Genomic instability is increased in patients with inflammatory bowel disease (IBD), yet whether it contributes directly to disease pathogenesis remains unclear. Here, we identify RADX, a structural antagonist to RAD51 and a key regulator of replication fork stability, as a critical suppressor of intestinal inflammation by limiting innate immune sensing of replication-associated DNA damage. RADX deficiency exacerbates experimental colitis, with macrophages serving as the principal mediators of this phenotype. Mechanistically, RADX competes with the DNA sensor IFI16 for binding to single-stranded DNA (ssDNA). Loss of RADX promotes ssDNA accumulation, triggering IFI16-dependent activation of NF-κB signaling and inflammasome assembly, thereby driving intestinal inflammation. Consistent with these findings, two RADX variants identified in patients with IBD associate with reduced RADX protein expression, increased DNA damage signaling, and elevated IL-1β levels. Pharmacological inhibition of RAD51 with RI-1 alleviated colitis in both wild-type and Radx-deficient mice. Together, these findings establish a mechanistic link between genome instability and intestinal inflammation, identify a RADX-IFI16 checkpoint that restrains pathogenic innate immune activation, and nominate modulation of replication stress as a therapeutic strategy for IBD. Genomic instability is observed in IBD, yet its pathogenic mechanism is unclear. Here, RADX, replication fork protector, is shown to suppress colitis by limiting ssDNA accumulation and IFI16-dependent inflammation, revealing a new therapeutic vulnerability. Genomic instability is observed in IBD, yet its pathogenic mechanism is unclear. Here, RADX, replication fork protector, is shown to suppress colitis by limiting ssDNA accumulation and IFI16-dependent inflammation, revealing a new therapeutic vulnerability.

Phase separation (PS) underpins compartmentalization in living cells, facilitating the formation of membraneless organelles and the regulation of cellular processes. Despite the increasingly pivotal role of engineering protein PS properties in the study and regulation of cellular physiological processes, manipulating PS ability through single amino acid alterations remains a challenge. Here, we develop phase separation scalpel (PScalpel), a machine learning-based tool identifying and recommends protein engineering strategies for directed changes in PS ability. Based on our biological experimental data, we apply transfer learning to achieve the feedback-driven optimization of specific protein prediction accuracy--markedly enhancing the predictive performance for TDP43, a neurodegenerative disease-associated protein. Furthermore, by engineering the crucial nucleic acid sensor cGAS as a model application, we successfully modulate its PS ability in the anticipated direction by altering a single amino acid, which subsequently optimizes its immune function and impacts the activity of engineered macrophages. Transcriptomic analysis of these cGAS-engineered macrophages further demonstrated that the immune function of macrophages can be altered by the manipulation of cGAS PS ability. In summary, PScalpel is an effective tool for guiding PS ability engineering, enabling targeted molecular and cellular modifications and providing nuanced methods for precise biomolecular engineering in future research.

The epigenome plays fundamental roles in diverse physiological processes, including the innate immune response to pathogen invasion. Dysregulation of epigenetic regulators leads to aberrant expression and activation of signaling cascades, thereby driving the development of disease. In this review, we discuss recent advances in how epigenetic regulators shape innate immune signaling networks, as well as the crosstalk between the epigenome and metabolites that have been identified as key regulatory molecules. We further highlight the critical functions of epigenetic modifiers in the pathogenesis of immune-related disorders, including sepsis and oral diseases, and summarize progress in therapeutic strategies targeting these regulatory factors.

METTL3, a key RNA N6-methyladenosine (m6A) methyltransferase, plays essential roles in cell fate regulation and tissue homeostasis, yet therapeutic strategies to enhance its activity remain unexplored. Here, we profile the S-palmitoylation landscape during embryonic stem cell differentiation and observe increased METTL3 S-palmitoylation at cysteine 376 during mesodermal commitment. This modification is catalyzed by ZDHHC24 and reversed by ABHD17A. METTL3 C376S mice exhibit cartilage defects and exacerbated osteoarthritis (OA). Through AI-guided screening, we identify Isoborneol as a small molecule that enhances METTL3 S-palmitoylation by disrupting its interaction with ABHD17A. Isoborneol treatment alleviates joint degeneration and preserves cartilage integrity in OA models. Mechanistically, S-palmitoylation promotes METTL3 condensate formation in proximity to ribosomes, facilitating its cytoplasmic spatial compartmentalization. This condensate state suppresses chaperone-mediated autophagy, thereby enhancing METTL3 protein stability. Our findings reveal S-palmitoylation as a regulatory mechanism governing METTL3 localization and turnover and establish a pharmacological strategy for restoring METTL3 activity in OA.

