Redox Biology

Reactive oxygen species (ROS) are central signaling molecules in many biological processes by inducing oxidative modifications of protein cysteine residues, including S-glutathionylation. Increasing evidence supports that ROS contribute to cancer progression via promoting cancer cell migration, invasion, and metastasis. Nevertheless, the protein targets of S-glutathionylation that regulate cancer cell motility remain ill-defined. In this study, we report on the redox regulation of ARHGEF7, a guanine nucleotide exchange factor highly expressed in metastatic cancer cells, that plays a major role in regulating cell migration. Our data demonstrates that ARHGEF7 is selectively glutathionylated at the highly conserved C312 residue in its PH domain, which is implicated in regulating its enzymatic activity. Breast cancer cell lines showed increased cell migration and invasion upon glutathionylation of ARHGEF7 at C312 in response to both oxidative stress and epidermal growth factor (EGF). Mechanistically, upon C312 glutathionylation, ARHGEF7 exhibited significantly enhanced binding to Rac1 and increased Rac1 recruitment to the cell membrane and lamellipodia. ARHGEF7 S-glutathionylation also increased its enzymatic rate of GDP-GTP nucleotide exchange, resulting in Rac1 activation. Consequently, ARHGEF7 C312 S-glutathionylation induced Rac1-PAK1 activation and their downstream pathways, including LIMK1 and MEK1, thereby enhancing migration and invasion. Our data reveal a new redox player in cell migration, with its potential implications for ROS-induced cancer progression.

Organisms need to be able to adapt to a changing environment in order to survive. The adaptive response invoked by a low dose of a stressor resulting in resistance to high levels of that stressor is known as hormesis and can even lead to lifespan extension of organisms. The exact mechanisms underlying stress-induced hormesis are unknown, although multiple studies pose mitochondria-derived Reactive Oxygen Species (ROS, e.g. H2O2) as an important contributor. Here we used chemo-genetic H2O2 production as a model to study ROS-dependent adaptive responses in a localization-dependent manner. We found that brief, sublethal H2O2 production at the nucleosomes provides p53-dependent resistance to a subsequent high dose of H2O2, whereas mitochondrial H2O2 production, surprisingly, does not. A multi-omics approach revealed that p53-induced hormesis is accompanied by metabolic rewiring that boosts reductive capacity, and that the increased stress resistance can mostly be attributed to its downstream target p21. Importantly, brief p53 stabilization also mounted protection against chemotherapy-induced DNA damage, suggesting that p53-dependent hormesis could be exploited to selectively protect healthy, p53-wildtype tissue from chemotherapy in the treatment of patients with p53 mutant tumors.

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cytc accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cytc levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.

Organismal survival depends on coordinated responses to oxidative stress and DNA damage. Using Caenorhabditis elegans, we investigate mul-1, a robust transcriptional target of ionizing radiation and reactive oxygen species. Although annotated as a mucin, MUL-1 is a small ShKT domain-containing protein belonging to an invertebrate expanded family of cysteine-rich proteins. mul-1 is selectively induced by oxidative stress, including IR, hydrogen peroxide (H2O2), Pseudomonas aeruginosa infection, or loss of the peroxiredoxin PRDX-2, via the p38 MAPK-ATF-7 pathway in intestinal cells. Loss of mul-1 and its paralogs increases ROS accumulation, oxidative stress sensitivity, and CEP-1/p53 dependent germ cell apoptosis. Combined deletion of mul-1 paralogs causes constitutive apoptosis, reduced fecundity, and compensatory activation of DAF-16/Foxo and SKN-1/Nrf2 stress response pathways. Together with genetic analysis of SYSM-1, these findings suggest MUL-1-like ShKT proteins buffer oxidative stress.

