Selenium-binding protein 1 (SELENBP1) catalyzes the oxidative conversion of methanethiol to hydrogen sulfide, hydrogen peroxide and formaldehyde. Methanethiol oxidase (MTO) activity of recombinant human SELENBP1 was shown to require copper (Cu) ions, whereas selenium (Se) was largely dispensable. However, the impact of Cu and Se on MTO activity in mammals has not been explored so far. To investigate whether SELENBP1 levels and MTO activity in mice are affected by dietary availability of trace elements, liver samples of C57BL/6JRj mice held on diets deficient in Cu and/or Se for eight weeks were analyzed, as compared to mice fed the control diet with adequate trace element supply. Dietary Cu deficiency always resulted in diminished hepatic MTO activity, as detected by methanethiol-derived production of hydrogen sulfide. The effect of Cu deficiency was more pronounced in male than in female mice. Se deficiency only slightly, and solely in males, lowered MTO activity. Another sex-specific effect was observed with hepatic SELENBP1 levels: whereas diets deficient in Cu and/or Se increased SELENBP1 levels in males, they were decreased in female mice under Cu-deficient conditions. In addition to dietary Cu deficiency, MTO activity and SELENBP1 levels were explored in a rat model of Wilson’s disease, which is characterized by hepatic Cu accumulation. Hepatic SELENBP1 levels were decreased in male but not in female rats under conditions of excess intrahepatic Cu. However, hepatic MTO activity was not altered in both sexes, if the liver was not severely damaged. Notwithstanding the relatively low number of 5-6 animals included in each group, our data allow for the conclusion that hepatic MTO activity depends on adequate dietary Cu supply, whereas excess Cu does not further enhance the MTO activity of SELENBP1.
Transfusion-related acute lung injury (TRALI) remains a life-threatening complication of blood transfusion, yet the macrophage-neutrophil-NETosis inflammatory amplification mechanism and effective interventions are still incompletely defined. Here, we examined a CD36/Fyn-CXCL-related axis in TRALI and evaluated a ROS-responsive platelet extracellular vesicle biomimetic nanosystem (PEV@SP@EG) as a targeted delivery strategy. In a murine TRALI model, exploratory single-cell transcriptomic analysis showed immune-cell remodeling and linked CD36 expression to M1-like macrophage features. CD36 knockdown or pharmacological inhibition of Fyn/NF-kappaB reduced M1 polarization markers, CXCL production, and ROS generation. Conditioned medium from CD36-overexpressing BMDMs promoted neutrophil migration and NETosis, whereas CXCL-CXCR2 blockade or CD36/Fyn inhibition weakened these responses. PEV@SP@EG showed ROS-triggered drug release and preferential lung enrichment. In vivo, PEV@SP@EG attenuated CD36/Fyn-NF-kappaB-CXCL-related inflammatory signaling, NET formation, and oxidative stress in TRALI lung tissue, with concomitant improvement in lung architecture and gas exchange. These findings support a role for macrophage CD36/Fyn signaling in TRALI-associated macrophage-neutrophil inflammatory crosstalk and suggest that PEV@SP@EG may provide a targeted nanotherapeutic delivery platform for TRALI intervention.
