Free Radical Biology and Medicine

The role of oxidative stress and the use of biochemical biomarkers in the severity of COVID-19 was evaluated through a literature review (2020-2021) using scientific search engines such as PubMed, Science Direct, and Google Scholar. The search was limited to articles published in Spanish or English that reported on COVID-19 and its relationship with oxidative stress, following PRISMA-2020 guidelines. The search terms included oxidative stress, COVID-19, SARS-CoV-2, oxidative biomarkers, and oxidative damage. 93.5% of the selected studies were from the year 2021. These studies evaluated both oxidative stress biomarkers and oxidative damage biomarkers in COVID-19 patients. The reviewed studies reinforce the strong association of SARS-CoV-2 with oxidative stress and demonstrate how SARS-CoV-2-induced ROS production and disruption of the antioxidant defense system trigger a pro-inflammatory environment and cause severe tissue damage. In 64.7% of the studies, a combination of oxidative stress biomarkers (antioxidant and oxidative damage biomarkers) was used to assess COVID-19 severity. The most commonly used antioxidant biomarkers were thiols and total antioxidant capacity, followed by glutathione. The most commonly used oxidative damage biomarkers were malondialdehyde and peroxides, followed by advanced oxidation protein products. COVID-19 leads to a decrease in the antioxidant defense system, reflected by a decrease in antioxidant biomarkers and an increase in oxidative damage biomarkers.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO{middle dot} bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required. HighlightsO_LITranscriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathways C_LIO_LIDisturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophy C_LIO_LIDUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex I C_LIO_LIFSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubes C_LIO_LIHypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidants C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/459509v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@16ae1a8org.highwire.dtl.DTLVardef@517caorg.highwire.dtl.DTLVardef@5d0734org.highwire.dtl.DTLVardef@183ef50_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDoxorubicin (DOX) remains a cornerstone in the treatment of various cancers. However, its clinical utilization is significantly hampered by dose-dependent cardiotoxicity. The generation of reactive oxygen species (ROS) constitutes the central component of the pathogenesis of DOX-induced cardiotoxicity. Activating transcription factor 4 (ATF4) has been demonstrated to exert a cardioprotective effect and augment cardiac antioxidative capacity in settings of heart failure. However, the role of ATF4 in DOX-induced cardiomyopathy (DIC) remains unknown. MethodsTo explore the role of ATF4 in DOX-induced cardiomyopathy, cardiac-specific ATF4 conditional heterozygous mice and AAV9 mediated ATF4 overexpression mouse models were utilized. Cardiac function was assessed by echocardiography. The upstream regulator and downstream mediator of ATF4 were evaluated using RNA-seq analysis and further verified using ChIP assay and luciferase reporter assay. ResultsWe found a substantial decrease in ATF4 expression levels in the heart of DIC mice. ATF4+/- mice exhibited a higher degree of susceptibility to DOX-induced cardiotoxicity in comparison with ATF4flox/flox mice, as evidenced by the manifestation of more severe cardiac dysfunction and a significantly earlier mortality rate. In contrast, cardiacc-specific overexpression ATF4 by AAV9 confers robust cardioprotection against DOX-induced cardiomyopathy. Mechanistically, we identified the upstream regulator of ATF4 as KLF16, which was significantly suppressed during DOX treatment. Further, the decrease of ATF4 led to a reduction in cystathionine {gamma}-lyase (CSE) transcription and hydrogen sulfide (H2S) production in the context of DOX-induced cardiotoxicity. ChIP and luciferase reporter assays revealed that ATF4 functioned as the transcription factor of the CSE gene, which is a key enzyme in the synthesis of H2S to counteract oxidative stress. Consistently, ROS scavengers or H2S donors was shown to mitigate the consequences of ATF4 deficiency. In contrast, the ectopic expression of ATF4 mitigated oxidative stress and apoptosis in DOX-induced cardiotoxicity, both in vivo and in vitro. ConclusionsOur study revealed a novel function of ATF4 in counteracting oxidative stress in DOX cardiotoxicity by promoting the transcription of CSE. ATF4 may represent a promising therapeutic target for the treatment of DOX-induced cardiomyopathy.