Reactive oxygen species (ROS) are key mediators of disease, yet accurate characterization in living systems remains challenging because current probes lack oxidation specificity and produce nonlinear, pH-dependent signals. Here we introduce Rosindol, a novel thioacetal-based fluorogenic probe that overcomes these limitations. Rosindol undergoes an umpolung oxidation in the presence of ROS to generate fluorescence, displaying dose-linear responses to H2O2, O2*-, OH*, and HOCl with minimal background signal. Unlike conventional probes, Rosindol is pH-independent, photostable, water soluble, and agnostic to glucose concentration, esterase expression, and ambient oxygen. Validation in human cells--including PMA-stimulated neutrophils and SOD knockout models--confirms accurate detection of cytosolic and mitochondrial ROS. In pancreatic cancer cells, Rosindol reveals a fourfold increase in mitochondrial O2*- generation capacity via Complex I of the electron transport chain. Glucose stimulation induces twofold higher ROS generation in malignant cells, highlighting a connection between Warburg metabolism and the etiology of oxidative stress in pancreatic cancer. These studies illustrate the utility of Rosindol to provide valuable insight to oxidative stress processes in complex biological environments.

Cu/Zn superoxide dismutase (SOD1) is canonically regarded as a superoxide scavenging antioxidant yet is paradoxically associated with multiple diseases. Here, we show that SOD1 catalyzes thiol oxidation to thiosulfinates (RS(O)SR), revealing a previously unrecognized copper mediated oxidant forming reaction in biology. Steady state kinetics demonstrate robust, O2 dependent thiol consumption, and ATR FTIR spectra of the SOD1-cysteine reaction display S-O bands ({approx}1042/1169 cm-1) identical to cysteine thiosulfinate, including matching pseudo-first order decay in excess cysteine. SOD1 generated thiosulfinates are potent electrophiles and oxidants, supported by dimedone trapping and O-atom transfer to TCEP and horseradish peroxidase, with alkaline lability consistent with thiosulfinate hydrolysis rather than H2O2. Exogenous thiosulfinates phenocopy SOD1 mediated thiol oxidation, including GSH depletion, protein sulfenylation and cytotoxicity. SOD1 inhibition strongly suppresses cysteine and homocysteine induced toxicity, demonstrating that thiol driven oxidative stress requires SOD1 activity. SOD1 overexpression in human cells triggers oxidative stress and reduces proliferation, an effect that is absent in a copper-deficient mutant. N-acetylcysteine treatment further amplified this SOD1-dependent oxidative stress. At lower levels, nanomolar thiosulfinates elicit a hormetic proliferative response and rescue SOD1 deficient growth, identifying a pro-growth signaling function mediated by basal thiosulfinate formation. Kinetic modelling indicates that thiol-thiosulfinate turnover can match basal superoxide dismutation, indicating that thiosulfinate synthesis is a major catalytic output of SOD1. These findings identify SOD1 as a thiol oxidizing enzyme that generates thiosulfinates, establishing a core sulfur-based oxidation pathway and revealing that two classical "antioxidants", SOD1 and thiols, together generate potent oxidants that link thiol metabolism to both cytotoxic and growth promoting outcomes. Significance StatementAlthough SOD1 is classically defined as an antioxidant, its association with diverse oxidative-stress-driven diseases suggests additional chemistry at work. Here we identify thiosulfinate synthesis as a major catalytic output of SOD1, revealing that the enzyme is not merely a superoxide detoxifier but a thiol-oxidizing catalyst. This activity provides a unifying mechanism for two long-standing biological paradoxes: the unexplained toxicity of elevated thiols and the pro-growth, pro-survival signaling linked to basal SOD1 activity. By establishing thiosulfinates as a central oxidative currency in cells, this work reframes SOD1 as a bifunctional oxidase that shapes both stress responses and proliferative programs.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation, with specific lipid species playing key roles in modulating susceptibility. Among these, ether lipids have shown conflicting effects, being linked to both protection and sensitization. Here, we dissect the relationship between lipid structure and ferroptosis sensitivity and explain how ether lipids exert context-dependent effects. Ether lipids can promote ferroptosis through a metabolic bias towards the accumulation of polyunsaturated acyl chains and ethanolamine head groups, whereas this pro-ferroptotic tendency is counterbalanced by the anti-ferroptotic vinyl ether moiety introduced by plasmanylethanolamine desaturase 1. We show that this protective effect is critical for preventing ferroptosis in hiPSC-derived neurons, which accumulate otherwise pro-ferroptotic ether lipids during differentiation. This effect is not solely due to its antioxidant properties but also stems from the reprogramming of mitochondrial respiration. The lack of vinyl ether bonds leads to multiple mitochondrial defects, including increased mitochondrial reactive oxygen species (ROS), lower membrane potential, and abnormal cristae structures. These findings indicate that vinyl ether bonds in ether lipids offer dual ferroptosis resistance by scavenging ROS and minimizing its production at the mitochondrial level. The disruption of this system in Caenorhabditis elegans leads to iron-induced death and impaired motility. Thus, our study reveals ether lipid structural remodeling as a key regulator of ferroptosis sensitivity in neurons.

