Redox Biology

Peroxiredoxin 6 (PRDX6) is a multifunctional enzyme with peroxidase, calcium independent phospholipase A2 (aiPLA2) and lysophospatidylcholine acyltransterase (LPCAT) activities. Although PRDX6 can repair peroxidised phospholipids and help prevent ferroptosis, its role in the adaptive response to physiological oxidative stress remains unclear. In this study PRDX6 function was investigated using myoblasts exposed to an acute low concentration of H2O2 and Caenorhabditis elegans subjected to a swimming intervention. Mild oxidative stress promoted myogenesis and mitochondrial turnover in myoblasts, while exercise enhanced activity and longevity in C. elegans. The adaptive responses were associated with increased mitochondrial localisation of PRDX6. In contrast, loss of PRDX6 under mild stress conditions resulted in increased mitochondrial lipid peroxidation, enhanced mitochondrial ER contact sites (MERCS), mitochondrial calcium accumulation, release of mitochondrial DNA and activation of innate immune signalling pathways. Similar phenotypes were induced by the ferroptosis activator erastin and rescued by the lipid peroxyl scavenger Ferrostatin-1, indicating lipid peroxidation was the key triggering event. Furthermore, inhibition of mitochondrial calcium uptake in C. elegans prevented calcium overload and attenuated inflammatory signalling. Together, the results identify PRDX6 as a conserved regulator of mitochondrial adaptation to physiological oxidative stress, functioning to limit mitochondrial lipid peroxidation and prevent excessive MERCS assembly, mitochondrial calcium dysregulation and inflammatory activation.

Redox signalling regulates development, tissue homeostasis, and organismal health. Hydrogen peroxide (H2O2) is a major signalling form of reactive oxygen species (ROS) that modulates protein activity through oxidation of redox-sensitive cysteines that is reversed by cellular reducing systems. Since H2O2 production, scavenging and reduction are spatially restricted, signalling specificity is strongly influenced by subcellular localisation. However, subcellular H2O2 dynamics in animal tissues remain poorly understood. To address this, we generated and validated Drosophila melanogaster lines expressing the ultrasensitive, ultrafast ratiometric H2O2 biosensor HyPer7 targeted to mitochondria, nucleus, cytosol, or plasma membrane. With its highly conserved metabolic and signalling pathways, tractable lifespan, and powerful genetic toolkit, Drosophila is an ideal model for studying redox biology. These new 'FlyPer' lines enable tissue-specific HyPer7 expression and high-resolution measurement of subcellular, in vivo H2O2 dynamics throughout the lifespan. Using FlyPer, we detected compartment-specific H2O2 dynamics during oxidative stress, ageing, wing disc development and embryogenesis, uncovering unexpected patterns of spatially and temporally regulated oxidation throughout the organism. Together, these findings establish FlyPer as a valuable toolkit for in vivo redox biology and suggest that compartmentalised redox dynamics are a fundamental yet still poorly understood layer of developmental programming.

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.

Polyaneuploid giant cells (PGCC), which occur more frequently in TP53-mutant tumors, are recognized as a driver of tumor recurrence and therapy resistance, but the mechanisms supporting their survival remain largely unknown. Our results indicate that polyaneuploid transition and subsequent PGCC maturation in drug-resistant phenotypes are associated with redox rewiring that shifts cellular homeostasis into mild pro-oxidative condition. These are accompanied by increased transcription of genes involved in protection against elevated reactive oxygen species and glutathione-dependent xenobiotic detoxification such as TXN, PRDX2/5, GPX1, and GSTP1/GSTO1. Functional studies provided evidence on the crucial role of Txn-Txnrd1 system in maintaining PGCC viability and their adaptation to increased level of reactive oxygen species. Pharmacological targeting of Txn or Txnrd1 as well as their silencing caused a decline in thiol content followed by further redox imbalance, which led to massive death of PGCC. Analysis of clinical datasets revealed direct and relatively strong link between transcription of TP53 and TXN or TXNRD1. Tumors with TP53low/TXNhigh or TP53low/TXNRD1high were associated with considerably poorer patient outcome, whereas elevated transcription of both TXN and TXNRD1 predicted reduced response to chemotherapy in glioblastoma and intestinal cancer. Concluding, Txn-Txnrd1 system enables PGCCs to tolerate pro-oxidative condition, thereby creating a therapeutically exploitable redox vulnerability of these cells, where Txnrd1 emerges as a potential target candidate to overcome PGCC-driven chemoresistance.