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where ~2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining a greater insight into oxidative stress regulation and pathways/processes being perturbed in an oxidizing environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/504013v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1917f1org.highwire.dtl.DTLVardef@1730cd0org.highwire.dtl.DTLVardef@4e4820org.highwire.dtl.DTLVardef@1615f4c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep redox profiling workflow results in stoichiometric quantification of thiol oxidation for > 18,000 Cys sites in muscle C_LIO_LIThiol redox changes were much more pronounced than protein abundance changes for the overlapping set of proteins C_LIO_LIRedox changes are most significant in coagulation and immune response pathways while abundance changes on bioenergetics pathways C_LI

The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase. C_FIG

Decreased ryanodine receptor type 1 (RyR1) protein is a hallmark of recessive RYR1-related myopathies (RyR1-RM), which are caused by recessive mutations in the RYR1 gene. It is not clear how the decrease in the RyR1 protein triggers muscular disorders. Furthermore, it is a hot topic whether a decrease in RyR1 protein levels can also occur during non-RYR1-related myopathies. In this study, we first show that reduced RYR1 transcripts are associated with various human myopathies, and that RyR1 protein levels are significantly decreased in muscle samples analysed in inflammatory myopathies (IM) and mitochondrial myopathies (MM), both of which are non-RYR1-RM. Secondly, proteomic data show that exclusive depletion of RyR1 protein in vitro recapitulates the common altered molecular pathways observed during myopathies. RyR1 protein depletion impairs ER-mitochondria tethering and Ca2+ transfer to mitochondria, decreases mitophagy genes and induces an accumulation of dysfunctional mitochondria. This phenomenon is also associated with altered lipid homeostasis with an increase in deleterious sphingolipid species. Finally, decreased RyR1 protein levels lead to an increase in the ER stress markers GRP78-Bip and CHOP in muscle cell in vitro, and in mouse and human muscles. Overall, our results indicate an important role of RyR1 protein depletion and ER stress in the pathogenesis of myopathies.

HIGHLIGHTSO_LID-PUFA diet prevents muscle atrophy in STZ-induced diabetic mice. C_LIO_LID-PUFA diet prevents muscle weakness depending on increased calcium release in STZ-induced diabetic mice. C_LIO_LID-PUFA diet may show a trend to decrease blood glucose in STZ-induced diabetic mice. C_LIO_LID-PUFA diet does not alter ferroptosis-related protein profiles including ACSL4, LPCAT3, ALOX12, and Gpx4. C_LI Oxidative stress and reactive oxygen species (ROS) have been linked to muscle atrophy and weakness. Diabetes increases the oxidative status of lipoproteins in nearly all tissues, including muscle tissues, but the role of lipid ROS on diabetes-induced muscle atrophy is not fully understood. Deuterium reinforced polyunsaturated fatty acids (D-PUFA) are more resistant to ROS-initiated chain reaction of lipid peroxidation than regular hydrogenated PUFA (H-PUFA). In this study, we tested the hypothesis that D-PUFA would protect muscle atrophy induced by diabetes driven by an accumulation of lipid hydroperoxides (LOOH). C57BL/6J mice were dosed with H-PUFA or D-PUFA for four weeks through dietary supplementation and then injected with streptozotocin (STZ) to induce insulin-deficient diabetes. After two weeks, muscles tissues were analyzed for individual muscle mass, force generating capacity and cross-sectional area. Skeletal muscle fibers from diabetic mice exhibited increased total ROS and LOOH. This was abolished by the D-PUFA supplementation regardless of accumulated iron. D-PUFA were found to be protective against muscle atrophy and weakness from STZ-induced diabetes. Prevention of muscle atrophy and weakness by D-PUFA might be independent of ACSL4/LPCAT3/15-LOX pathway. These findings provide novel insights into the role of LOOH in the mechanistic link between oxidative stress and diabetic myopathy and suggest a novel therapeutic approach to diabetes-associated muscle weakness.