Coenzyme A (CoA) is an essential cofactor required for numerous metabolic reactions, yet its ability to bind and regulate proteins remains poorly defined. Using a proteomic approach, we identified malic enzyme 2 (ME2) as a CoA-binding protein. ME2 uses NAD(P)+ to convert malate to pyruvate, generating NAD(P)H to support energy production and redox homeostasis. ME2 binds CoA at an allosteric site previously thought to bind NAD(P)+. Reduced CoA has minimal effect on ME2 activity, but the oxidized form, CoA disulfide, strongly activates ME2 by promoting ME2 tetramerization and a catalytically efficient closed conformation. Under oxidative stress, ME2 facilitates CoA disulfide formation, enhancing NADPH production and cellular defense against reactive oxygen species (ROS). Mice with ME2 mutation that cannot bind CoA show impaired muscle performance, elevated ROS, and mitochondrial dysfunction. These findings establish CoA as a redox-sensing cofactor, allowing cells to respond to ROS and promote mitochondrial metabolism, and expanding the function of this essential cofactor.

Supersulfides are emerging sulfur-containing signaling molecules involved in redox regulation, mitochondrial function, and protein S-sulfhydration. However, their dynamic behavior in living mammalian systems remains poorly understood because existing analytical methods require destructive sample preparation or lack sufficient intracellular applicability. Here, we developed a genetically encoded supersulfide-dependent translocation reporter (SuTR) for mammalian cells and in vivo imaging. Although the previously reported probe psGFP failed to respond to supersulfides in mammalian cells, fusion of psGFP with the sulfide-responsive transcriptional repressor (SqrR) generated SuTR, a novel reporter that exhibited supersulfide-dependent translocation from the nucleus to the cytoplasm. Na2S2 and Na2S induced dose-dependent cytosolic translocation of SuTR, whereas Na2S showed no effect. Fluorescence recovery after photobleaching (FRAP) analysis revealed accelerated fluorescence recovery shortly after supersulfide stimulation, and overexpression of the endogenous supersulfide-producing enzyme Cysteinyl-tRNA Synthetase 2 (CARS2) similarly altered reporter dynamics. Mutational analyses demonstrated that reporter responsiveness depends on the DNA-binding activity of SqrR. Furthermore, SuTR successfully detected supersulfide induction in mouse liver in vivo following Na2S administration. These findings establish SuTR as a genetically encoded reporter for monitoring supersulfide dynamics in mammalian cells and tissues. HighlightsO_LIWe developed SuTR, a genetically encoded supersulfide-dependent translocation reporter. C_LIO_LISupersulfides induce nuclear-to-cytoplasmic translocation of SuTR C_LIO_LIFRAP enables rapid detection of endogenous and exogenous supersulfide responses C_LIO_LISuTR activity depends on the DNA-binding function of SqrR C_LIO_LISuTR enables visualization of supersulfide dynamics in mouse liver in vivo C_LI