This study explores the pivotal role of NRF2 signaling in conferring resistance to chemotherapy and ferroptosis in medulloblastoma (MB), a highly malignant pediatric brain tumor. Using newly developed in vitro models of MB cells, resistant to standard chemotherapeutics (vincistine, etoposide, cisplatin, and cyclophosphamide), we observed that chemotolerant cells exhibit an enhanced antioxidant response. Specifically, we found higher levels of glutathione, compared to sensitive cells, and increased thioredoxin reductase activity, both key components in maintaining redox homeostasis. Furthermore, we identified a metabolic shift in resistant cells, marked by increased flux through the pentose phosphate pathway (PPP), which boosts NADPH production and supports the antioxidant defense mechanisms. This adaptive antioxidant response is largely mediated by hyperactivation of the NRF2 transcription factor and the consequent upregulation of a set of antioxidant genes, thus effectively reducing intracellular reactive oxygen species (ROS) levels and enhancing cells' ability to tolerate oxidative stress. Intriguingly, our results suggest that NRF2 activation not only supports the acquisition of chemotherapy resistance but also confers protection against ferroptosis induction. Indeed, resistant cells upregulate iron sequestration proteins and ferroptosis-suppressing genes, directly controlled by NRF2, thereby reducing vulnerability to lipid peroxidation-induced cell death. Inhibition of NRF2 in resistant cells increased their sensitivity to both chemotherapy and ferroptosis inducers and, more interestingly, its knockdown prevented sensitive cells from acquiring resistance. Overall, our study underscores the crucial role of NRF2-controlled redox homeostasis in mediating resistance mechanisms in MB, highlighting the therapeutic potential of targeting this pathway. Targeting NRF2 signaling may provide a novel approach to overcome therapy resistance and improve treatment outcomes for patients with this challenging cancer.
Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) affects millions of men, yet the immune circuits that sustain sterile prostatic inflammation and pain remain poorly defined. Here we identify a T cell-macrophage metabolic signaling axis that drives chronic prostatitis. Integrated analysis of mouse experimental autoimmune prostatitis, chronically inflamed human prostate tissue, single-cell transcriptomes and spatial profiles revealed a population of glycolytic, HIF-1α-activated inflammatory macrophages enriched in diseased prostate niches. T cell-derived CCL5 was markedly induced and spatially positioned adjacent to CCR5+ macrophages, where it licensed M1-like polarization, glycolytic remodeling and inflammatory transcription. Using T cell-specific Ccl5 knockout mice, we established CCL5 as a genetic driver of macrophage inflammation, prostate tissue injury and pelvic pain hypersensitivity, and these pathogenic features were further suppressed by CCL5 neutralization or pharmacological CCR5 blockade. Mechanistically, rmCCL5 stimulation promoted CCR5-sensitive ERK1/2 activation, PKM2 Ser37 phosphorylation and nuclear accumulation, enhanced the PKM2-HIF-1α interaction, and increased HIF-1α occupancy at the Il1b and Nos2 promoters. Targeting ERK1/2, PKM2 remodeling or HIF-1α collapsed this program and protected against prostatitis pathology. Together, these findings show how T cell-derived chemokine signals are translated into macrophage metabolic reprogramming and persistent inflammatory activation, nominating the CCL5-CCR5-ERK-PKM2/HIF-1α pathway as a genetically validated and pharmacologically tractable therapeutic axis in CP/CPPS.
Coordination of mitochondrial functional states with nuclear transcriptional programs remain critical for cellular adaptation. Here, we identify mitochondrial protein import capacity as a regulator of redox-dependent signalling in tumour cells. We show that the mitochondrial import motor component TIMM44, which is upregulated in angiogenesis-driven tumours, drives respiratory remodelling without increasing mitochondrial abundance. This mitochondrial reprogramming establishes a redox-competent state that activates ASK1-p38MAPK, resulting in SP1-dependent upregulation of VEGFA independently of HIF stabilization. Importantly, induction of mitochondrial biogenesis via PGC-1α fails to recapitulate this response despite increased active mitochondrial content, demonstrating that abundance of active mitochondria or generic metabolic activation is insufficient to drive this HIF-independent transcription. Collectively, our findings identify mitochondrial protein import process as an active signalling regulator rather than a passive housekeeping role, which links mitochondrial proteostasis to redox-sensitive kinase activation and transcriptional control. This work highlights a non-canonical mitochondrial signalling mechanism through which cells couple organelle remodelling to gene expression programs.
