RESUMO
Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma, and is usually associated with a pro-invasive phenotype. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against damage generated by reactive oxygen species (ROS) during melanogenesis stimulation. Our present results show that QSOX activity was two-fold higher in cells with stimulated melanogenesis when compared to control cells. Considering that glutathione (GSH) is one of the main factor responsible for controlling redox homeostasis in cells, this work also aimed to investigate the relationship between QSOX activity, GSH levels and melanogenesis stimulation in B16-F10 murine melanoma cell line. The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Interestingly, GSH-depleted cells without stimulation of melanogenesis kept high levels of viability, suggesting a possible adaptive mechanism of survival even under low GSH levels. They also showed lower extracellular activity of QSOX, and higher QSOX intracellular immunostaining, suggesting that this enzyme was less excreted from cells and corroborating with a diminished extracellular QSOX activity. On the other hand, cells under melanogenesis stimulation showed a lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by decreased cell viability after GSH-depletion, no alterations in QSOX extracellular activity, but higher QSOX nucleic immunostaining. We suggest that melanogenesis stimulation and redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, contributing to additional alterations of its metabolic adaptive response.
Assuntos
Melanoma , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Animais , Camundongos , Glutationa/metabolismo , Melanoma/metabolismo , Oxirredução , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismoRESUMO
The dissimilatory sulfite reductase enzyme has very characteristic active site where the substrate binds to an iron site, ligated by a siroheme macrocycle and a thiol directly connected to a [Fe4S4] cluster. This arrangement gives the enzyme remarkable efficiency in reducing sulfite and nitrite all the way to hydrogen sulfide and ammonia. For the first time we present a theoretical study where substrate binding modalities and activation are elucidated using active site models containing proton supply side chains and the [Fe4S4] cluster. Density functional theory (DFT) was deployed in conjunction with the energy decomposition scheme (as implemented in AMS), the quantum theory of atoms in molecules (QTAIM), and conceptual DFT (cDFT) descriptors. We quantified the role of the electrostatic interactions inside the active site created by the side chains as well as the influence of the [Fe4S4] cluster on the substrate binding. Furthermore, using conceptual DFT results we shed light of the activation process, thus, laying foundation for further mechanistic studies. We found that the bonding of the ligands to the iron complex is dominated by electrostatic interactions, but the presence of the [Fe4S4] cubane leads to substantial changes in electronic interaction. The spin state of the cubane, however, affects the binding energy only marginally. The conceptual DFT results show that the presence of the [Fe4S4] cubane affects the reactivity of the active site as it is involved in electron transfer. This is corroborated by an increase in the electrophilicity index, thus making the active site more prone to react with the ligands. The interaction energies between the ligand and the siroheme group are also increased upon the presence of the cubane group, thus, suggesting that the siroheme group is not an innocent spectator but plays an active role in the reactivity of the dSIR active site.
Assuntos
Proteínas Ferro-Enxofre , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Domínio Catalítico , Escherichia coli , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Ligantes , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismoRESUMO
Sulphate-reducing bacteria are commonly associated with biological causes of oil well souring. Biosulphetogenesis can directly affect oil quality and storage due to the accumulation of sulphides. In addition, these microorganisms can create bio-incrustation that can clog pipes. Sulphite reductase (SIR) is the enzyme responsible for converting ion sulphite into sulphide and several substances may interfere or control such activity. This interference can hinder growth of the sulphate-reducing bacteria and, consequently, it reduces sulphide accumulation in situ. This work focuses on molecular modelling techniques along with in vitro experiments in order to investigate the potential of two essential oils and one vegetable oil as main inhibitors of sulphite reductase activity. Docking simulation identified several substances present in Rosmarinus officinalis, Tea tree and Neem extractable oils as potential inhibitors of SIR. Substances present in Neem vegetable oil are the most potent inhibitors, followed by Rosmarinus officinalis and Tea tree essential oils. The Neem oil mixture showed a superior effectiveness in intracellular SIR inhibitory effects.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Simulação de Acoplamento Molecular , Óleos Voláteis/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Sulfatos/metabolismo , Óleo de Melaleuca/farmacologia , Bactérias/metabolismo , Rosmarinus/químicaRESUMO
Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease produced by the deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycans (GAGs) accumulation. Since currently available treatments remain limited and unspecific, novel therapeutic approaches are essential for the disease treatment. In an attempt to reduce treatment limitations, gene therapy rises as a more effective and specific alternative. We present in this study the delivery assessment of GALNS and sulfatase-modifying factor 1 (SUMF1) genes via HIV-1 derived lentiviral vectors into fibroblasts from MPS IVA patients. After transduction, we determined GALNS enzymatic activity, lysosomal mass change, and autophagy pathway impairment. Additionally, we computationally assessed the effect of mutations over the enzyme-substrate interaction and phenotypic effects. The results showed that the co-transduction of MPS IVA fibroblasts with GALNS and SUMF1 cDNAs led to a significant increase in GALNS enzyme activity and a reduction of lysosomal mass. We show that patient-specific differences in cellular response are directly associated with the set of mutations on each patient. Lastly, we present new evidence supporting autophagy impairment in MPS IVA due to the presence and changes in autophagy proteins in treated MPS IVA fibroblasts. Our results offer new evidence that demonstrate the potential of lentiviral vectors as a strategy to correct GALNS deficiency.