Melittin exhibits antitumor activity in cervical cancer models, yet the long non-coding RNA (lncRNA) response and associated regulatory networks remain poorly understood. Here, strand-specific RNA-seq data from melittin-treated and untreated U14 murine cervical cancer cells were analyzed to characterize melittin-responsive lncRNAs and explore their potential functional associations. A total of 28,162 lncRNAs were identified, including 27,307 known and 855 novel transcripts. Differential expression analysis revealed 404 differentially expressed lncRNAs (DElncRNAs), comprising 191 upregulated and 213 downregulated lncRNAs, w most of which were predicted to localize to the cytoplasm or nucleus. Cis-target analysis identified 52 neighboring mRNAs as putative targets of 46 DElncRNAs. Functional enrichment highlighted mitochondrial electron transfer and redox-related processes, including the mitochondrial electron transfer flavoprotein complex, electron-transferring-flavoprotein dehydrogenase activity, ubiquinone binding, and quinone binding. In parallel, melittin induced mitochondrial membrane depolarization and increased intracellular reactive oxygen species accumulation in U14 cells. Co-expression analysis further identified 138 lncRNAs co-expressed with 161 mRNAs, which were enriched in chromatin remodeling, DNA replication, and DNA repair. EdU incorporation decreased with increasing melittin concentrations, indicating suppression of DNA synthesis and proliferative activity. RT-qPCR analysis confirmed the expression trends of selected DElncRNAs. Collectively, these findings demonstrate extensive remodeling of the lncRNA landscape in melittin-treated U14 cells and suggest that melittin-responsive lncRNA-mRNA networks are associated with mitochondrial redox disruption and impaired DNA synthesis. This study provides a transcriptomic framework for identifying candidate lncRNA-mRNA regulatory axes underlying the antitumor response to melittin.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

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.

Tumour hypoxia is associated with increased invasiveness, metastasis, and drug resistance; however, its impact on drug-metabolising enzymes remains poorly understood. This study investigated the effect of hypoxia on the expression of selected aldehyde dehydrogenase (ALDH) isoforms (ALDH1A1, 1A2, 1A3, 1B1, 2, 3A1, and 7A1) in colorectal cancer (CRC) cells. CRC cell lines (HT29, DLD-1, SW480, and HCT116) were cultured under normoxic and hypoxic (0.1% O2) conditions, while HT29 and DLD-1 cells were additionally grown as multicellular spheroids (MCS). Expression of ALDH isoforms was assessed at the mRNA and protein levels. Functional studies included siRNA-mediated knockdown of ALDH1A1, ALDH3A1, and ALDH7A1, measurement of reactive oxygen species (ROS), and stable overexpression of ALDH7A1 in H1299 cells. ALDH7A1 was consistently upregulated at both transcript and protein levels in HT29 and DLD-1 cells exposed to hypoxia. Elevated ALDH7A1 expression was also observed in hypoxic regions of MCS and CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). Knockdown of ALDH7A1 in DLD-1 cells reduced proliferation, increased ALDH3A1 expression, and significantly elevated ROS levels, indicating a role in redox homeostasis and suggesting functional crosstalk between these isoforms. Conversely, stable overexpression of ALDH7A1 in H1299 cells markedly reduced ROS levels. Taken together, these findings identify ALDH7A1 as a hypoxia-responsive enzyme that promotes adaptation to oxidative stress and may contribute to CRC cell survival within the hypoxic tumour microenvironment.

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.