Cancer stem cells (CSCs) contribute to therapeutic resistance, metastatic progression, and tumor recurrence, yet the metabolic pathways that sustain their survival remain incompletely understood. Here, we identify 3-mercaptopyruvate sulfurtransferase (3-MST), a hydrogen sulfide–producing enzyme encoded by MPST, as a metabolic dependency of colorectal CSCs. 3-MST expression was increased in human colorectal tumors and cancer cell lines and strongly correlated with proliferative capacity. HCT116-derived CSCs exhibited elevated 3-MST expression, increased hydrogen sulfide and reactive sulfur species production, altered membrane rigidity, and a metabolically restrained phenotype characterized by low basal oxidative phosphorylation and glycolysis. Genetic depletion of 3-MST preferentially impaired CSC proliferation, spheroid formation, stem-like properties, and migration, with less pronounced effects in differentiated parental cells. Pharmacological inhibition of 3-MST reproduced these effects across CSCs derived from several colorectal cancer cell lines and induced near-complete suppression of mitochondrial respiration and glycolytic activity. 3-MST inhibition also increased membrane fluidity, promoted cell death, and reduced CSC-derived tumor growth in mice. Integrated transcriptomic, proteomic, metabolomic, and lipidomic analyses demonstrated coordinated disruption of mitochondrial carbon metabolism, respiratory-chain maintenance, lipid desaturation, and membrane phospholipid homeostasis. These changes were accompanied by accumulation of free fatty acids and diacylglycerols and activation of antioxidants, integrated stress-response, endoplasmic-reticulum-stress, apoptotic, and p53-associated pathways. Ferroptosis-related molecular signatures were also enriched. These findings identify 3-MST as a critical regulator of colorectal CSC bioenergetics and membrane homeostasis and reveal a therapeutically exploitable metabolic vulnerability in treatment-resistant colorectal cancer.
Reactive oxygen species (ROS) are central regulators of plant growth, development, and environmental adaptation, with hydrogen peroxide (H2O2) acting as a key signaling molecule. The ascorbate-glutathione cycle is the major antioxidant pathway that maintains H2O2 homeostasis, and monodehydroascorbate reductase (MDHAR) plays a critical role by regenerating reduced ascorbate (ASC) from monodehydroascorbate (MDHA). Although traditionally viewed as an ASC-recycling enzyme, increasing evidence indicates that MDHAR has broader functions in redox regulation. Recent studies have revealed that MDHAR contributes to stress responses, developmental regulation, and ROS signaling through mechanisms that cannot be fully explained by ASC recycling alone. In this review, we summarize recent advances in the structural characteristics, regulation, subcellular specialization, and evolution of plant MDHARs. We also discuss emerging non-canonical functions of MDHAR, alternative pathways for MDHA reduction, and the mechanistic basis for the contrasting effects of MDHAR manipulation on ASC accumulation. Finally, we propose that MDHAR functions as an integrative hub linking ascorbate metabolism with cellular redox networks and highlight key challenges and future opportunities for exploiting MDHAR to improve crop stress tolerance, productivity, and nutritional quality.
