RESUMO
The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (â¢NO) and superoxide (O2â¢-) produced by phagocytes contributes to its success as a human pathogen. Recombination of â¢NO and O2â¢- generates peroxynitrite (ONOO-), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward â¢NO and O2â¢- is well established, how Mtb responds to ONOO- remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both â¢NO and O2â¢-, which should combine to produce ONOO-. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO- levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO-. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe-4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe-4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe-S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO- in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO- and targeting Fe-S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).
Assuntos
Metabolismo Energético , Homeostase , Proteínas Ferro-Enxofre , Mycobacterium tuberculosis , Oxirredução , Ácido Peroxinitroso , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Humanos , Óxido Nítrico/metabolismo , Estresse Oxidativo , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/efeitos dos fármacos , Superóxidos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Tuberculose/microbiologia , Tuberculose/metabolismoRESUMO
Reactive oxygen and nitrogen species are small reactive molecules derived from elements in the airâoxygen and nitrogen. They are produced in biological systems to mediate fundamental aspects of cellular signaling but must be very tightly balanced to prevent indiscriminate damage to biological molecules. Small molecule probes can transmute the specific nature of each reactive oxygen and nitrogen species into an observable luminescent signal (or even an acoustic wave) to offer sensitive and selective imaging in living cells and whole animals. This review focuses specifically on small molecule probes for superoxide, hydrogen peroxide, hypochlorite, nitric oxide, and peroxynitrite that provide a luminescent or photoacoustic signal. Important background information on general photophysical phenomena, common probe designs, mechanisms, and imaging modalities will be provided, and then, probes for each analyte will be thoroughly evaluated. A discussion of the successes of the field will be presented, followed by recommendations for improvement and a future outlook of emerging trends. Our objectives are to provide an informative, useful, and thorough field guide to small molecule probes for reactive oxygen and nitrogen species as well as important context to compare the ecosystem of chemistries and molecular scaffolds that has manifested within the field.
Assuntos
Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismoRESUMO
Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of ß-amyloid (Aß), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aß nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aß1-42. Photonitrated Aß1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aß1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aß1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aß occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aß1-42.
Assuntos
Peptídeos beta-Amiloides , Luz , Fragmentos de Peptídeos , Tirosina , Peptídeos beta-Amiloides/metabolismo , Tirosina/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Humanos , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Camundongos , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
BACKGROUND: Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium. METHODS AND RESULTS: In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold (P<0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio (P<0.05). CONCLUSIONS: These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.
Assuntos
Ácido Eicosapentaenoico , Inflamação , Interleucina-6 , Óxido Nítrico , Fator de Necrose Tumoral alfa , Humanos , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Molécula 1 de Adesão Intercelular/metabolismo , Heme Oxigenase-1/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Cultivadas , Disponibilidade Biológica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácido Peroxinitroso/metabolismo , Mediadores da Inflamação/metabolismoRESUMO
