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.

Since NO can modulate mesenchymal cell function, we posit that NO can modulate gene expression associated with excitation-contraction coupling. Our study shows that treating asthma-derived HASMCs with a low dose of NO plus sGC stimulator BAY-41, in most cases sensitized smooth muscle sGC towards activation via an elevated sGC heterodimer and in some cases also improved sGC{beta}1, catalase, Cyb5r3 or Trx1 expression (n=24 non-asthma and n=25 asthma). Interestingly we found that majority of asthma HASMCs showed a marked downregulation of G6PD expression inducing a low GSH/GSSG ratio in asthma, and these findings were replicated in murine lungs of allergic asthma (OVA and CFA/HDM). Studies with HEK/COS-7 cells showed G6PD synergizing with hsp90 in enabling sGC heme-maturation. G6PD overexpression in HASMCs enhanced the sGC heterodimerization while silencing of endogenous G6PD abrogated it. Complementation of these cellular results with whole animal models of G6PD deficiency or overexpression provided verification to our findings. Mouse lung tissue from the humanized variant of G6PD deficiency, V68M (G6PD A-deficiency) showed significant downregulation in the sGC heterodimer, with a concomitant reduction in its NO heme-dependent activity, thereby showing that G6PD deficiency lowers sGC heme. Conversely, G6PD overexpressing mouse lung tissue displayed an elevated sGC heterodimer and also showed a robust G6PD-sGC{beta}1 interaction, suggesting G6PD to be involved in the heme-maturation of sGC{beta}1. While G6PD maintains the cell redox by generating NADPH, its new role in regulating sGC maturation links sGC dysfunction in asthma to G6PD deficiency and may potentially uncover new targets for asthma treatment.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

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

ObjectiveN-acetylcysteine (NAC) is a clinically available antioxidant with potential applications in trauma-induced hypermetabolic states, including burn injury and crush syndrome. However, its effects on heat-stressed skeletal muscle cells remain incompletely characterized. This study conducted a secondary analysis of a publicly available dataset to quantify NACs protective effects against heat-stress-induced cellular damage. MethodsWe re-analyzed a publicly available dataset (Lu J, 2024, Mendeley Data, doi:10.17632/wffrtcgbnx.1) containing 21 observations across three conditions: Control (n=3), Heat Stress only (HS, n=3), and HS with NAC at five doses (0.5-8.0 mM, n=3 per dose). The primary outcome was the protective ratio [(HS+NAC - HS) / (Control - HS)], where 1.0 indicates complete protection. Statistical analyses included one-way ANOVA, post-hoc t-tests with Bonferroni correction, Cohens d effect sizes, and bootstrap confidence intervals. ResultsHeat stress significantly reduced cell viability by 56.3% (Control: 100.0 {+/-} 12.2 vs HS: 43.7 {+/-} 5.1; t(4)=7.37, p=0.002, Cohens d=6.02). NAC demonstrated a biphasic dose-response with maximal protection at 2.0 mM (66.7 {+/-} 14.4), yielding a protective ratio of 0.409 (95% CI: 0.146-0.675), representing 40.9% protection against heat stress damage. The comparison between HS and HS+NAC (2.0 mM) showed a large effect size (Cohens d = 2.12) but did not reach statistical significance (p = 0.060) due to the small sample size. One-way ANOVA confirmed overall group differences (F(2,18)=32.39, p<0.001, 2=0.783). ConclusionsNAC provides partial protection against heat stress-induced skeletal muscle cell damage at 2.0 mM, with a large effect size suggesting clinical relevance despite limited statistical power. These preliminary findings support further investigation of NAC as an adjunct therapy in trauma-induced hypermetabolic states. All analysis code is provided for reproducibility.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

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.

The persistence of P. aeruginosa infections is largely driven by the secretion of several factors during invasion, including the redox-active phenazine pyocyanin (PYO), which promotes biofilm formation and oxidative stress. Biofilms contribute to chronic infections and antibiotic resistance, limiting the efficacy of conventional therapies. We found that a synthetic compound, I-152, a conjugate of N-acetyl-L-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-{beta}-mercaptoethylamine; SMEA), effectively restored colistin susceptibility against P. aeruginosa by altering biofilm nanomechanical properties. These perturbations in matrix integrity were associated with I-152s ability to hinder the phenazine redox cycle, shifting PYO to a reduced state and promoting chemical interactions (S-conjugates). The compound decreased PYO accumulation in bacterial cultures and PYO-generated reactive oxygen species (ROS) in macrophage cells. Together with PYO, LPS is another driver of ROS-dependent inflammatory signaling in the host, which leads to an uncontrolled cytokine response and organ damage, especially in patients with cystic fibrosis. I-152 treatment downregulated the expression of LPS-induced inflammatory cytokines, i.e., IL-6 and TNF-, in bone marrow-derived macrophages (BMDM) isolated from transgenic CFTR-/- and CFTR+/+ mice. Consistently, I-152 partially counteracted the inflammatory response in the P. aeruginosa LPS-induced acute lung injury murine model. Taken together, these results support I-152 as an adjunctive treatment for P. aeruginosa respiratory infections through a dual mechanism: combating antimicrobial resistance in biofilms and dampening host inflammation in the respiratory system.