Severe or recurrent acute kidney injury (AKI) is a critical risk factor for chronic kidney disease (CKD) progression, characterized by irreversible fibrosis and limited therapeutic options. Maladaptive repair in proximal tubular epithelial cells (PTECs) during AKI-to-CKD progression is crucial, with the oxidative stress (OS)-ferroptosis axis emerging as a potential therapeutic target. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of redox balance and ferroptosis, is essential for cellular homeostasis, but its role in PTECs maladaptive repair is unclear. In this study, we employed Nrf2 knockout (KO) mice to establish an AKI-to-CKD model through bilateral ischemia-reperfusion injury (bIRI). Nrf2 deficiency significantly exacerbated renal fibrosis, OS markers (H2O2, NOX4, 8-OHdG), and ferroptosis indicators (4-HNE, MDA, ACSL4), while concurrently suppressing antioxidant enzyme activity (SOD, GPx) and the expression of maladaptive repair-related genes (Vcam1, Irf8, etc.). Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were significantly elevated in KO mice. We found increased oxidative and inflammatory markers in CKD patients' serum and urine, highlighting the role of OS in disease progression. Treatment with 6K, a Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 protein-protein interaction (PPI) inhibitor, markedly improved renal function, suppressed OS and ferroptosis, and upregulated adaptive repair-related genes and downregulated maladaptive repair-related genes in bIRI mice. Compound 6K exhibited a kidney-targeted distribution profile, with an area under the concentration-time curve (AUC) kidney/AUC blood ratio of 1.01, suggesting its potential for targeted treatment of kidney diseases. In human kidney-2 (HK-2) cells, 6K activated the Nrf2-GPX4 axis, thereby alleviated RSL3-induced suppression of GPX4 expression, reduced reactive oxygen species (ROS) accumulation and lipid peroxidation, and mitigated ferroptosis. Furthermore, 6K treatment significantly delayed fibrosis progression in bIRI and unilateral ureteral obstruction (UUO) models. In summary, our findings demonstrate that Nrf2 deficiency exacerbates AKI-CKD progression through redox imbalance and ferroptosis-mediated maladaptive repair. Targeting Keap1-Nrf2 with 6K protects against renal injury and fibrosis, highlighting its therapeutic potential for kidney diseases.
Many age-related neurodegenerative disorders are marked by progressive defects in cellular energy metabolism and protein homeostasis that converge on mitochondrial and lysosomal dysfunction. TLDc domain-containing proteins, such as OXR1, NCOA7, and related family members, have emerged as crucial modulators of organellar physiology and cellular stress responses. Growing evidence indicates that TLDc proteins physically interact with vacuolar ATPases (V-ATPases) to modulate their assembly and catalytic activity, linking TLDc function directly to the maintenance of lysosomal and Golgi lumen pH. This organellar pH homeostasis, in turn, is fundamental to intracellular iron handling and metabolic regulation, processes essential for mitochondrial bioenergetics, lysosomal functions, and cellular viability. Lysosomes maintain an acidic lumen via V-ATPase proton pumping, counterbalanced by specific ion channels, including TMEM175. This acidic environment is required for ferric iron reduction and subsequent release into the cytosol; when acidification fails, cells develop cytosolic iron deficiency, mitochondrial defects, pseudohypoxia via HIF-1α activation, and inflammation. Conversely, iron flux from lysosomes to mitochondria depends on acidic conditions and direct organelle contact, as exemplified by BDH2-driven siderophore transport, a V-ATPase-dependent but not TLDc-regulated process, which supports mitochondrial bioenergetics and sustains lysosomal acidity. Iron and pH dysregulation synergize to drive ferroptosis, lipid peroxidation, and neurotoxicity. Emerging studies link lysosomal deacidification and iron dyshomeostasis to the pathogenesis of major neurodegenerative diseases. These mechanisms collectively shape neuronal resilience, survival, and aging trajectories. This review integrates recent insights into how TLDc proteins coordinate organellar pH regulation and iron homeostasis and discusses how disruption of these interconnected pathways contributes to age-related neurodegeneration.
The Keap1-Nrf2 system plays a central role in cellular defense against oxidative stress. Structural information on full-length Keap1 is essential for understanding the molecular basis of this regulation. However, its overall architecture has remained elusive due to pronounced conformational flexibility. In this study, we performed single-particle cryo-electron microscopy (cryo-EM) analysis of full-length Keap1 and found multiple particle conformations accompanied by severe preferred orientation in vitrified ice. To address these problems, we developed an analytical system that focuses on measuring the inter-domain distance between the two DC domains of the Keap1 homodimer. Using this approach, we identified the change of inter-domain distance distributions of Keap1 induced by Nrf2 binding or its inhibition, suggesting that these conformational changes are associated with Nrf2 regulation. Furthermore, through integration of this system with Keap1 deletion mutants, three flexible regions within Keap1 are found to contribute substantially to the conformational flexibility of the Keap1 homodimer. Together, these findings provide structural insights into the dynamic changes of the Keap1-Nrf2 system that would contribute to the development of Keap1-targeted therapeutics.
