RESUMO
Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.
Assuntos
Interações Hospedeiro-Patógeno , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , NADPH Oxidases/metabolismo , Oxirredução , Transporte ProteicoRESUMO
Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 µg/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by ~40%), whereas PDIred diminished (by ~60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by ~70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (>80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re)cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer.
Assuntos
Membrana Celular/enzimologia , Citosol/enzimologia , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Superóxidos/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Bacitracina/farmacologia , Membrana Celular/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Mutação de Sentido Incorreto , NADPH Oxidases/genética , Oxirredução/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
We focused on the effect of mild hyperhomocysteinemia (HHcy) on the development of atherosclerosis, using apolipoprotein E-deficient (apoE(-/-)) and normal mice. Mice received diets enriched in methionine with low or high levels of folate, B(12) and B(6) (diets B and C, respectively), and diet only with low levels of folate, B(12) and B(6) (diets D), to induce mild HHcy. Normal mice fed on diets B, C and D presented mild HHcy, but they did not develop atherosclerotic lesions after 24 weeks of diet. In addition, increased endoplasmic reticulum stress was present in normal mice fed on diet B, compared to others groups. ApoE(-/-) mice fed on diet B for 20 weeks presented the greatest atherosclerotic lesion area at the aortic sinus than other groups. These results suggest that the methionine may have a toxic effect on endothelium, and the B-vitamins addition on diet may have a protective effect in the long term, despite the increase on homocysteine levels. Mild HHcy accelerated the development of atherosclerosis in apoE(-/-) mice, and supplementation with B-vitamins is important for prevention of vascular disease, principally in the long term.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/complicações , Hiper-Homocisteinemia/complicações , Animais , Apolipoproteínas E/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Dieta , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Ácido Fólico/farmacologia , Hiper-Homocisteinemia/induzido quimicamente , Hiper-Homocisteinemia/fisiopatologia , Masculino , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.
Assuntos
Complexo de Golgi/enzimologia , Leishmania , Leishmaniose/enzimologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fagocitose , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Antibacterianos , Brefeldina A/farmacologia , Cricetinae , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidoresRESUMO
Cellular mechanisms governing redox homeostasis likely involve their integration with other stresses. Endoplasmic reticulum (ER) stress triggers complex adaptive or proapoptotic signaling defined as the unfolded protein response (UPR), involved in several pathophysiological processes. Since protein folding is highly redox-dependent, convergence between ER stress and oxidative stress has attracted interest. Evidence suggests that ROS production and oxidative stress are not only coincidental to ER stress, but are integral UPR components, being triggered by distinct types of ER stressors and contributing to support proapoptotic, as well as proadaptive UPR signaling. Thus, ROS generation can be upstream or downstream UPR targets and may display a UPR-specific plus a nonspecific component. Enzymatic mechanisms of ROS generation during UPR include: (a) Multiple thiol-disulfide exchanges involving ER oxidoreductases including flavooxidase Ero1 and protein disulfide isomerase (PDI); (b) Mitochondrial electron transport; (c) Nox4 NADPH oxidase complex, particularly Nox4. Understanding the roles of such mechanisms and how they interconnect with the UPR requires more investigation. Integration among such ROS sources may depend on Ca(2+) levels, ROS themselves, and PDI, which associates with NADPH oxidase and regulates its function. Oxidative stress may frequently integrate with a background of ER stress/UPR in several diseases; here we discuss a focus in the vascular system.
Assuntos
Transporte de Elétrons/fisiologia , Retículo Endoplasmático/enzimologia , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/fisiologia , Ativação Enzimática , Homeostase , Isoenzimas/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico , Doenças Vasculares/metabolismoRESUMO
Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII.
Assuntos
Isoenzimas/metabolismo , Mitocôndrias/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Angiotensina II/farmacologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Isoenzimas/genética , Mitocôndrias/efeitos dos fármacos , Dados de Sequência Molecular , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/genética , Óxidos de Nitrogênio/metabolismo , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Reação em Cadeia da Polimerase , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Vasoconstritores/farmacologiaRESUMO
Assessment of low-level superoxide in nonphagocytic cells is crucial for assessing redox-dependent signaling pathways and the role of enzymes such as the NADPH oxidase complex. However, most superoxide probes present inherent limitations. Particularly, assessment of dihydroethidium (DHE) fluorescence is limited regarding a lack of possible quantification and simultaneous detection of its two main products: 2-hydroxyethidium, more specific for superoxide, and ethidium, which reflects H2O2-dependent pathways involving metal proteins. HPLC separation and analysis of those two main products have been described. This chapter reports procedures used for the validation of superoxide measurements in vascular system. Superoxide assessment was performed for cultured cells and tissue fragments incubated with DHE, followed by acetonitrile extraction and HPLC run, with simultaneous fluorescence detection of 2-hydroxyethidium and ethidium and ultraviolet detection of remaining DHE. It also describes procedures for DHE-based NADPH oxidase activity assays using HPLC or fluorometry. Such methods can enhance accuracy and allow better quantitation of vascular superoxide measurements.
Assuntos
Técnicas de Química Analítica/métodos , Etídio/análogos & derivados , NADPH Oxidases/fisiologia , Superóxidos/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Etídio/metabolismo , Humanos , OxirreduçãoRESUMO
Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDI's unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.
Assuntos
Músculo Liso Vascular/enzimologia , NADPH Oxidases/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Doenças Vasculares/etiologia , Endotélio Vascular/enzimologia , Humanos , Músculo Liso Vascular/citologia , Oxirredução , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Transdução de Sinais , Compostos de Sulfidrila/químicaRESUMO
Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.