Oxidation of free methionine plays important roles in cellular redox homeostasis, yet its accurate quantification has been hindered by methodological challenges. Here, we introduce free Methionine Oxidation by Blocking (fMObB), a mass spectrometry-based method that enables accurate measurement of the fractional oxidation of free methionines. Applying fMObB to Escherichia coli, we quantify free methionine oxidation under basal and oxidative stress conditions, and in strains lacking methionine sulfoxide reductases. We find that during oxidative stress, free methionines exhibit higher oxidation levels than protein-bound methionines and that methionine sulfoxide reductases play a central role in maintaining reduced free methionine pools. Together, this work establishes fMObB as a generalizable strategy for probing free methionine redox states in cellular systems.

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.

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.

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

Alzheimers disease (AD) is increasingly recognized as a systemic disorder in which peripheral metabolic and redox dysfunction affects neurovascular injury and amyloid pathology. However, the lipid-redox mechanisms linking adipose tissue dysfunction to AD are less explored. Here, we applied an integrated multi-omics and imaging approach to investigate adipose lipid remodelling that leads to nitric oxide (NO)-derived nitro-fatty acid modifications and their effects on {beta}-amyloid and VEGF in the hippocampus of APP/PS1 mice. Targeted lipidomics revealed broad suppression of lysophospholipids and membrane phospholipids (LPC, PC, PE, PG) in gonadal white adipose tissue (gWAT), consistent with impaired membrane turnover and mitochondrial lipid deficiency. Untargeted lipidomics demonstrated accumulation of ceramides, triacylglycerols, monoacylglycerols, and phosphatidic acids, indicating lipotoxicity and disrupted lipid flux. Oxidized lipid mediator profiling showed increased 13-HODE, 12-HETE, and 14-HDHA. Further, Raman microscopy mapping revealed a shift from protective nitro-oleic acid toward increased nitration of polyunsaturated fatty acids. These lipid abnormalities coincided with increased adipose expression of redox and inflammatory markers, including NOX4 and TNF-, and impaired mitochondrial redox metabolism assessed by fluorescence lifetime imaging (FLIM). Citrulline and nitrite treatments partially normalized adipose lipid-redox signatures. Citrulline restored phospholipid remodeling and nitro-oleic acid signaling, whereas nitrite preferentially enhanced stress-associated signaling lipids. Importantly, both interventions reduced hippocampal {beta}-amyloid burden and restored VEGF expression, with citrulline producing the strongest neurovascular rescue. These findings identify adipose lipid-redox imbalance as a systemic contributor to neurovascular pathology in AD and highlight NO-directed metabolic modulation as a strategy to mitigate disease-associated lipid dysfunction.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Text AbstractIn response to constant homeostatic threats, organisms have developed complex regulatory networks to monitor cellular functions and restore normal function. Here, we identify MDT-15 and its effectors, the fatty acid desaturases FAT-5, FAT-6, and FAT-7, as activators of the Ethanol and Stress Response (ESRE) mitochondrial surveillance pathway. Our data show that box C/D snoRNPs, which were previously linked to ESRE activation, also regulate FAT-6 and FAT-7 protein levels. Notably, knockdown of mdt-15 or fib-1, a component of box C/D snoRNP complex, increased accumulation of the mitophagic activator PINK-1, the first step in licensing mitophagy, suggesting a relationship between ESRE surveillance and mitophagic activation. Supplementation with downstream unsaturated fatty acid products of FAT-6 and FAT-7 enhanced ESRE and mitophagic activation, but did not affect UPRmt. Since fatty acids activated ESRE and PINK-1 in wild-type and mutant genetic backgrounds, they are likely to act via a mechanism independent of FAT-6 and FAT-7 function. Our results provide insight into a novel interplay between box C/D snoRNPs, MDT-15, and fatty acids in the regulation of mitochondrial surveillance and mitophagy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/656193v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@92f749org.highwire.dtl.DTLVardef@a8f496org.highwire.dtl.DTLVardef@51b28dorg.highwire.dtl.DTLVardef@1a15374_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.