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1.
Proc Natl Acad Sci U S A ; 121(42): e2320187121, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39382994

RESUMO

Canopy shade enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs) to boost auxin synthesis in the cotyledons. Auxin, together with local PIFs and their positive regulator CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), promotes hypocotyl growth to facilitate access to light. Whether shade alters the cellular redox status thereby affecting growth responses, remains unexplored. Here, we show that, under shade, high auxin levels increased reactive oxygen species and nitric oxide accumulation in the hypocotyl of Arabidopsis. This nitroxidative environment favored the promotion of hypocotyl growth by COP1 under shade. We demonstrate that COP1 is S-nitrosylated, particularly under shade. Impairing this redox regulation enhanced COP1 degradation by the proteasome and diminished the capacity of COP1 to interact with target proteins and to promote hypocotyl growth. Disabling this regulation also generated transversal asymmetries in hypocotyl growth, indicating poor coordination among different cells, which resulted in random hypocotyl bending and predictably low ability to compete with neighbors. These findings highlight the significance of redox signaling in the control of diffuse growth during shade avoidance.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Hipocótilo , Espécies Reativas de Oxigênio , Ubiquitina-Proteína Ligases , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Espécies Reativas de Oxigênio/metabolismo , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Óxido Nítrico/metabolismo , Ácidos Indolacéticos/metabolismo , Luz , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Oxirredução , Transdução de Sinais
2.
Nitric Oxide ; 147: 26-41, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38614230

RESUMO

Nitric oxide (NO) acts in different physiological processes, such as blood pressure control, antiparasitic activities, neurotransmission, and antitumor action. Among the exogenous NO donors, ruthenium nitrosyl/nitro complexes are potential candidates for prodrugs, due to their physicochemical properties, such as thermal and physiological pH stability. In this work, we proposed the synthesis and physical characterization of the new nitro terpyridine ruthenium (II) complexes of the type [RuII(L)(NO2)(tpy)]PF6 where tpy = 2,2':6',2″-terpyridine; L = 3,4-diaminobenzoic acid (bdq) or o-phenylenediamine (bd) and evaluation of influence of diimine bidentate ligand NH.NHq-R (R = H or COOH) in the HSA/DNA interaction as well as antiviral activity. The interactions between HSA and new nitro complexes [RuII(L)(NO2)(tpy)]+ were evaluated. The Ka values for the HSA-[RuII(bdq)(NO2)(tpy)]+ is 10 times bigger than HSA-[RuII(bd)(NO2)(tpy)]+. The sites of interaction between HSA and the complexes via synchronous fluorescence suppression indicate that the [RuII(bdq)(NO2)(tpy)]+ is found close to the Trp-241 residue, while the [RuII(bd)(NO2)(tpy)]+ complex is close to Tyr residues. The interaction with fish sperm fs-DNA using direct spectrophotometric titration (Kb) and ethidium bromide replacement (KSV and Kapp) showed weak interaction in the system fs-DNA-[RuII(bdq)(NO)(tpy)]+. Furthermore, fs-DNA-[RuII(bd)(NO2)(tpy)]+ and fs-DNA-[RuII(bd)(NO)(tpy)]3+ system showed higher intercalation constant. Circular dichroism spectra for fs-DNA-[RuII(bd)(NO2)(tpy)]+ and fs-DNA-[RuII(bd)(NO)(tpy)]3+, suggest semi-intercalative accompanied by major groove binding interaction modes. The [RuII(bd)(NO2)(tpy)]+ and [RuII(bd)(NO)(tpy)]3+ inhibit replication of Zika and Chikungunya viruses based in the nitric oxide release under S-nitrosylation reaction with cysteine viral.