There are no licensed vaccines for human cytomegalovirus (HCMV), and current antiviral drugs that target viral proteins are toxic and prone to resistance. Targeting host pathways essential for virus replication provides an alternate strategy that may reduce opportunities for drug resistance to occur. Oxidative stress is triggered by numerous viruses including HCMV. Peroxynitrite is a reactive nitrogen species that is formed during oxidative stress. Herein, we identified that HCMV rapidly induces the generation of intracellular peroxynitrite upon infection in a manner partially dependent upon xanthine oxidase generation. Peroxynitrite promoted HCMV infection in both cell-free and cell-associated infection systems in multiple cell types. Inhibiting peroxynitrite within the first 24 hours of infection prevented HCMV replication and peroxynitrite promoted cell entry and pp65 translocation into the host cell nuclei. Furthermore, using the murine cytomegalovirus model, we demonstrated that antagonizing peroxynitrite significantly reduces cytomegalovirus replication and pathogenesis in vivo. Overall, our study highlights a proviral role for peroxynitrite in CMV infection and implies that RNS and/or the mechanisms that induce their production could be targeted as a novel strategy to inhibit HCMV infection. IMPORTANCE: Human cytomegalovirus (HCMV) causes significant disease in individuals with impaired or immature immune systems, such as transplant patients and after congenital infection. Antiviral drugs that target the virus directly are toxic and are susceptible to antiviral drug resistance due to virus mutations. An alternate strategy is to target processes within host cells that are required by the virus for replication. Herein, we show that HCMV infection triggers a highly reactive molecule, peroxynitrite, during the initial stages of infection. Peroxynitrite was required for the initial entry of the virus into the cell and promotes virus replication in multiple cell types, suggesting a broad pro-viral function. Importantly, targeting peroxynitrite dramatically inhibited cytomegalovirus replication in cells in the laboratory and in mice, suggesting that therapeutic targeting of this molecule and/or the cellular functions it regulates could represent a novel strategy to inhibit HCMV infection.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Modelos Animais de Doenças , Ácido Peroxinitroso , Internalização do Vírus , Replicação Viral , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacologia , Animais , Camundongos , Citomegalovirus/fisiologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Internalização do Vírus/efeitos dos fármacos , Humanos , Infecções por Citomegalovirus/virologia , Replicação Viral/efeitos dos fármacos , Muromegalovirus/fisiologia , Muromegalovirus/efeitos dos fármacos , Linhagem Celular , Estresse OxidativoRESUMO
Acute lung injury (ALI) is a serious pulmonary inflammatory disease resulting from excessive reactive oxygen species (ROS) which could cause the damage of the alveolar epithelial cells and capillary endothelial cells. Peroxynitrite, as one of short-lived reactive oxygen species, is closely related to the process of ALI. Thus, it is important to monitor the fluctuation of peroxynitrite in living system for understanding the process of ALI. Herein, the novel mitochondria-targeted fluorescent probe BHMT was designed to respond to peroxynitrite and pH with distinct fluorescence properties respectively. The absorption spectrum of the probe BHMT exhibited a notable red shift as the pH value declined from 8.8 to 2.6. Upon reaction with peroxynitrite, BHMT had a significant increase of fluorescence intensity (63-fold) with maintaining a detection limit of only 43.7 nM. Furthermore, BHMT could detect the levels of endogenous peroxynitrite and image the intracellular pH in ratiometric channels utilizing cell imaging. In addition, BHMT was successfully applied to revealing the relationship between the peroxynitrite and the extent of ALI. Thus, these results indicated the probe BHMT could be a potential tool for diagnosing the early stage of ALI and revealed the peroxynitrite was likely to be a crucial therapeutic target in ALI treatment.
Assuntos
Lesão Pulmonar Aguda , Corantes Fluorescentes , Mitocôndrias , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/análise , Lesão Pulmonar Aguda/diagnóstico por imagem , Lesão Pulmonar Aguda/metabolismo , Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Humanos , Animais , Concentração de Íons de Hidrogênio , Camundongos , Imagem Óptica , MasculinoRESUMO
Although the labile iron pool (LIP) biochemical identity remains a topic of debate, it serves as a universal homeostatically regulated and essential cellular iron source. The LIP plays crucial cellular roles, being the source of iron that is loaded into nascent apo-iron proteins, a process akin to protein post-translational modification, and implicated in the programmed cell death mechanism known as ferroptosis. The LIP is also recognized for its reactivity with chelators, nitric oxide, and peroxides. Our recent investigations in a macrophage cell line revealed a reaction of the LIP with the oxidant peroxynitrite. In contrast to the LIP's pro-oxidant interaction with hydrogen peroxide, this reaction is rapid and attenuates the peroxynitrite oxidative impact. In this study, we demonstrate the existence and antioxidant characteristic of the LIP and peroxynitrite reaction in various cell types. Beyond its potential role as a ubiquitous complementary or substitute protection system against peroxynitrite for cells, the LIP and peroxynitrite reaction may influence cellular iron homeostasis and ferroptosis by changing the LIP redox state and LIP binding properties and reactivity.