Background HFpEF is characterized by microvascular rarefaction and endothelial metabolic dysfunction, but the mechanisms linking these abnormalities remain unclear. Methods HFpEF was induced in mice by a high-fat diet plus L-NAME. Whole-heart lactylome profiling identified altered lactylation sites. Mechanistic studies included endothelial-targeted AAV9 manipulation and NNT-WT/NNT-K1079R rescue in vivo, complemented by experiments in primary cardiac microvascular endothelial cells. Results Endothelial HSPA1A was markedly reduced in HFpEF. Endothelial-targeted HSPA1A knockdown aggravated diastolic dysfunction, microvascular rarefaction and exercise capacity, whereas HSPA1A overexpression alleviated these features. Mechanistically, HSPA1A interacted with BAG2 to restrain CHIP-dependent HIF-1α ubiquitination, thereby stabilizing HIF-1α and sustaining endothelial glycolysis and lactate availability. Endothelial HIF-1α knockdown attenuated HSPA1A-mediated protection in vivo. Consistent with impaired lactate availability, lactate levels and protein lactylation were reduced in HFpEF hearts, and NNT-K1079 was identified as a prominently decreased lactylation site. Lactate increased NNT lactylation, whereas p300 inhibition or knockdown attenuated this response. Compared with NNT-WT, the NNT-K1079R mutant showed reduced lactate responsiveness, impaired redox defense and angiogenic function. To test whether NNT-K1079 mediates HSPA1A protection, NNT epistasis and rescue experiments were performed. Endothelial NNT knockdown weakened HSPA1A-mediated protection, whereas NNT-WT, but not NNT-K1079R, restored microvascular density and diastolic function. In turn, loss of NNT-K1079 lactylation disrupted mitochondrial ROS/ATP homeostasis, thereby suppressing HSF1-dependent HSPA1A transcription. Conclusions HSPA1A sustains NNT-K1079 lactylation and mitochondrial redox homeostasis through the BAG2–CHIP–HIF-1α–glycolysis/lactate pathway. In turn, NNT-K1079 lactylation sustains HSPA1A expression, forming a protective feedback loop whose disruption contributes to microvascular rarefaction and diastolic dysfunction in HFpEF.
Myocardial infarction (MI) initiates a wound-healing response where immune cells shape inflammation, tissue repair, and long-term remodeling. Although CD4+ T cells are increasingly recognized as contributors to post-MI healing, the transcriptional reprogramming defining their early pro-reparative functions remains incompletely resolved. Here, RNA sequencing of cardiac CD4+ T cells isolated 1 week after MI found that a substantial post-MI transcriptional fraction lay outside canonical cytokine-induced T helper subset polarization, including type 1 T helper cell (Th1), Th2, Th17, nature-occurring CD4+ regulatory T cell (nTreg), and peripherally induced Treg (iTreg) reference transcriptomic programs. Instead, this response was organized into a distinct CD4+ Th tissue injury-polarized (CD4+/TIP) transcriptomic module enriched for extracellular matrix organization, adhesion, vascular, and developmental programs, with a coordinated downregulated arm involving RNA metabolism, chromatin regulation, and protein catabolic processes. Within the CD4+/TIP population, MI induced and polarized at least 10 transcriptionally distinct CD4+ Th subsets at 1 week post-MI. Integration with curated transcription factors, epigenetic, reduction-oxidation (redox), and unfolded protein response (UPR) datasets identified a stress-adaptive architecture, in which regulatory subsets and the CD4+/TIP population shared redox attenuation features, while the CD4+/TIP state showed the strongest coupling to reparative tissue interaction programs, predominant Activating Transcription Factor 6 (ATF6)-aligned UPR structure, restrained proteostasis related outputs, and an innate-adjacent immune and secretome signature enriched for complement-associated, inflammatory recruitment, and extracellular communication genes. These findings identify a specific MI-associated CD4+ T-cell transcriptomic state during early MI inflammation and tissue repair. They establish a coordinated framework in which redox control, UPR, and tissue-injury-polarized CD4+ T-cell immune programs converge outside traditional Th and Treg lineages, offering new targets for CD4+ T-cell-mediated tissue repair after MI.