Assuntos
Antivirais , DNA , Rutênio , Humanos , DNA/metabolismo , DNA/química , Rutênio/química , Rutênio/farmacologia , Antivirais/farmacologia , Antivirais/química , Antivirais/metabolismo , Ligantes , Animais , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Piridinas/química , Piridinas/farmacologia , Iminas/química , Iminas/farmacologia , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo
3.
Nitric Oxide ; 146: 1-9, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38428514

RESUMO

BACKGROUND: Cannabidiol (CBD) is the second most abundant pharmacologically active component present in Cannabis sp. Unlike Δ-9-tetrahydrocannabinol (THC), it has no psychotomimetic effects and has recently received significant interest from the scientific community due to its potential to treat anxiety and epilepsy. CBD has excellent anti-inflammatory potential and can be used to treat some types of inflammatory and neuropathic pain. In this context, the present study aimed to evaluate the analgesic mechanism of cannabidiol administered systemically for the treatment of neuropathic pain and determine the endogenous mechanisms involved with this analgesia. METHODS: Neuropathic pain was induced by sciatic nerve constriction surgery, and the nociceptive threshold was measured using the paw compression test in mice. RESULTS: CBD produced dose-dependent antinociception after intraperitoneal injection. Selective inhibition of PI3Kγ dose-dependently reversed CBD-induced antinociception. Selective inhibition of nNOS enzymes reversed the antinociception induced by CBD, while selective inhibition of iNOS and eNOS did not alter this antinociception. However, the inhibition of cGMP production by guanylyl cyclase did not alter CBD-mediated antinociception, but selective blockade of ATP-sensitive K+ channels dose-dependently reversed CBD-induced antinociception. Inhibition of S-nitrosylation dose-dependently and completely reversed CBD-mediated antinociception. CONCLUSION: Cannabidiol has an antinociceptive effect when administered systemically and this effect is mediated by the activation of PI3Kγ as well as by nitric oxide and subsequent direct S-nitrosylation of KATP channels on peripheral nociceptors.


Assuntos
Analgésicos , Canabidiol , Classe Ib de Fosfatidilinositol 3-Quinase , Canais KATP , Neuralgia , Óxido Nítrico Sintase Tipo I , Óxido Nítrico , Transdução de Sinais , Animais , Canabidiol/farmacologia , Canais KATP/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Analgésicos/farmacologia , Analgesia
4.
Biol Res ; 56(1): 51, 2023 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-37773178

RESUMO

BACKGROUND: Nitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown. RESULTS: We report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases. CONCLUSIONS: S-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Adesão Celular , Células Endoteliais , Molécula 1 de Adesão de Célula Vascular/metabolismo , Óxido Nítrico/metabolismo , Melanoma Maligno Cutâneo
5.
Molecules ; 28(16)2023 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-37630415

RESUMO

Cancer involves a series of diseases where cellular growth is not controlled. Cancer is a leading cause of death worldwide, and the burden of cancer incidence and mortality is rapidly growing, mainly in developing countries. Many drugs are currently used, from chemotherapeutic agents to immunotherapy, among others, along with organ transplantation. Treatments can cause severe side effects, including remission and progression of the disease with serious consequences. Increased glycolytic activity is characteristic of cancer cells. Triosephosphate isomerase is essential for net ATP production in the glycolytic pathway. Notably, some post-translational events have been described that occur in human triosephosphate isomerase in which functional and structural alterations are provoked. This is considered a window of opportunity, given the differences that may exist between cancer cells and their counterpart in normal cells concerning the glycolytic enzymes. Here, we provide elements that bring out the potential of triosephosphate isomerase, under post-translational modifications, to be considered an efficacious target for treating cancer.


Assuntos
Neoplasias , Triose-Fosfato Isomerase , Humanos , Triose-Fosfato Isomerase/genética , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Ciclo Celular , Proliferação de Células
6.
Int J Pharm ; 640: 122998, 2023 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-37127139

RESUMO

The co-therapy of common chemotherapeutics with nitric oxide (NO), an endogenous signaling molecule, is proposed as an alternative to sensitize cancer cells and enhance treatments' efficacy. Herein, we have synthesized cisplatin-releasing zinc oxide nanoparticles (ZnO/CisPt NPs), which promoted a sustained and pH targeted release, able to release a higher amount of CisPt at tumor microenvironment conditions. This material was combined with a chronic NO treatment, at low concentration, in prostate cancer cells (PC3). NO treatment enhanced the S-NO concentration in PC3 cells, suggesting the nitrosylation or transnitrosylation processes enhancement, which are directly related to S-NO binding to proteins, function alterations and cancer cells death. Indeed, these mechanisms directly impacted the cytotoxic effect of ZnO/CisPt NPs, inducing a 30 % higher viability reduction of PC3 cells after NO treatment, along with a higher selectivity index when compared to normal human fibroblasts (FN1).