Assuntos
Ferro , Oxirredução , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Ferro/metabolismo , Humanos , Ferroptose/efeitos dos fármacos , Animais , Peróxido de Hidrogênio/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacosRESUMO
Drug-induced liver injury (DILI) poses a severe threat to public health. Endoplasmic reticulum (ER) stress contributes significantly to DILI pathogenesis, with peroxynitrite (ONOO-) identified as a pivotal indicator. However, the temporal and spatial fluctuations of ONOO- associated with ER stress in the pathogenesis of DILI remain unclear. Herein, a novel ER-specific near-infrared (NIR) probe (QM-ONOO) with aggregation-induced emission (AIE) features for monitoring ONOO- fluctuations in DILI was elaborately constructed. QM-ONOO exhibited excellent ER-targeting specificity, a large Stoke's shift, and a low detection limit (26.9 nM) toward ONOO-. QM-ONOO performed well in imaging both exogenous and endogenous ONOO- in HepG2 cells. Furthermore, molecular docking calculations validated the ER-targeting mechanism of QM-ONOO. Most importantly, using this probe allowed us to intuitively observe the dynamic fluctuations of ONOO- during the formation and remediation processes of DILI in the acetaminophen (APAP)-induced mouse model. Consequently, this work provides a promising tool for in-depth research of ONOO- associated pathological processes in DILI.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Retículo Endoplasmático , Corantes Fluorescentes , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/química , Humanos , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Corantes Fluorescentes/química , Retículo Endoplasmático/metabolismo , Camundongos , Células Hep G2 , Acetaminofen/toxicidade , Acetaminofen/efeitos adversos , Técnicas Biossensoriais/métodos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Simulação de Acoplamento Molecular , Imagem Óptica/métodosRESUMO
T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Ácido Peroxinitroso , Linfócitos T Reguladores , Ácido Peroxinitroso/metabolismo , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Esclerose Múltipla/metabolismo , Esclerose Múltipla/imunologia , Camundongos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/imunologia , Humanos , Receptores de Interleucina-2/metabolismo , Feminino , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , Células Th17/imunologia , Células Th17/metabolismo , Masculino , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Hepatic fibrosis (HF) is caused by persistent inflammation, which is closely associated with hepatic oxidative stress. Peroxynitrite (ONOO-) is significantly elevated in HF, which would be regarded as a potential biomarker for the diagnosis of HF. Research has shown that ONOO- in the Golgi apparatus can be overproduced in HF, and it can induce hepatocyte injury by triggering Golgi oxidative stress. Meanwhile, the ONOO- inhibitors could effectively relieve HF by inhibiting Golgi ONOO-, but as yet, no Golgi-targetable fluorescent probe available for diagnosis and assessing treatment response of HF through sensing Golgi ONOO-. To this end, we reported a ratiometric fluorescent probe, Golgi-PER, for diagnosis and assessing treatment response of HF through monitoring the Golgi ONOO-. Golgi-PER displayed satisfactory sensitivity, low detection limit, and exceptional selectivity to ONOO-. Combined with excellent biocompatibility and good Golgi-targeting ability, Golgi-PER was further used for ratiometric monitoring the Golgi ONOO- fluctuations and screening of ONOO- inhibitors from polyphenols in living cells. Meanwhile, using Golgi-PER as a probe, the overexpression of Golgi ONOO- in HF and the treatment response of HF to the screened rosmarinic acid were precisely visualized for the first time. Furthermore, the screened RosA has a remarkable therapeutic effect on HF, which may be a new strategy for HF treatment. These results demonstrated the practicability of Golgi-PER for monitoring the occurrence, development, and personalized treatment response of HF.
Assuntos
Corantes Fluorescentes , Complexo de Golgi , Cirrose Hepática , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Corantes Fluorescentes/química , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/diagnóstico por imagem , Humanos , Complexo de Golgi/metabolismo , Células Hep G2 , Animais , Estresse Oxidativo/efeitos dos fármacos , Ácido Rosmarínico , Limite de DetecçãoRESUMO
Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.
Assuntos
Capsicum , Catalase , Frutas , Óxido Nítrico , Proteínas de Plantas , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Capsicum/enzimologia , Capsicum/metabolismo , Catalase/metabolismo , Catalase/genética , Frutas/crescimento & desenvolvimento , Frutas/genética , Frutas/metabolismo , Frutas/enzimologia , Frutas/efeitos dos fármacos , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Ácido Peroxinitroso/metabolismoRESUMO
A near-infrared fluorescent probe (TX-P) for detecting peroxynitrite is constructed. The probe has a near-infrared emission (725 nm), large Stokes shift (125 nm) and excellent sensitivity and selectivity. In addition, TX-P can be used to visualize ONOO- in living cells, image ONOO- in paw edema mice and evaluate anti-inflammatory drugs.
