.


Asunto(s)
Proteínas Fúngicas/análisis , Regulación Fúngica de la Expresión Génica , Interacciones Huésped-Patógeno , Estrés Oxidativo , Paracoccidioides/patogenicidad , Fosfoproteínas/análisis , Proteoma/análisis , Humanos , Espectrometría de Masas , Transducción de Señal
17.
Microbes Infect ; 7(1): 55-65, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15716071

RESUMEN

Paracoccidioides brasiliensis is the dimorphic fungus responsible for human paracoccidioidomycosis (PCM). We previously observed that P. brasiliensis isolates bearing highly polymorphic PbGP43 of genotype A (Pb2, Pb3 and Pb4) were phylogenetically distant from the others. The PbGP43 gene encodes an immune dominant diagnostic antigen (gp43), and its polymorphism reflects broader genetic diversity in the species. In the present study, we observed that isolates with PbGP43 of genotype A showed low virulence when inoculated in B10.A mice by the intraperitoneal, intratracheal and intravenous routes. In vitro studies detected sharp and prolonged down-regulation of PbGP43 in Pb3 (and not in Pb18 or Pb339) as a result of heat shock at 42 degrees C and temperature shift to prompt mycelium to yeast transition, which was, however, not disturbed. Differences in transcriptional regulation are possibly a consequence of mutations in the PbGP43 promoter region, which we here show to be more polymorphic in genotype A isolates. As opposed to Pb3's rapid adaptation to in vitro culture conditions after isolation from the lung, Pb12, the most aggressive isolate tested here, showed slow growth and phase transition in vitro. Interestingly, animals that were highly infected by Pb12 produced small amounts of anti-gp43 antibodies. That was apparently due to down-regulation in PbGP43 expression. We present the first evidence of transcriptional regulation of gp43 expression, but our results suggest that gene expression is also regulated at the protein and/or secretion levels.


Asunto(s)
Antígenos Fúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Glicoproteínas/metabolismo , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/metabolismo , Paracoccidioidomicosis/microbiología , Animales , Anticuerpos Antifúngicos/análisis , Antígenos Fúngicos/biosíntesis , Antígenos Fúngicos/genética , Enfermedad Crónica , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Paracoccidioides/genética , Paracoccidioidomicosis/patología , Polimorfismo Genético , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virulencia
18.
Antioxid Redox Signal ; 18(3): 221-38, 2013 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-22894707

RESUMEN

AIMS: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS: Ras signaling pathway stimulation by 50 and/or 100 µM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 µM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.


Asunto(s)
Proliferación Celular , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , S-Nitrosoglutatión/farmacología , Animales , Bradiquinina/farmacología , Células COS , Señalización del Calcio , Membrana Celular/enzimología , Chlorocebus aethiops , Cisteína/análogos & derivados , Cisteína/metabolismo , Activación Enzimática , Aparato de Golgi/enzimología , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Óxido Nítrico/fisiología , Oxidación-Reducción , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/metabolismo , Procesamiento Proteico-Postraduccional , S-Nitrosotioles/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo
19.
Mol Cancer Res ; 9(11): 1471-8, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21900362

RESUMEN

Phage-display peptide libraries have been widely used to identify specific peptides targeting in vivo tumor cells and the tumor vasculature and playing an important role in the discovery of antitumor bioactive peptides. In the present work, we identified a new melanoma-homing peptide, (-CVNHPAFAC-), using a C7C phage-display library directed to the developing tumor in syngeneic mice. Phage were able to preferentially target melanoma in vivo, with an affinity about 50-fold greater than that with normal tissue, and the respective synthesized peptide displaced the corresponding phage from the tumor. A preferential binding to endothelial cells rather than to melanoma cells was seen in cell ELISA, suggesting that the peptide is directed to the melanoma vasculature. Furthermore, the peptide was able to bind to human sonic hedgehog, a protein involved in the development of many types of human cancers. Using a new peptide approach therapy, we coupled the cyclic peptide to another peptide, HTMYYHHYQHHL-NH(2), a known antagonist of VEGFR-2 receptor, using the GYG linker. The full peptide CVNHPAFACGYGHTMYYHHYQHHL-NH(2) was effective in delaying tumor growth (P < 0.05) and increasing animal survival when injected systemically, whereas a scramble-homing peptide containing the same antagonist did not have any effect. This is the first report on the synthesis of a tumor-homing peptide coupled to antiangiogenic peptide as a new anticancer therapeutics.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Biblioteca de Péptidos , Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Animales , Sistemas de Liberación de Medicamentos , Humanos , Masculino , Melanoma Experimental/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Péptidos/química , Péptidos/farmacocinética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Especificidad por Sustrato
20.
Redox Rep ; 16(1): 27-37, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21605496

RESUMEN

We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27B(PTPα)) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27B(PTPα)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27B(PTPα)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.


Asunto(s)
Apoptosis , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Caspasas/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , L-Lactato Deshidrogenasa/efectos de los fármacos , Óxido Nítrico/metabolismo , Fosfatidilserinas/análisis , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Propidio/análisis , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Transfección
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