Assuntos
Nanopartículas Metálicas , Nanopartículas , Neoplasias da Próstata , Óxido de Zinco , Masculino , Humanos , Óxido de Zinco/química , Óxido Nítrico , Nanopartículas/toxicidade , Neoplasias da Próstata/tratamento farmacológico , Cisplatino/farmacologia , Nanopartículas Metálicas/química , Microambiente Tumoral
7.
Front Physiol ; 13: 864948, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35431975

RESUMO

Connexins are a family of proteins that can form two distinct types of channels: hemichannels and gap junction channels. Hemichannels are composed of six connexin subunits and when open allow for exchanges between the cytoplasm and the extracellular milieu. Gap junction channels are formed by head-to-head docking of two hemichannels in series, each one from one of two adjacent cells. These channels allow for exchanges between the cytoplasms of contacting cells. The lens is a transparent structure located in the eye that focuses light on the retina. The transparency of the lens depends on its lack of blood irrigation and the absence of organelles in its cells. To survive such complex metabolic scenario, lens cells express Cx43, Cx46 and Cx50, three connexins isoforms that form hemichannels and gap junction channels that allow for metabolic cooperation between lens cells. This review focuses on the roles of Cx46 hemichannels and gap junction channels in the lens under physiological conditions and in the formation of cataracts, with emphasis on the modulation by posttranslational modifications.

8.
Biochim Biophys Acta Proteins Proteom ; 1870(3): 140754, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34995802

RESUMO

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cisteína/química , Óxido Nítrico/química , Proteínas Tirosina Fosfatases/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Biotina/metabolismo , Catálise , Cristalografia por Raios X/métodos , Cisteína/metabolismo , Humanos , Espectrometria de Massas/métodos , Estrutura Molecular , Óxido Nítrico/metabolismo , Oxirredução , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Yersinia enterocolitica/metabolismo
9.
Am J Physiol Heart Circ Physiol ; 321(6): H1083-H1095, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34652985

RESUMO

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.


Assuntos
Músculos Abdominais/irrigação sanguínea , Adesão Celular , Células Endoteliais/efeitos dos fármacos , Leucócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Técnicas de Cocultura , Células Endoteliais/enzimologia , Ativação Enzimática , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fatores de Tempo
10.
Antioxidants (Basel) ; 10(4)2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33918310

RESUMO

The cardioprotective effects of nitric oxide (NO) have been described through S-nitrosylation of several important proteins in the mitochondria of the cardiomyocyte. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of S-nitrosothiols by producing denitrosylation, thus limiting the cardioprotective effect of NO. The effect of GSNOR inhibition on the damage by cardiac ischemia-reperfusion is still unclear. We tested the hypothesis that pharmacological inhibition of GSNOR promotes cardioprotection by increasing the levels of protein S-nitrosylation. In a model of ischemia-reperfusion in isolated rat heart, the effect of a GSNOR inhibitor, 5-chloro-3-(2-[4-ethoxyphenyl) (ethyl) amino]-2-oxoethyl)-1H-indole-2-carboxylic acid (C2), was investigated. Ventricular function and hemodynamics were determined, in addition to tissue damage and S-nitrosylation of mitochondrial proteins. Hearts treated with C2 showed a lower release of myocardial damage marker creatine kinase and a reduction in the infarcted area. It also improved post-ischemia ventricular function compared to controls. These results were associated with increasing protein S-nitrosylation, specifically of the mitochondrial complexes III and V. The pharmacological inhibition of GSNOR showed a concentration-dependent cardioprotective effect, being observed in functional parameters and myocardial damage, which was maximal at 1 µmol/L, associated with increased S-nitrosylation of mitochondrial proteins. These data suggest that GSNOR is an interesting pharmacological target for cardiac reperfusion injury.

11.
J Proteomics ; 231: 104020, 2021 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-33096306

RESUMO

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Matriz Extracelular , Proteoma , Proteômica
12.
Redox Biol ; 38: 101769, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33126056

RESUMO

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II.