Assuntos
Edema , Corantes Fluorescentes , Ácido Peroxinitroso , Animais , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Camundongos , Edema/diagnóstico por imagem , Edema/tratamento farmacológico , Edema/induzido quimicamente , Raios Infravermelhos , Humanos , Imagem Óptica , Células RAW 264.7 , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/uso terapêuticoRESUMO
Peroxynitrite, a short-lived and reactive oxidant, emerges from the diffusion-controlled reaction between the superoxide radical and nitric oxide. Evidence shows that peroxynitrite is a critical mediator in physiological and pathological processes such as the immune response, inflammation, cancer, neurodegeneration, vascular dysfunction, and aging. The biochemistry of peroxynitrite is multifaceted, involving one- or two-electron oxidations and nitration reactions. This minireview highlights recent findings of peroxynitrite acting as a metabolic mediator in processes ranging from oxidative killing to redox signaling. Selected examples of nitrated proteins (i.e., 3-nitrotyrosine) are surveyed to underscore the role of this post-translational modification on cell homeostasis. While accumulated evidence shows that large amounts of peroxynitrite participates of broad oxidation and nitration events in invading pathogens and host tissues, a closer look supports that low to moderate levels selectively trigger signal transduction cascades. Peroxynitrite probes and redox-based pharmacology are instrumental to further understand the biological actions of this reactive metabolite.
Assuntos
Oxirredução , Ácido Peroxinitroso , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/química , Humanos , Animais , Transdução de SinaisRESUMO
Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.
Assuntos
Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Ácido Peroxinitroso , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Mycobacterium tuberculosis/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Over-consumption of drugs can result in drug-induced liver damage (DILI), which can worsen liver failure. Numerous studies have shown the significant role ferroptosis plays in the pathophysiology of DILI, which is typified by a marked imbalance between the generation and breakdown of lipid reactive oxygen species (ROS). The content of peroxynitrite (ONOO-) rapidly increased during this process and was thought to be a significant marker of early liver injury. Therefore, the construction of fluorescence probe for the detection and imaging of ONOO- holds immense importance in the early diagnosis and treatment of ferroptosis-mediated DILI. RESULTS: We designed a probe DILI-ONOO based on the ICT mechanism for the purpose of measuring and visualizing ONOO- in ferroptosis-mediated DILI processes and associated studies. This probe exhibited significant fluorescence changes with good sensitivity, selectivity, and can image exogenous and endogenous ONOO- in cells with low cytotoxicity. Using this probe, we were able to show changes in ONOO- content in ferroptosis-mediated DILI cells and mice models induced by the intervention of acetaminophen (APAP) and isoniazid (INH). By measuring the concentration of ferroptosis-related indicators in mice liver tissue, we were able to validate the role of ferroptosis in DILI. It is worth mentioning that compared to existing alanine transaminase (ALT) and aspartate aminotransferase (AST) detection methods, this probe can achieve early identification of DILI prior to serious liver injury. SIGNIFICANCE: This work has significant reference value in researching the relationship between ferroptosis and DILI and visualizing research. The results indicate a strong correlation between the progression of DILI and ferroptosis. Additionally, the use of DILI-ONOO shows promise in investigating the DILI process and assessing the effectiveness of medications in treating DILI.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Ferroptose , Corantes Fluorescentes , Ácido Peroxinitroso , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Ferroptose/efeitos dos fármacos , Animais , Ácido Peroxinitroso/metabolismo , Camundongos , Corantes Fluorescentes/química , Humanos , Acetaminofen/toxicidade , Imagem Óptica , Camundongos Endogâmicos C57BL , Masculino , Isoniazida/química , Raios InfravermelhosRESUMO
BACKGROUND: Acute kidney injury (AKI) poses a severe risk to public health, mostly manifested by damage and death of renal tubular epithelial cells. However, routine blood examination, a conventional approach for clinical detection of AKI, is not available for identifying early-stage AKI. Plenty of reported methods were lack of early biomarkers and real time evaluation tools, which resulted in a vital challenge for early diagnosis of AKI. Therefore, developing novel probes for early detection and assessment of AKI is exceedingly crucial. RESULTS: Based on ESIPT mechanism, a new fluorescent probe (MEO-NO) with 2-(2'-hydroxyphenyl) benzothiazole (HBT) derivatives as fluorophore has been synthesized for dynamic imaging peroxynitrite (ONOO-) levels in ferroptosis-mediated AKI. Upon the addition of ONOO-, MEO-NO exhibited obvious fluorescence changes, a significant Stokes shift (130 nm) and rapid response (approximately 45 s), and featured exceptional sensitivity (LOD = 7.28 nM) as well as high selectivity from the competitive species at physiological pH. In addition, MEO-NO was conducive to the biological depth imaging ONOO- in cells, zebrafish, and mice. Importantly, MEO-NO could monitor ONOO- levels during sorafenib-induced ferroptosis and CP-induced AKI. With the assistance of MEO-NO, we successfully visualized and tracked ONOO- variations for early detection and assessment of ferroptosis-mediated AKI in cells, zebrafish and mice models. SIGNIFICANCE AND NOVELTY: Benefiting from the superior performance of MEO-NO, experimental results further demonstrated that the levels of ONOO- was overexpressed during ferroptosis-mediated AKI in cells, zebrafish, and mice models. The developed novel probe MEO-NO provided a strong visualization tool for imagining ONOO-, which might be a potential method for the prevention, diagnosis, and treatment of ferroptosis-mediated AKI.