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-pecific Vhl knockout mouse (RodVhl), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type-enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod{Delta}Vhl mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic 13C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

i.Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-{beta} (TGF-{beta}) signaling leading to excessive extracellular matrix (ECM) deposition. The fibroblast-to-myofibroblast-transition (FMT) and metabolic reprogramming of lung fibroblasts (HLFs) are essential to IPF pathogenesis, yet the connection between nutrient metabolism and fibrogenesis remains poorly defined. The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a nutrient-sensitive enzyme that adds O-GlcNAc moieties to substrates. We previously showed that loss of OGT reverses bleomycin-induced pulmonary fibrosis in mice. Here, using unbiased kinomics, we show that pharmacologic inhibition of OGT suppressed non-canonical TGF-{beta}-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-{beta}-induced phosphorylation of p38, but not ERK or JNK, was reduced by OGT blockade. Furthermore, p38 itself was O-GlcNAc-modified, which enhanced its phosphorylation and promoted downstream phosphorylation of the NADPH oxidase subunit, p47phox. Inhibition of OGT, p38, or p47phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47phox-dependent H2O2 production. HighlightsO_LIUsing PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-{beta} MAPKs and multiple CDKs. C_LIO_LIOGT blockade selectively attenuates p38 phosphorylation, despite TGF-{beta}-induced substrate redundancy with ERK and JNK. C_LIO_LIWe provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. C_LIO_LIThe study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47phox, therefore regulating NOX-dependent H2O2 production. C_LIO_LIBlocking OGT or inhibiting p38/p47phox dramatically reduces TGF-{beta}-driven H2O2 production in human lung fibroblasts. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/728188v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1da33c7org.highwire.dtl.DTLVardef@7d8001org.highwire.dtl.DTLVardef@15ee0adorg.highwire.dtl.DTLVardef@1f0a89d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Peroxiredoxin 2 (Prdx2) mediates redox signaling by transferring oxidative equivalents to target proteins such as STAT3, a redox-sensitive transcription factor implicated in inflammation and cancer. Although this interaction has been demonstrated in cells, reconstituting the Prdx2:STAT3 complex in vitro remains challenging due to its transient and redox-dependent nature. Here we test various conditions to stabilize the complex between taggless Prdx2 and the core fragment of STAT3 (CF-STAT3), including oxidants, detergents, the facilitator Annexin A2, anaerobic environments, and CovalX crosslinking. Complex formation was assessed via mass photometry, analytical size-exclusion chromatography (SEC), SEC-MALS, and electron microscopy (EM). No stable complex was observed under standard conditions. Anaerobic environments briefly stabilized the interaction, but cryo-EM could not resolve the structure. CovalX crosslinking yielded short-lived but homogeneous complexes. We found that Prdx2 is highly susceptible to hyperoxidation at its peroxidatic cysteine, particularly in the presence of DTT or excess H2O2, resulting in loss of function. Maintaining non-reducing conditions during purification preserved Prdx2 in an oxidation-competent state, promoting formation of the disulfide bond between the peroxidatic and resolving cysteines and thereby enabling reproducible detection of a weak complex with CF-STAT3. Our findings establish a framework for studying redox-relay protein complexes in vitro and highlight the importance of oxidation state management during protein handling.

Punicalagin, an ellagic acid polyphenol from pomegranate, has been proposed as an antagonist of protein disulfide isomerase (PDI) and endoplasmic reticulum resident protein 57 (ERp57), thiol oxidoreductases that regulate protein folding and extracellular thrombotic signaling. Here, biochemical oxidase and reductase assays on PDI show that punicalagin inhibits both activities with micromolar potency, thereby extending earlier work that described only disulfide reductase inhibition. In parallel, thiol labeling of catalytic cysteines revealed no change in the redox state, supporting a noncovalent, allosteric of inhibition. Molecular docking and molecular dynamics simulations showed that punicalagin binds stably and preferentially to defined sites on the Nterminal domains of PDI through extensive hydrogen bonding and van der Waals contacts, which is an alternative binding mode to previously reported C-terminal binding. Finally, artificial intelligence-driven network analysis identified PDI as a high-confidence target of punicalagin and related galloylated polyphenols, alongside additional signaling proteins. Together, these findings provide further mechanistic framework for punicalagin-mediated antagonism of PDI and highlight galloylated polyphenols as promising scaffolds for protein disulfide isomerase-targeted therapeutics. HighlightsO_LIPunicalagin, a galloylated polyphenol, antagonizes not only the reductase activity but also the oxidase activity of protein disulfide isomerase C_LIO_LIProtein disulfide isomerase inhibition by punicalagin is through N-terminal binding C_LIO_LIPunicalagin inhibits conformationally rather than catalytic cysteine modification C_LIO_LIArtificial intelligence network analysis reveals pathway inhibition by punicalagin C_LI