Glyoxal (GO) is a small, highly reactive molecule that is produced naturally in cells during normal metabolism and can also come from processed foods and oxidative stress. Because of its high reactivity, glyoxal can modify DNA and proteins to form harmful products called advanced glycation end-products (AGEs), which have been linked to diseases such as diabetes, cancer, and aging. Although glyoxal is known to modify DNA and proteins, it is not well understood whether it can form DNA-protein crosslinks (DPCs), a type of DNA damage in which proteins become permanently attached to DNA. In this study, we investigated glyoxal induced DPC formation in HeLa cells using biochemical assays and mass spectrometry-based proteomics experiments. We observed that glyoxal exposure elevated cellular DPC formation in a concentration- and time-dependent manner. Cells with reduced SPRTN expression accumulated higher levels of DPCs, suggesting that SPRTN plays an important role in repairing glyoxal induced DNA damage. Proteomics experiments revealed 469 proteins exhibited elevated DNA association in glyoxal-treated samples, including histones and other proteins involved in chromatin organization, DNA replication, DNA repair, and gene expression. In-vitro experiments confirmed that glyoxal can directly crosslink DNA with histone proteins. Overall, this study provides the first evidence that glyoxal forms DNA-protein crosslinks in human cells. These findings provide a foundation for future studies on the chemical structure, biological effects and repair of glyoxal induced DNA-protein crosslinks and their possible role in human disease.

Elephant seals are remarkable breath-hold divers, capable of remaining submerged for up to two hours during diving bouts. These dives entail repeated, extreme hypoxia/reoxygenation events that would induce severe lipid peroxidation and tissue dysfunction in most mammals. Here, we show that primary vascular endothelial cells derived from elephant seals possess an intrinsic resistance to lipid peroxidation. Comparative transcriptomic and lipidomic profiling across seal, human, and sheep cells identified ferroptosis - an iron-dependent, lipid peroxidation-driven cell death pathway - as uniquely regulated in seal cells following hydroperoxide exposure. Mechanistically, seal cells exhibit robust baseline expression of acyl-CoA synthetase long-chain family member 3 (ACSL3), alongside rapid, seal-specific induction of the sole mammalian iron exporter, ferroportin (SLC40A1). Functional validation using genetic and pharmacological approaches revealed that seal cells are naturally enriched in monounsaturated fatty acids and triglycerides and utilize lipid droplet biogenesis and active iron export as dual protective axes to evade lipid peroxidation. Together, these findings show that elephant seal cells employ a coordinated cytoprotective network of lipid remodeling and iron handling to withstand the severe challenges of deep diving. SIGNIFICANCE STATEMENTDeep-diving marine mammals repeatedly experience extreme hypoxia-reoxygenation events that would induce severe oxidative damage in most terrestrial mammals. However, vascular cells derived from seals naturally resist lipid peroxidation, a major driver of ischemia-reperfusion injury. Here, we show that elephant seal endothelial cells evade lipid peroxidation through two complementary mechanisms: lipid droplets that sequester peroxidation-prone phospholipids, and rapid iron export that limits lipid peroxide formation. These findings reveal naturally evolved cellular strategies that protect against vascular oxidative stress, offering new insights into physiological resilience against ischemia-reperfusion injury.

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

Cysteine oxidation analyses require the preservation of the redox state present at harvest and quantitative scaling to relate oxidation to protein copy numbers, rather than only providing fractional oxidation data. Here, we present ReCap, a Redox Capture workflow combining Oxi-DIA, an enrichment-free isotope-encoded DIA workflow, with Oxi-Stop, a simple oxygen-exclusion strategy for cryopreserved tissue. In mouse brains, Oxi-DIA quantified 17,809 cysteine sites belonging to 6,085 protein groups in every sample, enabling matched measurements of residue-resolved oxidation and protein abundance. Atmospheric oxygen exposure during 14 days of cryopreservation distorted the measured cysteine redox state. The resultant increase of an estimated 5.3176 x 1011 {micro}g-1 oxidised cysteine molecules was mitigated by Oxi-Stop, which minimised exogenous oxidation during cryopreservation. Copy-number scaling altered the interpretation of cysteine oxidation values. Although cysteine oxidation was detected across 2,371 sites and 1,439 proteins, 20 sites on abundant proteins accounted for 44% of the oxidised signal. ReCap advances redox proteomics from providing a site catalogue into a biologically weighted map of redox information, revealing cysteine oxidation as a sparse, ordered and quantitatively concentrated signal.