Assuntos
Angiotensina II , Nitritos , Angiotensina II/farmacologia , Animais , Anti-Hipertensivos , Células HEK293 , Humanos , Óxido Nítrico , Proteína Quinase C , Ratos
13.
Front Physiol ; 11: 595526, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33281627

RESUMO

Leukocyte recruitment is one of the most important cellular responses to tissue damage. Leukocyte extravasation is exquisitely regulated by mechanisms of selective leukocyte-endothelium recognition through adhesion proteins in the endothelial cell surface that recognize specific integrins in the activated leukocytes. A similar mechanism is used by tumor cells during metastasis to extravasate and form a secondary tumor. Nitric oxide (NO) has been classically described as an anti-inflammatory molecule that inhibits leukocyte adhesion. However, the evidence available shows also a positive role of NO in leukocyte adhesion. These apparent discrepancies might be explained by the different NO concentrations reached during the inflammatory response, which are highly modulated by the expression of different nitric oxide synthases, along the inflammatory response and by changes in their subcellular locations.

14.
Cancers (Basel) ; 12(9)2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32825247

RESUMO

Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.

15.
Int J Mol Sci ; 21(15)2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32751416

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs causing its lateralization to the plasma membrane where Cx43 forms hemichannels. We tested the hypothesis that in DMD cardiomyopathy, increased oxidative stress is associated with the formation and activation of Cx43 hemichannels. For this, we used mdx mice as a DMD model and evaluated cardiac function, nitroso-redox changes and Cx43 hemichannels permeability. Mdx hearts presented increased NADPH oxidase-derived oxidative stress and increased Cx43 S-nitrosylation compared to controls. These redox changes were associated with increased Cx43 lateralization, decreased cardiac contractility and increased arrhythmic events. Pharmacological inhibition of NADPH oxidase using apocynin (one month) reduced systemic oxidative stress and reversed the aforementioned changes towards normal, except Cx43 lateralization. Opening of Cx43 hemichannels was blocked by apocynin treatment and by acute hemichannel blockade with carbenoxolone. NADPH oxidase inhibition also prevented the occurrence of apoptosis in mdx hearts and reversed the ventricular remodeling. These results show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. These changes lead to apoptosis and cardiac dysfunction and were prevented by NADPH oxidase inhibition.


Assuntos
Conexina 43/fisiologia , Distrofia Muscular de Duchenne/metabolismo , Miocárdio , Acetofenonas/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Miocárdio/metabolismo , Miocárdio/patologia , NADPH Oxidases/antagonistas & inibidores , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
16.
Front Plant Sci ; 11: 970, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32695132

RESUMO

Nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. Nitrate reductase (NR) not only mediates the reduction of NO3 - to NO2 - but also reduces NO2 - to NO, a relevant pathway for NO production in higher plants. Herein, we hypothesized that sugarcane plants supplied with more NO3 - as a source of N would produce more NO under water deficit. Such NO would reduce oxidative damage and favor photosynthetic metabolism and growth under water limiting conditions. Sugarcane plants were grown in nutrient solution and received the same amount of nitrogen, with varying nitrate:ammonium ratios (100:0 and 70:30). Plants were then grown under well-watered or water deficit conditions. Under water deficit, plants exhibited higher root [NO3 -] and [NO2 -] when supplied with 100% NO3 -. Accordingly, the same plants also showed higher root NR activity and root NO production. We also found higher photosynthetic rates and stomatal conductance in plants supplied with more NO3 -, which was associated with increased root growth. ROS accumulation was reduced due to increases in the activity of catalase in leaves and superoxide dismutase and ascorbate peroxidase in roots of plants supplied with 100% NO3 - and facing water deficit. Such positive responses to water deficit were offset when a NO scavenger was supplied to the plants, thus confirming that increases in leaf gas exchange and plant growth were induced by NO. Concluding, NO3 - supply is an interesting strategy for alleviating the negative effects of water deficit on sugarcane plants, increasing drought tolerance through enhanced NO production. Our data also provide insights on how plant nutrition could improve crop tolerance against abiotic stresses, such as drought.

17.
Front Microbiol ; 11: 1184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582109

RESUMO

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.

18.
Front Microbiol, v. 11, 1184, jun. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4834

RESUMO

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.

19.
Front Microbiol, v. 11, 1184, jun. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4126

RESUMO

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.

20.
Front Physiol ; 10: 988, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440166

RESUMO

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

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