Mammalian single-stranded DNA binding protein 1 (SSB1) has been established as an essential component of genome stability in both human cells and mice. Moreover, SSB1 was recently implicated in cytoplasmic stress response by its involvement in Ras GTPase-activating protein-binding protein 1 (G3BP1)-containing cytoplasmic stress granules (SGs) upon various forms of stress. Here, we generated and analyzed human cellular knockout and rodent ischemia-reperfusion (I/R) models to define SSB1s roles in cytoplasmic stress response. Analysis of wild-type as well as SSB1 and G3BP1 knockout human retinal pigment epithelial (RPE-1) cells shows stress-specific incorporation of SSB1 into SGs and a negative regulator role for SSB1 in SG dynamics under sublethal stress conditions. We find that SSB1 knockout measurably increases cellular sensitivity to oxidative stress but does not alter cell proliferation following mild acute stress. Moreover, we detect SSB1 efflux from the nucleus upon stress that is dependent upon the presence of G3BP1 in a stress-specific manner. In addition, using mouse and rat models we observe significant upregulation and robust cytoplasmic granulation of SSB1 upon renal ischemia-reperfusion stress, establishing SSB1s involvement in complex organismal stress response in vivo. Together, our data demonstrate active involvement of SSB1 in cytoplasmic response to cellular stress and acute kidney injury, with implications for targeting stress response functions in cancerous versus non-cancerous contexts. HIGHLIGHTSO_LISSB1 is incorporated into cytoplasmic stress granules and negatively regulates stress granule assembly under sublethal stress conditions C_LIO_LISSB1 shows stress- and G3BP1-dependent nuclear efflux C_LIO_LISSB1 is upregulated and undergoes apical granulation in renal epithelial cells during renal ischemia-reperfusion injury C_LI

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-linked cancer with limited therapeutic options and a dismal 5-year survival rate of [~]5%. While aberrant production of reactive oxygen and nitrogen species (ROS/RNS) is a hallmark of MPM, targeted approaches to exploit these redox vulnerabilities remain scarce. Here, using the MOSAIC multimodal cancer patient atlas, we identify Peroxiredoxin 5 (PRDX5) as being significantly upregulated in the epithelioid subtype of MPM. We show that MPM cells exhibit enhanced resistance to nitrosative and oxidative stress compared to healthy mesothelial cells, a phenotype correlated with basal PRDX5 expression. Next, utilising a machine learning guided discovery pipeline, we identified three putative allosteric pockets in PRDX5 and conducted a virtual screen of 3.6 million compounds. High-throughput biochemical validation of 452 candidates yielded 36 non-covalent hits, including sub-micromolar inhibitors. These findings establish PRDX5 as a novel, subtype specific therapeutic target in MPM and provide a chemical framework for the development of next-generation redox-modulating oncology treatments.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a mitochondrial lipid storage myopathy characterized by impaired fatty acid {beta}-oxidation, mitochondrial dysfunction, and progressive neuromuscular and cardiac disease. MADD is most commonly caused by pathogenic variants in electron transfer flavoprotein dehydrogenase (ETFDH), which encodes electron transfer flavoprotein-ubiquinone oxidoreductase (Etf-QO), a critical redox enzyme that transfers electrons from acyl-CoA dehydrogenases to the mitochondrial electron transport chain. Defective Etf-QO activity disrupts electron flow, promotes reactive oxygen species (ROS) production, and impairs cellular energy metabolism, linking abnormal lipid oxidation to oxidative stress-mediated tissue damage. To investigate the role of redox imbalance in MADD pathogenesis, we generated CRISPR/Cas9 knock-in Drosophila melanogaster models carrying patient-relevant Etf-QO missense mutations (L127R, S296C, and L399F; corresponding to human L138R, S307C, and L409F) within conserved FAD- and ubiquinone-binding domains. Mutant flies developed progressive locomotor impairment, reduced muscle performance, and marked lipid droplet accumulation in skeletal muscle, cardiac tissue, and fat bodies, indicating systemic defects in mitochondrial lipid utilization. Cardiac analyses demonstrated reduced fractional shortening, prolonged heart period, and increased arrhythmia index, consistent with metabolic cardiomyopathy associated with mitochondrial oxidative stress. In vivo respirometry revealed significantly decreased oxygen consumption, reflecting impaired oxidative phosphorylation. At the molecular level, mutant flies exhibited elevated ROS levels and ATP depletion, accompanied by increased expression of AMPK, PGC-1, and Tfam, suggesting activation of energy stress signaling and compensatory mitochondrial biogenesis. Importantly, endurance exercise significantly improved locomotor and cardiac function while reducing lipid accumulation and oxidative stress. Together, these findings establish a redox-centered in vivo model of MADD and identify oxidative stress as a major driver of disease pathology and a potential therapeutic target.