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase HvFdR (HVO_2345; fdr), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. HvFdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, HvFdR mediates reverse electron transfer to NADP, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, HvFdR bound-FAD exhibits a low midpoint redox potential (-413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for HvFdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of HvFdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H2O2 production under aerobic conditions. Together, these findings establish HvFdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.

Nitric oxide (NO) is a key signaling molecule that regulates diverse physiological responses, including adaptation to hypoxia in plants. Although stabilization of group-VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors under low O2 is known to be facilitated by the N-dragon pathway, whether NO directly mediates N-terminal cysteine (Cys) oxidation to regulate ERFVII stability remains unresolved. Here, we combined genetic, computational, and structural approaches to investigate NOs role in proteasomal degradation of ERFVII during flooding stress. Arabidopsis mutants with elevated S-Nitrosoglutathione (GSNO) levels compromised ERFVII activation during dark submergence but increased expression of genes encoding N-degron pathway enzymes. Although direct detection of in vivo NO-mediated S-nitrosylated proteins remains technically challenging, the GPS-SNO 1.0 tool predicted the conserved N-terminal second Cys residue as a high-confidence S-nitrosylation site. Structural modeling using the Schrodinger Suite 2024-4 further revealed that conversion of the Cys thiol (Cys-SH) to S-nitrosothiol (Cys-SNO) induced notable conformational changes in ERFVII TFs. Molecular docking further demonstrated that Cys-SNO-modified ERFVII peptides exhibited stronger binding affinities and altered interaction networks with N-degron pathway enzymes, supporting a role for NO-mediated structural remodeling in ERFVII degradation. Elevated GSNO also disrupted energy balance efficiency for overcoming O2 deficiency, altered the expression of sugar starvation-responsive genes, and impaired ATP binding capacity of Cys-SNO ERFVII proteins, as evidenced by docking and molecular dynamics simulations. Collectively, these results support a model in which NO-mediated modification of the conserved N-terminal Cys promotes ERFVII degradation, thereby linking NO signaling to hypoxia-responsive transcriptional regulation and metabolic adaptation during flooding stress.

Mitochondria-targeted modulation of redox homeostasis has emerged as a promising strategy for controlling pathological cell proliferation. Here, we investigate the effects of Mitocurcumin in a Yorkie-driven intestinal stem cell tumor model in Drosophila. Using an integrative, genetically tractable approach combining in silico molecular modelling with in vivo functional analyses, we identify thioredoxin reductase (TrxR) as a conserved redox-associated target of Mitocurcumin. Docking and molecular dynamics simulations predict a stable interaction of Mitocurcumin with both Drosophila and mammalian TrxR homologs. Functionally, Mitocurcumin treatment reduces mitotic activity, elevates reactive oxygen species (ROS) selectively within escargot-positive intestinal stem cell population, enhances apoptosis in the tumor-bearing guts, and causes significant mitochondrial membrane depolarization. These cellular effects coincide with dose-dependent regression of Yorkie-induced intestinal hyperplasia. Despite mitochondrial functional impairment, mitochondrial morphology remains largely preserved, suggesting primary disruption of redox buffering rather than structural collapse. Metabolomic profiling of these guts further reveals remodelling of energy metabolism consistent with adaptive responses to oxidative stress. Importantly, Mitocurcumin alleviates tumor-associated organismal bloating and significantly extends lifespan indicating a previously uncharacterized systemic, organism-wide response to Mitocurcumin treatment in an in vivo scenario. Collectively, our findings establish TrxR-mediated redox regulation as a critical vulnerability in Yorkie-driven hyperproliferation and highlight the utility of Drosophila as an integrative in vivo platform for evaluating mitochondria-targeted bioactive molecules.