Assuntos
Condroitina Sulfatases , Fibroblastos/metabolismo , Vetores Genéticos , HIV-1 , Mucopolissacaridose IV , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Transdução Genética , Condroitina Sulfatases/biossíntese , Condroitina Sulfatases/genética , Terapia Genética , Células HEK293 , Humanos , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/metabolismo , Mucopolissacaridose IV/terapia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genéticaRESUMO
The incidence of prostate cancer (PCa) is increasing, and it is currently the second most frequent cause of death by cancer in men. Despite advancements in cancer therapies, new therapeutic approaches are still needed for treatment-refractory advanced metastatic PCa. Cross-species analysis presents a robust strategy for the discovery of new potential therapeutic targets. This strategy involves the integration of genomic data from genetically engineered mouse models (GEMMs) and human PCa datasets. Considering the role of antioxidant pathways in tumor initiation and progression, we searched oxidative stress-related genes for a potential therapeutic target for PCa. First, we analyzed RNA-sequencing data from Pb-Cre4; Ptenf/f mice and discovered an increase in sulfiredoxin (Srxn1) mRNA expression in high-grade prostatic intraepithelial neoplasia (PIN), well-differentiated adenocarcinoma (medium-stage tumors), and poor-differentiated adenocarcinoma (advanced-stage prostate tumors). The increase of SRXN1 protein expression was confirmed by immunohistochemistry in mouse prostate tumor paraffin samples. Analyses of human databases and prostate tissue microarrays demonstrated that SRXN1 is overexpressed in a subset of high-grade prostate tumors and correlates with aggressive PCa with worse prognosis and decreased survival. Analyses in vitro showed that SRXN1 expression is also higher in most PCa cell lines compared to normal cell lines. Furthermore, siRNA-mediated downregulation of SRXN1 led to decreased viability of PCa cells LNCaP. In conclusion, we identified the antioxidant enzyme SRXN1 as a potential therapeutic target for PCa. Our results suggest that the use of specific SRXN1 inhibitors may be an effective strategy for the adjuvant treatment of castration-resistant PCa with SRXN1 overexpression.
Assuntos
Terapia de Alvo Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Gradação de Tumores , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Estresse Oxidativo/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Prognóstico , Próstata/patologia , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de SobrevidaRESUMO
Candida glabrata is an opportunistic fungal pathogen that can cause life-threatening infections in immunocompromised patients. To ensure a successful infection, C. glabrata has evolved a variety of strategies to avoid killing within the host. One of these strategies is the resistance to oxidative stress. Here we show that the sulfiredoxin Srx1 and the peroxiredoxins, Tsa1 and Tsa2, are implicated in the oxidative stress response (OSR) and required for virulence. We analyzed null mutations in SRX1, TSA1 and TSA2 and showed that TSA2 and SRX1 are required to respond to oxidative stress. While TSA1 expression is constitutive, SRX1 and TSA2 are induced in the presence of H2O2 in a process dependent on H2O2 concentration and on both transcription factors Yap1 and Skn7. Msn2 and Msn4 are not necessary for the regulation of SRX1, TSA1 and TSA2. Interestingly, TSA1 and TSA2, which are localized in the cytoplasm, are induced in the presence of neutrophils and required for survival in these phagocytic cells.