Background Alkaloids from Macleaya cordata have demonstrated to possess anti-tumor effects against various cancer types, but their efficacy on GBM remains unexplored. In this study we investigated the anti-GBM activity of 6-Met and its underlying mechanisms. Methods GBM cell proliferation was assessed using CCK-8, RTCA, and colony formation assays in vitro. A xenograft mode used to investigate the anti-tumor efficacy of 6-Met. Apoptosis and ROS were quantified by Flow cytometry. GSH content was determined using a commercial GSH/GSSG assay kit. In parallel, the activities of GSS, GCLC, and GR, were quantified by corresponding commercial assay kits. Cellular bioenergetics was evaluated by Seahorse XFe96 analyzer. Ubiquitination analysis was conducted by immunoprecipitation (IP) using an anti-ubiquitin antibody. Finally, expression changes were analyzed by Western blotting and quantitative reverse transcription PCR. Results Our results revealed that 6-Met significantly suppressed GBM cell growth in vitro and transplanted tumor growth in vivo. Additionally, 6-Met induced caspase-dependent apoptosis in GBM cells. Mechanistically, 6-ME suppressed GSS expression by enhancing its ubiquitination, resulting in impaired GSH synthesis and subsequent ROS accumulation. The elevated ROS levels further disrupted mitochondrial function by damaging Fe-S clusters, as evidenced by reduced expression of Fe-S cluster-associated proteins, ultimately impairing mitochondrial respiration. Concurrently, ROS activation suppressed the mTOR signaling via modulation of its phosphorylation. Notably, exogenous GSH supplementation and ectopic GSS expression mitigates 6-Met cytotoxicity in GBM cells. Furthermore, exogenous expression of GSS abrogated 6-Met-induced transplanted tumor growth inhibition in vivo. Clinically, we observed that GSS expression was significantly elevated in GBM tissues compared to normal brain tissues, and lower GSS expression correlated with better patient prognosis, highlighting its potential as a prognostic marker. Conclusion Collectively, these findings establish that 6-Met inhibits GBM tumor growth by disrupting GSS-dependent glutathione synthesis, thereby promoting oxidative stress, impairing mitochondrial respiration and mTOR signaling, and ultimately triggering apoptosis.
Human selenoprotein S (selenos) is part of the integrated cellular stress response and linked to protein quality control and signaling pathways. Consequently, genetic polymorphisms of selenos are associated with increased risks for diabetes, dyslipidemia, and cardiovascular diseases. Determining the specific roles of selenos in these cellular pathways and diseases has been challenging, as selenos associates with a wide range of protein complexes. Thus, to map the cellular functions of selenos and uncover their interconnections, we used affinity purification and in vivo crosslinking to stabilize transient protein interactions, followed by proteomics to record the resulting selenos interactome. Through mapping of selenos protein partners, we found evidence that selenos associates with complexes responsible for the insertion of membrane proteins into the endoplasmic reticulum (ER) bilayer and their connected quality control components. Furthermore, selenos is also part of metabolic, trafficking, and mitochondrial pathways. Notably, proteins involved in translation preferentially associate with selenos when its C-terminal intrinsically disordered segment containing the redox-active motif is accessible. Together, these results identify the C-terminal redox loop of selenos as a central interaction hub connecting translation with ER membrane protein biogenesis and quality control.
