Intracellular peroxynitrite perturbs redox balance, bioenergetics, and Fe-S cluster homeostasis in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (•NO) and superoxide (O2•-) produced by phagocytes contributes to its success as a human pathogen. Recombination of •NO and O2•- generates peroxynitrite (ONOO-), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward •NO and O2•- 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 •NO and O2•-, 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).

Endoplasmic reticulum-targeted fluorescent probe with aggregation-induced emission features for imaging peroxynitrite in drug-induced liver injury model.

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.

Eicosapentaenoic Acid Improves Endothelial Nitric Oxide Bioavailability Via Changes in Protein Expression During Inflammation.

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.

Red-Light-Photosensitized Tyrosine 10 Nitration of ß-Amyloid1-42 Diverts the Protein from Forming Toxic Aggregates.

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.

Cytomegalovirus-induced peroxynitrite promotes virus entry and contributes to pathogenesis in a murine model of infection.

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.

The Ubiquity of the Reaction of the Labile Iron Pool That Attenuates Peroxynitrite-Dependent Oxidation Intracellularly.

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.

Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

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.

A near-infrared fluorescent probe for imaging peroxynitrite levels in paw edema mice and drug evaluation.

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.

Visulization of peroxynitrite variation for accurate diagnosis and assessing treatment response of hepatic fibrosis using a Golgi-targetable ratiometric fluorescent probe.

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.

Peroxynitrite reduces Treg cell expansion and function by mediating IL-2R nitration and aggravates multiple sclerosis pathogenesis.

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.

A novel ESIPT fluorescent probe for early detection and assessment of ferroptosis-mediated acute kidney injury via peroxynitrite fluctuation.

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.

Peroxynitrite imaging in ferroptosis-mediated drug-induced liver injury with a near-infrared fluorescence probe.

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.

Peroxynitrite: a multifaceted oxidizing and nitrating metabolite.

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.

Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

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.

Multifunctional fluorescent probe for simultaneous revealing Cys and ONOO- dynamic correlation in the ferroptosis.

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.

Visualizing the crucial roles of plasma membrane and peroxynitrite during abdominal aortic aneurysm using two-photon fluorescence imaging.

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.

A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

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.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

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.

Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation.

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits ß5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and ß5. Peroxynitrite induced a dose-dependent decrease in PAC1 and ß5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

Development of bishydrazide-based fluorescent probes for the imaging of cellular peroxynitrite (ONOO-) during ferroptosis.

Peroxynitrite (ONOO-) is a critical ROS in living systems, and could induce lipid peroxidation which is the driver of ferroptotic cell death. Therefore, precise and rapid detection of cellular ONOO- is critical for the deep study of the biological functions of ONOO- during ferroptosis. Herein, we developed fluorescent probes (Rh-1, Rh-2 and Rh-3) for the rapid detection of intracellular ONOO- during ferroptosis. These probes used bishydrazide groups as the reactive sites for ONOO-. The response of these probes to ONOO- resulted in the production of the emissive xanthene fluorophore, providing a marked enhancement in the fluorescence intensity at 561 nm. The probe Rh-3 exhibited prominent selectivity and sensitivity towards ONOO-. Bioimaging experiments suggested that Rh-3 could be applied to image exogenous and endogenous ONOO- in living cells. By fluorescence imaging, it was demonstrated that erastin-induced ferroptosis caused increased levels of the endogenous ONOO-, and ferrostatin-1 (Fer-1) and vitamin E (VE) could markedly inhibit the excessive production of ONOO- during ferroptosis in living cells.