Assuntos
Candida glabrata/genética , Candida glabrata/patogenicidade , Proteínas Fúngicas/genética , Estresse Oxidativo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Candida glabrata/enzimologia , Proteínas Fúngicas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Neutrófilos/microbiologia , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , VirulênciaRESUMO
Quiescent and contractile VSMC can switch to proliferative and migratory phenotype in response to growth factors and cytokines, an effect underscored by Nox family NADPH oxidases, particularly Nox1. We previously showed that quiescin/sulfhydryl oxidase 1 (QSOX1) has a role in neointima formation in balloon-injured rat carotid. Here, we investigated the intracellular redox mechanisms underlying these effects in primary VSMC. Our results show that exogenous incubation with wild type QSOX1b (wt QSOX), or with secreted QSOX1, but not with the inactive C452S QSOX 1b (C452S QSOX) or secreted inactive C455S QSOX1, induces VSMC migration and chemotaxis. PEG-catalase (PEG-CAT) prevented, while PEG-superoxide dismutase (PEG-SOD) increased migration induced by wt QSOX. Moreover, wt QSOX-induced migration was abrogated in NOX1-null VSMC. In contrast, both wt QSOX and C452S QSOX, and both secreted QSOX1 and C455S QSOX1, induce cell proliferation. Such effect was unaltered by PEG-CAT, while being inhibited by PEG-SOD. However, QSOX1-induced proliferation was not significantly affected in NOX1-null VSMC, compared with WT VSMC. These results indicate that hydrogen peroxide and superoxide mediate, respectively, migration and proliferation. However, Nox1 was required only for QSOX1-induced migration. In parallel, QSOX1-induced proliferation was independent of its redox activity, although mediated by intracellular superoxide.
Assuntos
Movimento Celular , Músculo Liso Vascular/citologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Animais , Proliferação de Células , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Espaço Intracelular/metabolismo , Camundongos , NADPH Oxidase 1/metabolismo , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
In the last years, H2S has been recognized as a signaling molecule in mammals, which can synthesize and catabolize (by oxidation) such species. The latter process is accelerated by a sulfide:quinone oxidoreductase (SQR, E.C. 1.8.5.4), a flavin-dependent sulfide oxidase (FDSO). FDSOs catalyze electron transfer from H2S to an acceptor in catalytic cycles involving two phases: (I) reduction of FAD by H2S (SH-) and (II) electron transfer from FADH- to the electron acceptor. The first step of FAD reduction consists on the reaction of SH- with a catalytic disulfide at the active site of the enzyme, to yield a thiolate and a persulfide in the protein. This step is ca. 106 times faster than the analogous reaction with low-molecular-weight disulfides (LMWDs) and the causes of such extraordinary acceleration remain unknown. Using the IEF-PCM(ε ≈ 10)/M06-2X-D3/6-31+G(d,p) level of theory, we have modeled the reaction of SH- with a disulfide as located in a representative model of the active site extracted from a prokaryotic SQR, assessing the effects of partial covalent interactions (PCIs) between the leaving sulfur atom and flavin ring on the activation Gibbs free-energy barrier at 298 K (∆G298K). To also evaluate the importance of entropic penalties on the first step, we have modeled at the same level of theory the reaction of (bis)hydroxyethyl disulfide in aqueous solution, a LMWD for which experimental data is available. Our results show that PCIs between the leaving sulfur atom and the flavin group only have a minor effect (∆G298K reduced by 1.6 kcal mol-1) while compensating entropic penalties could have a much larger effect (up to 8.3 kcal mol-1). Finally, we also present here a first model of some of further steps in the phase I of the catalytic cycle as in mammalian FDSOs, providing some light about their detailed mechanism. Graphical abstract .
Assuntos
Teoria da Densidade Funcional , Flavinas/metabolismo , Sulfeto de Hidrogênio/metabolismo , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Acidithiobacillus/enzimologia , Biocatálise , Domínio Catalítico , Dissulfetos/metabolismo , Entropia , OxirreduçãoRESUMO
Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 µg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Western Blotting , Linhagem Celular Tumoral , Citocinas/biossíntese , Regulação para Baixo , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/metabolismo , Mieloma Múltiplo/imunologia , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologiaRESUMO
The present study investigated the antioxidant effect of a new class of quinoline derivatives (a-d) on assays in vitro. Lipid peroxidation, thiol peroxidase-like and free radical scavenging activities were determined to evaluate antioxidant activity of compounds. Thiol oxidase-like and δ-aminolevulinate dehydratase activities were performed as a toxicological parameter. A second objective of this study was to evaluate the in vivo antinociceptive effect of the compound with better antioxidant effect and without toxic effects in a model of nociception induced by formalin in mice. In liver, at 100 µM, compound a reduced the lipid peroxidation to the control levels, while compounds c and d partially reduced it. In brain, only compound d partially reduced the lipid peroxidation at 50 and 100 µM. Compound b did not have an effect on the lipid peroxidation. Thiol peroxidase-like and free radical scavenging activities are not involved in the antioxidant mechanisms of these compounds. Compounds did not present thiol oxidase-like activity and effect on the δ-aminolevulinate dehydratase. In vivo experiments showed that compound a caused an inhibition of licking time in the first and second phases, and edema formation induced by formalin. In conclusion, quinoline derivative without selenium presented better in vitro antioxidant effect and in vivo antinociceptive activity.