T lymphocytes are key components in adaptive immunity and their activation naturally involves mitochondrial-derived oxygen species (mtROS). In particular, H2O2 has been implicated as an important signaling molecule regulating major T cell functions. H2O2 targets the oxidation status of functional cysteine residues but knowledge if and where this happens in T cell signaling networks is widely missing. This study aimed to identify mtROS-sensitive processes in activated primary human CD4+ T cells. By using a thiol-specific redox proteomic approach we examined the oxidation state of 4784 cysteine-containing peptides of ex vivo stimulated T cells from healthy individuals. Upon activation, a shift in oxidation was observed at catalytic cysteine residues of peroxiredoxins (PRDX5 & PRDX6), and T cells were found to maintain their global thiol-redox homeostasis. In parallel, a distinct set of 88 cysteine residues were found to be differentially oxidized upon T cell activation suggesting novel functional thiol switches. In mitochondria, cysteine oxidations selectively modified regulators of respiration (NDUFA2, NDUFA8, and UQCRH) confirming electron leakage from electron transport complexes I and III. The majority of oxidations occurred outside mitochondria and enriched sensitive thiols at regulators of cytoskeleton dynamics (e.g. CYFIP2 and ARPC1B) and known immune functions including the non-receptor tyrosine phosphatase PTPN7. Conversely, cysteine reduction occurred predominantly at transcriptional regulators and sites that coordinate zinc-binding in zinc-finger motifs. Indeed, fluorescence microscopy revealed a colocalization of zinc-rich microenvironments and mitochondria in T cells suggesting mtROS-dependent zinc-release of identified transcriptional regulators including ZFP36, RPL37A and CRIP2. In conclusion, this study complements knowledge on the mtROS signaling network and suggests zinc-dependent thiol switches as a mechanism of how mtROS affects transcription and translation in T cells.

Palmitic acid is the most abundant saturated fatty acid in human serum. In cell culture systems, palmitate overload is considered a toxic stimulus, and promotes lipid accumulation, insulin resistance, endoplasmic reticulum stress, oxidative stress, as well as cell death. An increased supply of fatty acids has also been shown to change the predominant form of the mitochondrial network, although the metabolic effects of this change are still unclear. Here, we aimed to uncover the early bioenergetic outcomes of lipotoxicity. We incubated hepatic PLC/PRF/5 cells with palmitate conjugated to BSA and followed real-time oxygen consumption and extracellular acidification for 6 hours. Palmitate increased glycolysis as soon as 1 hour after the stimulus, while oxygen consumption was not disturbed, despite overt mitochondrial fragmentation and cellular reductive imbalance. Palmitate only induced mitochondrial fragmentation if glucose and glutamine were available, while glycolytic enhancement did not require glutamine, showing it is not dependent on morphological changes. NAD(P)H levels were significantly abrogated in palmitate-treated cells. Knockdown of the mitochondrial NAD(P) transhydrogenase or addition of the mitochondrial oxidant-generator menadione in control cells modulated ATP production from glycolysis. Indeed, using selective inhibitors, we found that the production of superoxide/hydrogen peroxide at the IQ site of electron transport chain complex I is associated with the metabolic rewiring promoted by palmitate, while not changing mitochondrial oxygen consumption. In conclusion, we demonstrate that increased glycolytic flux linked to mitochondrially-generated redox imbalance is an early bioenergetic result of palmitate overload and lipotoxicity.

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O {middle dot}- and H O and nitrite ({middle dot}NO -) reductase activity leading to NO. Since these products express important, and often diametrically opposite, biological activity consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ({middle dot}NO) formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2{middle dot}- and NO generation compared to the WT, whilst the Ile703Val increases UA and {middle dot}NO formation, but not O2{middle dot}-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also suggest that targeting enzyme activity to enhance the NO2--reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease. HighlightsO_LIMutations of xanthine oxidoreductase modulate both its expression and activity C_LIO_LIThe His1221Arg natural mutation increases xanthine oxidoreductase activity C_LIO_LIRaised xanthine oxidoreductase activity coupled with increased availability of nitrite substrate leads to increased NO provision C_LI