Transcription factors often exhibit a striking paradox: they function as tumor suppressors in one context and promote oncogenesis in another. The underlying mechanisms of this context dependence have remained elusive. We propose a novel conceptual framework, systemic redox switching, to resolve this paradox. Based on our research on upstream stimulatory factor 2 (USF2) and convergent observations on other factors, our model suggests that redox regulation is network-embedded rather than driven by discrete cysteine switches. We propose that transcription factors occupy distinct regulatory regimes (homeostatic, adaptive, and survival states) which are connected by threshold-like, hysteretic transitions. Beyond classical graded input–output views, this framework explicitly posits discrete, hysteretic regime transitions at the transcriptional-network level and links them to a minimal dynamical model of the USF2–TFEB–NRF2–redox motif. These transitions convert continuous redox inputs into distinct changes in promoter occupancy and transcriptional programs. USF2 exemplifies a kinase-integrated, non-canonical switch that decodes mitochondrial and autophagy signals via phosphorylation (e.g., Ser155) and context-dependent cooperation with NRF2 and HIFs. Extending this logic to diverse archetypes (NRF2, HIFs, FOXOs, c-MYC, and AHR) demonstrates the framework's generalizability. The model is experimentally tractable; targeted perturbations of primary sensing modules (e.g., KEAP1 mutation, PHD inhibition, or USF2 phosphorylation disruption) should predictably alter regime transitions. By reframing paradoxical behaviors as controlled state transitions, this framework provides a unifying, network-level understanding with direct implications for targeting therapies in cancer, metabolic diseases, and age-related pathologies.
BACKGROUND:Heart failure (HF) progression is closely linked to oxidative stress. 5-Oxoproline (5-OP), a product of glutathione degradation, is normally metabolized by 5-oxoprolinase (OPLAH) but accumulates when the gamma-glutamyl cycle is disrupted. Here, we investigated the clinical characteristics of circulating 5-OP, its proteomic correlates, and the associations to outcome in HF. METHODS:In serum of 823 BIOSTAT-CHF patients, 5-OP was quantified by validated liquid chromatography-mass spectrometry and analyzed for associations with clinical outcomes. Proteomic correlates were identified across 355 OLINK proteins using stability selection with Minimax Concave Penalty regression. Mechanistic context was evaluated in a multi-comorbidity, large-animal cardio-kidney-metabolic (CKM) model with regional OPLAH assessment. RESULTS:Higher 5-OP was associated with worse renal function (eGFR declining across 5-OP tertiles, 67.9 to 60.2 mL/min/1.73 m2; p = 0.0012) and higher all-cause mortality (HR 1.55, 95% CI 1.11-2.17, p = 0.010). Per SD increase in log-5-OP, risk for the 2-year composite endpoint increased (HR 1.27, 95% CI 1.05-1.53), with broadly similar associations across CKD strata (interaction p = 0.63). TGF-α was the most robust proteomic correlate (π = 0.70; empirical permutation p = 0.001). In CKM swine, circulating 5-OP was elevated and renal cortical OPLAH protein, but not cardiac OPLAH, was selectively reduced, consistent with a renal contribution to systemic 5-OP elevation. CONCLUSION:Circulating 5-OP identifies HF patients at higher risk and is robustly associated with TGF-α. In a translational swine model, selective loss of renal cortical OPLAH provides tissue context supporting a renal contribution to systemic 5-OP elevation in cardiorenal syndrome.