Oncogenic KRAS mutations drive tumorigenesis by promoting pro-survival signaling and metabolic reprogramming, including the maintenance of redox balance to evade oxidative stress. A key mechanism involves the upregulation of the xCT cystine/glutamate antiporter, which sustains glutathione (GSH) synthesis and protects cells from oxidative damage and ferroptosis. While it is known that the ETS1-ATF4 complex mediates transcriptional upregulation of xCT, the upstream regulators linking KRAS signaling to this axis remain to be fully defined. Here, we demonstrate that oncogenic KRAS signaling induces the GSK3{beta}-mediated proteasomal degradation of the tumor suppressor CCDC6. We show that CCDC6 acts as a negative regulator of the xCT-promoting transcription factor ATF4 by directly interacting with it and preventing its recruitment to the xCT promoter. Consequently, KRAS-driven CCDC6 degradation disinhibits ATF4, leading to increased xCT expression, elevated intracellular GSH, and enhanced resistance to ferroptosis. Crucially, pharmacological inhibition of CCDC6 turnover using proteasome, GSK3{beta}, or specific KRAS mutant inhibitors (Sotorasib, Adagrasib, HRS4642) restored CCDC6 protein levels and robustly sensitized KRAS-mutated cells to ferroptosis-inducing agents like Sulfasalazine. Furthermore, validation in preclinical models and human colorectal cancer samples revealed that CCDC6 protein levels are predominantly downregulated in KRAS-mutant cases This work uncovers a novel KRAS/CCDC6/xCT signaling axis that mediates ferroptosis resistance in KRAS-mutated cancers. Moreover, it identifies CCDC6 turnover as a critical vulnerability and a promising therapeutic target to enhance the efficacy of ferroptosis-inducing agents.