Assuntos
Injúria Renal Aguda , Ferroptose , Corantes Fluorescentes , Ácido Peroxinitroso , Peixe-Zebra , Ferroptose/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Ácido Peroxinitroso/metabolismo , Injúria Renal Aguda/induzido quimicamente , Animais , Camundongos , Humanos , Imagem Óptica , Estrutura Molecular , Diagnóstico PrecoceRESUMO
Ferroptosis is a type of lipid peroxidation-induced apoptosis brought on by imbalances in iron metabolism and redox. It involves both the thiol-associated anti-ferroptosis pathway and the excessive buildup of reactive oxygen species (ROS), which stimulates the ferroptosis pathway. Determining the precise control mechanism of ferroptosis requires examining the dynamic connection between reactive sulfur species (RSS) and ROS. Cysteine (Cys) and peroxynitrite (ONOO-) are highly active redox species in organisms and play dynamic roles in the ferroptosis process. In this study, a coumarin dye was conjugated with specific response sites for Cys and ONOO-, enabling the simultaneous detection of Cys and ONOO- through the green and red fluorescence channels, respectively (λem = 498 nm for Cys and λem = 565 nm for ONOO-). Using the probe LXB, we monitored the changes in Cys and ONOO- levels in the ferroptosis pathway induced by erastin. The results demonstrate a significant generation of ONOO- and a noticeable decrease in intracellular Cys levels at the beginning upon erastin treatment and finally maintains a relatively low level. This study presents the first probe to investigate the intracellular redox modulation and control between Cys and ONOO- during ferroptosis, providing valuable insights into the potential mutual correlation between Cys and ONOO- in this process.
Assuntos
Cisteína , Ferroptose , Corantes Fluorescentes , Ácido Peroxinitroso , Ferroptose/efeitos dos fármacos , Corantes Fluorescentes/química , Cisteína/metabolismo , Cisteína/análise , Humanos , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/metabolismo , Espectrometria de Fluorescência , Oxirredução , Piperazinas/farmacologia , Piperazinas/química , Cumarínicos/química , Cumarínicos/farmacologiaRESUMO
Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.
Assuntos
Corantes Fluorescentes , Estresse Oxidativo , Ácido Peroxinitroso , Superóxidos , Células PC12 , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/metabolismo , Animais , Ratos , Estresse Oxidativo/efeitos dos fármacos , Corantes Fluorescentes/química , Superóxidos/metabolismo , Superóxidos/análise , Imagem ÓpticaRESUMO
Peroxynitrite (ONOO-) and cell plasma membrane (CPM) are two key factors in cell pyroptosis during the progression of abdominal aortic aneurysm (AAA). However, their combined temporal and spatial roles in initiating AAA pathogenesis remain unclear. Herein, we developed a two-photon fluorescence probe, BH-Vis, enabling real-time dynamic detection of CPM and ONOO- changes, and revealing their interplay in AAA. BH-Vis precisely targets CPM with reduced red fluorescence intensity correlating with diminished CPM tension. Concurrently, a blue shift of the fluorescence signal of BH-Vis occurs in response to ONOO- offering a reliable ratiometric detection mode with enhanced accuracy by minimizing external testing variables. More importantly, two photon confocal imaging with palmitic acid (PA) and ganglioside (GM1) manipulation, which modulating cell pyroptosis, showcases reliable fluorescence fluctuations. This groundbreaking application of BH-Vis in a mouse AAA model demonstrates its significant potential for accurately identifying cell pyroptosis levels during AAA development.
Assuntos
Aneurisma da Aorta Abdominal , Membrana Celular , Imagem Óptica , Ácido Peroxinitroso , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/patologia , Ácido Peroxinitroso/metabolismo , Animais , Camundongos , Membrana Celular/metabolismo , Membrana Celular/química , Humanos , Corantes Fluorescentes/química , Piroptose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Masculino , FótonsRESUMO
KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.