Heart failure (HF) remains a leading cause of morbidity and mortality worldwide. A hallmark of HF progression is profound metabolic remodeling accompanied by mitochondrial dysfunction in cardiomyocytes. Impaired mitochondrial oxidative phosphorylation, excessive reactive oxygen species (ROS) production, and disrupted redox homeostasis collectively drive oxidative damage and compromise mitochondrial integrity, ultimately leading to contractile failure, for which no viable strategies currently exist. Although mitochondrial dysfunction is now recognized as a central driver of HF pathogenesis, the upstream molecular regulators that initiate or amplify these defects remain incompletely understood. Here, we identify myocilin as a fibroblast-derived mediator that drives cardiomyocyte mitochondrial dysfunction and ROS production in HF. Myocilin was consistently upregulated in patients with HF and in murine HF models induced by transverse aortic constriction and isoproterenol, and was predominantly expressed in cardiac fibroblasts. In vivo study using male mice showed that myocilin overexpression exacerbated cardiac dysfunction and fibrosis, whereas genetic ablation markedly alleviated pathological remodeling. Using transwell systems and recombinant protein stimulation, we found that fibroblast-derived myocilin impaired mitochondrial function in cardiomyocytes, as evidenced by reduced ATP production, increased ROS, and loss of membrane potential. Mechanistically, myocilin directly interacted with SLC3A2, the heavy chain that pairs with SLC7A11 to form the cystine/glutamate antiporter, on cardiomyocytes and promoted its degradation, thereby impairing cystine uptake. This led to glutathione depletion and redox imbalance, subsequently triggering ferroptosis-associated mitochondrial dysfunction in cardiomyocytes. Collectively, these findings identify a fibroblast-cardiomyocyte signaling axis in which myocilin disrupts cardiomyocyte metabolic homeostasis. Targeting the myocilin-SLC3A2 pathway may represent a potential therapeutic strategy for HF.
Nitric oxide (NO) is a pleiotropic free radical that functions as a master regulator of gene expression, and its sustained production within the tumor microenvironment reshapes the epitranscriptomic state of cancer cells. We previously demonstrated that NO inhibits the m6A mRNA demethylases FTO and ALKBH5 through dinitrosyliron complex formation while leaving the methyltransferase METTL3 intact, a demethylase-specific perturbation that increases global m6A on mRNA. Here, integrating m6A-RIP-seq and RNA-seq from triple-negative breast cancer cells, we show that chronic NO does not produce the uniform hypermethylation anticipated from demethylase inhibition. Instead, it redistributes m6A on mRNA, enriching the 5'UTR and coding sequence while depleting the 3'UTR and departing from the canonical stop-codon and 3'UTR topology. We found that the position of m6A, rather than its intensity or mere presence, shapes the outcome, in part by determining which reader protein is predicted to recognize it. In parallel, NO drives a canonical NF-κB and inflammatory transcriptional program. The transcriptional program is independent of the m6A methylome in both which genes respond and how strongly they respond, ruling out a linear methylome-to-transcriptome cascade; even so, m6A position remains associated with the direction of change among responding transcripts. The 3'UTR is the primary site of m6A loss and shows a suggestive computational link to miRNA-mediated regulation. Sense-antisense coordination reinforces the transcriptional response without bridging the two programs. These findings demonstrate that NO not only increases m6A abundance, but it also rewrites the m6A positional code, establishing spatial reprogramming of the epitranscriptome as a previously unrecognized mode of gene regulation.
Chemotherapy resistance remains a significant challenge in colorectal cancer (CRC) treatment, with disrupted redox balance playing a central role. Here we identify CHTOP as a key regulator of oxidative stress and chemoresistance in CRC. Mechanistically, CHTOP promotes NRF2 transcriptional activity by recruiting the SENP3-containing 5FMC complex to deSUMOylate NRF2, thereby sustaining HO-1 expression and redox balance. Notably, CHTOP expression itself is tightly controlled by a feedback mechanism. The p52 isoform of PSIP1 inhibits CHTOP expression by interfering with HNRNPH1-mediated splicing, leading to CHTOP degradation via nonsense-mediated decay (NMD). Conversely, elevated oxidative stress stabilizes SENP3, which promotes deSUMOylation and degradation of p52, thereby relieving p52-mediated suppression of CHTOP expression. This establishes an oxidative stress-SENP3-p52-CHTOP feedback loop that fine-tunes CHTOP levels. In 5-FU-resistant CRC cells, CHTOP downregulation shifts cells into an elevated oxidative stress state, which correlates with reduced 5-FU sensitivity. Notably, either restoring CHTOP expression or further depleting CHTOP disrupts this redox balance and resensitizes resistant cells to 5-FU. These findings suggest that modulating CHTOP expression may offer a therapeutic strategy to overcome chemoresistance in CRC through redox regulation.