YbbN/CnoX are proteins that display a Trx domain linked to a tetratricopeptide (TPR) domain, which are involved in protein-protein interactions and protein folding processes. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl (bleach). EcYbbN contains a SQHC motif within the Trx domain and displays no thiol-disulfide oxidoreductase activity. EcYbbN also presents a second Cys residue at Trx domain (Cys63) 24 residues away from SQHF motif that can form mixed disulfides with substrates. Here, we compared EcYbbN with two other YbbN proteins: from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). While EcYbbN displays two Cys residues along a SQHC[N24]C motif; XfYbbN and PaYbbN present two and three Cys residues in the CAPC[N24]V and CAPC[N24]C motifs, respectively. These three proteins are representatives of evolutionary conserved YbbN subfamilies. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (5,5'-dithiobis-(2-nitrobenzoic acid) = DTNB); and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that in vivo these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km=1.27 x 107 M-1.s-1), like the canonical XfTrx (XfTsnC). Furthermore, EcYbbN (as described before) and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function when the SQHC was replaced by CQHC. In contrast, the XfYbbN C40A mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. Finally, we generated a P. aeruginosa strain with the ybbN gene deleted, which did not present increased sensitivity to heat shock or to oxidants or to reductants. Altogether, our results suggest that different YbbN/CnoX proteins display distinct properties and activities, depending on the presence of the three conserved Cys residues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/034579v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@6970f0org.highwire.dtl.DTLVardef@75da52org.highwire.dtl.DTLVardef@15067c9org.highwire.dtl.DTLVardef@1cdd97d_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- CXXC motif is required for the thiol-disulfide reductase activity of YbbN proteins. - XfYbbN and PaYbbN display thiol-disulfide oxidoreductase activity - The affinities of XfTrxR for XfYbbN and XfTsnC (canonical Trx) are comparable - XfYbbN and EcYbbN, but not PaYbbN, display holdase activity induced by hypochlorous acid - Engineering EcYbbN/CnoX by inserting a Cys residue in the SQHC motif resulted in a gain of function (thiol-disulfide oxidoreductase activity) and abolished the HOCl-induced holdase activity.

Hydrogen peroxide (H2O2) and lipid hydroperoxides (LOOH) are initiators and transducers of inter- and intra-cellular signaling in response to diverse environmental, pathological and developmental cues. The accumulation of both H2O2 and LOOH is often temporally and spatially coincident in tissues, but it is unknown if this coincidence extends to subcellular compartments. If distinct accumulation of different peroxides occurs at this smaller spatial scale, then it would be an important factor in signaling specificity. Fusion of the redox-sensitive (ro)GFP2 to the Saccharomyces cerevisiae (yeast) OXIDANT RECEPTOR PEROXIDASE1 (ORP1), also known as GLUTATHIONE PEROXIDASE3 (GPX3), created a now widely used biosensor that is assumed to detect H2O2 in vivo. This is despite monomeric GPX enzymes, such as ORP1/GPX3, possessing wide peroxide substrate specificities. Consequently, we confirmed in vitro that roGFP2-ORP1 is not only oxidized by H2O2, but also by phospholipid fatty acid peroxides generated in lecithin-derived liposomes by lipoxygenase-catalyzed peroxidation. This led us to doubt that roGFP2-ORP1 in vivo is specific for H2O2. To address this issue of peroxide specificity, we constructed a modified biosensor called roGFP2-synORP1. This version has greatly diminished reactivity towards phospholipid fatty acid peroxides but retains high sensitivity for H2O2. These two roGFP2-based biosensors, targeted to chloroplasts, cytosol and the nucleus, were quantitatively imaged in parallel in Nicotiana benthamiana abaxial epidermal cells experiencing high light- and herbicide-induced photo-oxidative stress. From differential patterns of oxidation of these probes, we inferred that the chloroplasts accumulated both peroxide types. In contrast, LOOH and H2O2 accumulated exclusively in the cytosol and nucleus respectively. Therefore, this suggests that the signalling networks initiated by different peroxides will have a distinct spatial component.

Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme important for the reduction of toxic lipid peroxide products. Previous studies revealed the importance of mouse Gpx4 in protecting cardiomyocytes from ferroptosis and, subsequently, the development of cardiovascular disease. In this paper, we investigate the transcriptional consequences of cardiac-specific deletion of Gpx4 in mice and compare this response with that observed in human cardiomyopathy. The findings in this study highlight the importance of GPX4 in maintaining both structural and functional stability of the heart and identify key pathway changes resulting from excessive ferroptosis in cardiac tissue. By overlapping common transcriptional programs perturbed in this animal model and human cardiomyopathy, our findings identify putative mechanisms through which ferroptosis contributes to the development and progression of heart disease. These studies may help guide future cardiovascular therapeutics targeting ferroptosis-dependent pathways.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS due to a lack of disease models. Here, we studied oxidative the stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations. mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided in 46 years-old-men (C1), 46 years-old-women (C2) or 26/27 years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA). Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect separation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA analysis. Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1, and healthy controls that can be used as therapeutic targets for ALS drug development. Highlights Lymphoblasts from ALS patients present altered redox properties Redox profiling evidenced total SOD and glutathione reductase activities, SOD1 protein levels, DCF fluorescence, catalase and Nrf2 transcripts, and tert-butyl hydroperoxide cytotoxicity, as discriminant features for experimental groups. Lymphoblast redox profiles may be helpful for patient stratification and precision medicine

Hydrogen peroxide (H2O2) functions as a secondary messenger in cellular redox signaling, acting mainly via oxidation of protein thiols. Its spatially and temporally regulated activity within cells is essential for maintaining proper redox balance, and disruptions in these patterns can lead to oxidative stress and various related pathologies. Redox proteomics, which examines the impact of H2O2 at the proteome level, typically focuses only on thiol oxidation, overlooking broader proteomic alterations and the significance of subcellular localization in these redox processes. In this study, we address these open questions by combining chemogenetics with Thermal Proteome Profiling (TPP) to map global proteome response to compartmentalized H2O2 production. We identified hundreds of proteins with altered thermostability and/or abundance upon localized H2O2 generation in the cytosol, nucleus, and the ER lumen, highlighting their roles in cellular responses to localized H2O2. We identified proteins such as MAP2K1, PARK7, TRAP1, and UBA2 to be highly sensitive to localized H2O2 production. Furthermore, we validated their altered thermostability and found that these changes are controlled via dysregulated protein-protein interactions. This study provides a valuable resource for researchers exploring redox-mediated signal transduction and offers novel insights that could be harnessed in treating oxidative stress-induced pathologies.

Mitochondrial creatine kinase (mtCK) regulates the "fast" export of phosphocreatine to support cytoplasmic phosphorylation of ADP to ATP which is more rapid than direct ATP export. Such "creatine-dependent" phosphate shuttling is attenuated in several muscles, including the heart, of the D2.mdx mouse model of Duchenne muscular dystrophy at only 4 weeks of age. However, the degree to which creatine-dependent and -independent systems of phosphate shuttling progressively worsen or potentially adapt in a hormetic manner throughout disease progression remains unknown. Here, we performed a series of proof-of-principle investigations designed to determine how phosphate shuttling pathways worsen or adapt in later disease stages in D2.mdx (12 months of age). We also determined whether changes in creatine-dependent phosphate shuttling are linked to alterations in mtCK thiol redox state. In permeabilized muscle fibres prepared from cardiac left ventricles, we found that 12-month-old male D2.mdx mice have reduced creatine-dependent pyruvate oxidation and elevated complex I-supported H2O2 emission (mH2O2). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mH2O2 was lowered suggesting that impairments in the faster mtCK-mediated phosphocreatine export system resulted in compensation of the alternative slower pathway of ATP export. The apparent impairments in mtCK-dependent bioenergetics occurred independent of mtCK protein content but were related to greater thiol oxidation of mtCK and a more oxidized cellular environment (lower GSH:GSSG). Next, we performed a proof-of-principle study to determine whether creatine-dependent bioenergetics could be enhanced through chronic administration of the mitochondrial-targeting, ROS-lowering tetrapeptide, SBT-20. We found that 12 weeks of daily treatment with SBT-20 (from day 4 to [~]12 weeks of age) increased respiration and lowered mH2O2 only in the presence of creatine in D2.mdx mice without affecting calcium-induced mitochondrial permeability transition activity. In summary, creatine-dependent mitochondrial bioenergetics are attenuated in older D2.mdx mice in relation to mtCK thiol oxidation that seem to be countered by increased creatine-independent phosphate shuttling as a unique form of mitohormesis. Separate results demonstrate that creatine-dependent bioenergetics can also be enhanced with a ROS-lowering mitochondrial-targeting peptide. These results demonstrate a specific relationship between redox stress and mitochondrial hormetic reprogramming during dystrophin deficiency with proof-of-principle evidence that creatine-dependent bioenergetics could be modified with mitochondrial-targeting small peptide therapeutics.

Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. The up-regulated APRT found in wound skin correlated with the demands of repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. LC-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. These data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. Our study provided an explanation for the up- regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation.

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in tafazzin and characterized by loss of cardiolipin and severe cardiomyopathy. Mitochondrial superoxide/H2O2 production has been implicated in the cardiomyopathy observed in different BTHS models. There are at least 11 mitochondrial sites that produce superoxide/H2O2 at significant rates. Which of these sites generate oxidants at excessive rates in BTHS is unknown. Here, we measured the maximum capacity of superoxide/H2O2 production from each site in mitochondria isolated from heart and skeletal muscle of the tafazzin knockdown mice (tazkd) at 3, 7 and 12 months of age. Strikingly, the superoxide/H2O2 production capacities of these sites were overall indistinguishable between tazkd mice and their wildtype littermates across the time points analyzed. The only exception was site GQ in glycerol phosphate dehydrogenase, which was increased in the skeletal muscle of 7 months old tazkd mice. Mitochondrial superoxide/H2O2 production was also measured ex vivo during the oxidation of a complex mixture of substrates mimicking either heart or skeletal muscle cytosol and was found to be indistinguishable between wildtype and tazkd mice. However, we consistently measured decreased FAD-linked respiration in mitochondria isolated from tazkd mice. We conclude that the maximum capacity and ex vivo rates of superoxide/H2O2 production were not increased in mitochondria isolated from heart and skeletal muscle of tazkd mice, despite reduced oxidative capacity. Therefore, it seems unlikely that mitochondrial oxidants contribute to the development of cardiomyopathy in tazkd mice. These observations raise questions about the involvement of mitochondrial oxidants in BTHS pathology.

Redox homeostasis is an integral part of many cellular processes, and its perturbation is associated with conditions such as diabetes, aging, and neurodegenerative disorders. Redox homeostasis or redox potential in organelles is maintained within a particular range to facilitate the organelle specific cellular redox reactions. Previous studies using yeast, cell systems, and nematodes have demonstrated that the Endoplasmic Reticulum (ER) has a more oxidizing environment while the cytosol exhibits a reducing redox potential. However, we know very little about how universal this phenomenon is. We created transgenic zebrafish (Danio rerio) lines with roGFP sensors targeted to the ER and cytosol for studying physiological redox potential at the systems level. In the process, we also characterized the ER-targeting signal sequence in D. rerio for the first time. Measurements of the redox state in live embryos found that the endoplasmic reticulum exhibits deviations from its expected oxidizing redox state in different regions of the developing embryos. The ER is far more reducing than expected in certain tissues of the embryo. Cytosol also exhibited unexpected redox states in some parts of the embryo. Notably, the brain showed regions with unexpected redox states in both the ER and the cytosol. Tissue-specific differences in ER-redox potential became even more evident in a transgenic line expressing the more sensitive roGFPiE variant. Thus, live monitoring of redox potential across the developing zebrafish embryos revealed unanticipated redox states of the ER that will require new biological definitions.

Myocardial infarction (MI) is a serious cardiovascular problem that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic-reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation-reduction states that have been a subject of attention in the food industry, specifically for meat consumers. However, rarely if ever, have the myoglobin optical properties been used to understand the pathology of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation-reduction states of myoglobin in the ischemic area of the myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it aligns with the spectral profile of myoglobin. Under ischemia-reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 hours post-reperfusion and decreases with cardioprotection. These results were correlated with MI measurements by Late Gadolinium Enhancement MRI. In conclusion, this seminal work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.