The intracellular environment Salmonella confronts during infection is characterized by multiple redox stressors including reactive oxygen species (ROS) and copper (Cu) ions. Under these conditions, alternative systems of thiol oxidoreductases such as the Cu induced Scs system are required to protect and repair periplasmic proteins. The scsABCD operon encodes three Dsb-like enzymes, ScsB, ScsC, and ScsD, and an accessory protein, ScsA. These proteins are required both for Cu resistance and H2O2 tolerance. ScsB and ScsC function analogously to the canonical DsbD/DsbC redox pair of thiol oxidoreductases. The absence of ScsC was shown to affect the folding/activity of periplasmic proteins involved in amino acid transport and redox homeostasis. Here, we focus in ScsD, the least characterized member of this system. Upon Cu-induced expression, ScsD localizes to the inner membrane, enabling its predicted C-terminal Dsb-like domain to be exposed to the periplasm. Functional analysis indicates that ScsD exists in a reduced state in the Salmonella envelope and serves as a redox partner of ScsB. ScsD exhibits in vivo disulfide reductase activity and restores a deficient disulfide reduction pathway in Salmonella. Similar to ScsC and ScsB, ScsD binds Cu(I) via the Cys residues of its Dsb-like domain; however, this metal interaction appears to lack relevance in Cu detoxification as no impact on intracellular Cu levels was observed. Our results define ScsD as a specialized membrane-bound thiol-disulfide reductase in the Salmonella envelope and highlight the versatility of the Scs system in maintaining periplasmic proteostasis when canonical pathways are compromised by host-imposed Cu stress. ImportanceCopper is a key component of the innate immune system, serving as a primary defense against pathogens like Salmonella. Copper overload targets the bacterial envelope, specifically attacking protein sulfhydryl groups. This causes protein misfolding and inactivation, disrupting essential processes like metabolism, transport and virulence. To survive this stress and restore thiol homeostasis, Salmonella utilizes the scsABCD operon. While the ScsB-ScsC redox pair is well-documented and some protein substrates identified, the role of ScsD remains undefined. This work characterizes ScsD as an inner-membrane-anchored thiol reductase and a new redox partner for ScsB. The ScsD/ScsB pair expands the bacteriums protein quality control capacity, allowing Salmonella to maintain envelope homeostasis within the hostile, copper-rich environment of the host.

Hydropersulfides have gained attention in cell biology as excellent nucleophiles and membrane-protective radical scavengers. They form perthiyl radicals, which terminate radical chain reactions through self-recombination, leading to the formation of polysulfides. It is currently unknown how polysulfides are subsequently reduced again in non-enzymatic or enzymatic metabolic pathways. Here we used stopped-flow kinetic measurements in combination with mass spectrometry to show that the model class I glutaredoxin from the malaria parasite Plasmodium falciparum (PfGrx) rapidly reduces the polysulfides glutathione trisulfide (GS3G) and glutathione tetrasulfide (GS4G), yielding the glutathionylated enzyme and the corresponding glutathione hydropersulfide GSSH and hydrotrisulfide GS3H. The second-order rate constants of these enzymatic reductions [≥]107 M-1s-1 are even slightly higher than for glutathione disulfide (GSSG). In contrast, PfGrx was inactive or only moderately active using cystine or cysteine trisulfide as oxidants. GSSH and GS3H are further reduced by PfGrx with second-order rate constants on the order of 106-107 M-1s-1, yielding the glutathionylated enzyme as well as hydrogen sulfide (H2S) and hydrogen disulfide (H2S2), respectively. Thus, glutaredoxins specifically recognize the glutathione moiety of glutathione (hydro)polysulfides and glutathione hydropersulfide. Due to the rapid reduction of glutathionylated glutaredoxins by reduced glutathione (GSH), glutathione (hy-dro)per/polysulfides are efficiently converted to GSSG and H2S or the corresponding hydrogen polysulfides. As a consequence, the steady-state concentration of glutathione (hydro)per/polysulfides should be tightly controlled in subcellular compartments containing active glutaredoxins and high GSH concentrations.

Careful balance of the redox status of the embryo and reduction of oxidative stress is crucial in early development. Here we show that the culture of preimplantation mouse embryos in the conditionally non-essential amino acid L-proline (Pro) increases the intracellular concentration of the potent antioxidant glutathione as shown by staining of 2-cell, 4-cell and 8-cell embryos with tetrafluoroterephthalonitrile (4F-2CN). Further, liquid-chromatography/mass spectrometry showed increased GSH levels in all Pro-treated preimplantation stages of development compared to controls. The GSH:GSSG ratio also showed a Pro-dependent increase. Overall, our results indicate that the beneficial effect of Pro in preimplantation embryo culture is due to the reduction in oxidative stress mediated through an increase in cellular GSH concentration.