Mitophagy is the process of selective autophagic clearance of damaged mitochondria and is closely implicated in neurodegenerative disease. PTEN-induced kinase 1 (PINK1) and a RBR E3 ubiquitin-protein ligase (Parkin) constitute a positive feedback loop in mitophagy initiation. It is known that reactive oxygen species (ROS) modulate mitophagy, while the exact regulatory mechanism remains largely elusive. Here, we found that exogenously applied ROS effectively block mitophagy induced by acute mitochondrial damage agents, which could be reversed by antioxidants. Mechanistically, ROS activate poly(ADP-ribose) polymerase 1 (PARP1), and suppression of PARP1 eliminates the inhibitory effect of ROS on mitophagy. Notably, PARP1 directly interacts with PINK1 and mediates its PARylation at residue E417, thereby negatively regulating PINK1 function. Collectively, our study identifies PARylation as a new form of post-translational modification of PINK1 and reveals a novel mechanism underlying the regulatory role of ROS in mitophagy by PARP1 activation and PARylation of PINK1. In briefGao et al. demonstrate that exogenous ROS inhibit mitophagy. Mechanistically, ROS activate PARP1, which mediates PARylation of PINK1, a central regulator of mitophagy, leading to its functional impairment. This study reveals a novel regulatory mechanism of ROS on mitophagy through PARP1 activation and identifies PARylation as a novel form of post-translational modification of PINK1. HighlightsO_LIROS block PINK1-Parkin-mediated mitophagy. C_LIO_LIROS activate PARP1. C_LIO_LIPARP1 suppression eliminates the inhibitory effect of ROS on mitophagy. C_LIO_LIPARylation of PINK1 by PARP1 impairs its activity and mitophagy. C_LI

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eyes optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical AbstractIllustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733742v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@1ef3a7eorg.highwire.dtl.DTLVardef@e9293dorg.highwire.dtl.DTLVardef@192729forg.highwire.dtl.DTLVardef@1a32cb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin HvFdx (HVO_2995) and its flavin-dependent oxidoreductase HvFdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii. Genetic and biochemical analyses established HvFdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of HvFdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, HvFdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of HvFdx was found to stimulate electron flow from HvFdR as measured by an anaerobic NADPH [->] HvFdR [->] HvFdx [->] DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with HvFdx lysine acetylation and optimal growth of H. volcanii. Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii.

Vacuolar ATPases (V-ATPases) are highly conserved multi-subunit proton pumps that drive the acidification of intracellular vesicles, especially endosomes and lysosomes. By regulating progressive acidification of the endolysosomal pathway, V-ATPase activity impacts signaling transduction pathways both positively (e.g., internalization and activation of receptor-ligand complexes in endosomes) and negatively (e.g., degradation of pathway mediators in lysosomes). While the role of V-ATPases in human neurodegenerative diseases and cancer has been extensively studied, the requirement for these proteins in intestinal restitution remains poorly understood. Here, we use Drosophila to study the role of V-ATPases in regulating intestinal-injury and repair driven by excessive oxidative stress. We find that RNAi driven depletion of multiple subunits of the V-ATPase complex suppressed oxidative stress-induced lethality. By contrast, depletion of the main lysosomal catabolic enzyme in Drosophila (Cathepsin-D) had no effect. On a cellular level, these effects map to absorptive enterocytes (ECs) of the Drosophila intestine. Molecular analysis of intestines following injury by oxidative stress compared to uninjured controls reveals increased cell death, increased JNK-pathway activity, and increased IMD/NF-{kappa}B pathway signaling reporter expression compared to uninjured controls. Depletion of Vha44 (subunit C of the V1 complex) was sufficient to suppress the increased cell death, JNK pathway, and IMD/NF-{kappa}B pathway markers induced by oxidative stress in the intestine. Furthermore, overexpression of the MAP3K TAK1 enhanced death, JNK pathway and IMD/NF-{kappa}B pathway activation in a Vha44 dependent manner. These findings suggest that inhibition of V-ATPase activity can protect against intestinal injury caused by excessive oxidative stress. On a molecular level, we find that attenuation of endolysosomal acidification dampens pro-apoptotic JNK and IMD/NF-{kappa}B pathways, highlighting endosomal acidification as a potential amplifier of excessive oxidative stress.