Acute severe ulcerative colitis (ASUC) now imposes an increasing global burden, yet lacks broadly effective therapeutic options. While organoid transplantation represents a promising approach for intestinal injuries, its efficacy for ASUC treatment remains suboptimal. Here, we elucidate the intrinsic mechanism of RIPK1 involvement in necroptosis initiation, and further develop an organoid-based dual-axis therapeutic paradigm for ASUC. We identified that RIPK1 undergoes PIAS1-catalyzed SUMO1 modification at lysine 305, which promotes its compartmentalization within phase-separated structures, thereby serving as nucleation platforms for accelerating RIPK3 amyloid fibril assembly. Interfering with phase separation of RIPK1 suppresses necroptosis in intestinal cells and colonic organoids in vitro, as well as alleviates intestinal injury and reduces mortality in vivo. Notably, while colonic organoid transplantation showed limited therapeutic efficacy in ASUC, a synergistic therapy combining necroptosis blockade and organoid transplantation effectively reduced inflammatory damage and enhanced epithelial regeneration by reprogramming the intestinal microenvironment. These findings suggest that the SUMO1-RIPK1 axis functions as a druggable checkpoint governing necroptotic cell fate and presents a clinically actionable strategy to potentiate regenerative medicine paradigms in ASUC pathogenesis.

How liver metastases evade immunity remains unclear. A new study published in Nature Metabolism reveals that DHHC17-driven laminin 511 palmitoylation and its subsequent secretion blunts neutrophil-mediated killing of metastatic cancer cells, uncovering a druggable immune checkpoint in the liver.

This study aimed to investigate the cardioprotective role and underlying mechanisms of the endogenous metabolite mesaconate in both acute and chronic cardiovascular disease models, with a focus on its regulation of macrophage inflammation. Transcriptomic profiling and functional validation in bone marrow-derived macrophages suggested GPR35 as a potential target of mesaconate. Further mechanistic studies revealed that mesaconate alleviates macrophage ferroptosis via GPR35, thereby contributing to its anti-inflammatory effects. In a myocardial ischemia/reperfusion model, mesaconate significantly attenuated cardiac injury, improved systolic function, and reduced infarct size, while myeloid-specific GPR35 knockdown abolished these protective effects. In aged mice, mesaconate ameliorated cardiac hypertrophy and vascular dysfunction, effects closely associated with suppressed macrophage inflammation. Collectively, this study demonstrates for the first time that mesaconate alleviates macrophage inflammation by activating GPR35 to suppress ferroptosis, confers GPR35-dependent cardioprotection in ischemic injury, and mitigates aging-related cardiovascular pathology, highlighting mesaconate as a promising therapeutic candidate for inflammatory cardiovascular diseases.

Metabolic pathways determine cellular fate and function; however, the exact roles of metabolites in host defence against influenza virus remain undefined. Here we employed pharmacological inhibition and metabolomics analysis to show that the metabolic pathways of oxaloacetate (OAA) are integrated with antiviral responses to influenza virus. Cytosolic malate dehydrogenase 1 senses intracellular OAA to undergo dimerization and functions as a scaffold to recruit the transcription factor ETS2 for phosphorylation by the kinase TAOK1 at serine 313. The phosphorylated ETS2 translocates into the nucleus and supports optimal expression of TBK1, an indispensable activator of type I interferon responses. OAA supplementation provides a broad-spectrum antiviral ability, and OAA deficiency caused by Acly genetic ablation decreases antiviral immunity and renders mice more susceptible to lethal H1N1 virus infection. Our results uncover a signalling pathway through cellular OAA sensing that links metabolism and innate immunity to coordinate defence against viral challenge.

Neonatal sepsis (NS) is highly likely to cause death; however, early diagnosis of NS is still a great challenge. This study aimed to determine the diagnostic values of IL-6, IL-8, and serum amyloid A (SAA) in NS patients. C-Reactive protein (CRP), procalcitonin (PCT), interleukin (IL)-6, IL-8, and SAA were detected in 120 infants with NS (60 premature infants [NS-PIs] and 60 term infants [NS-TIs]). Sixty noninfected premature infants and 60 noninfected term infants composed the control group. Receiver operating characteristic (ROC) curves were used to determine the sensitivity and specificity of IL-6, IL-8, and SAA alone and in combination with CRP and PCT. The area under the curve (AUC) was calculated to evaluate the diagnostic value. The clinical characteristics of the subjects were recorded. The expression of CRP, PCT, IL-6, IL-8, and SAA was upregulated in patients with NS compared with control subjects. When the SAA cut-off value was 10.18 mg/L, the greatest AUC for the diagnosis of NS-PIs was for SAA (AUC = 0.833, 95% CI 0.762-0.905, P < 0.001). When the CRP cut-off value was 9.562 mg/L, the smallest AUC for the diagnosis of NS-PIs was for CRP (AUC = 0.776, 95% CI 0.684-0.867, P < 0.001). When the IL-8 cut-off value was 52.03 pg/mL, the greatest AUC for the diagnosis of NS-TIs was for IL-8 (0.821). When the IL-8 cut-off value was 52.03 pg/mL, the greatest AUC for the diagnosis of NS-TIs was for IL-8 (AUC = 0.821, 95% CI 0.745-0.898, P < 0.001). When the CRP cut-off value was 13.18 mg/L, the smallest AUC for the diagnosis of NS-TIs was for CRP (AUC = 0.762, 95% CI 0.667-0.857; P < 0.001). Additionally, according to the AUC value, the best combination was SAA and PCT for NS-PI diagnosis, and the best combination was PCT and IL-6 for NS-TI. In conclusion, compared with PCT and CRP, IL-6, IL-8, and SAA are better diagnostic biomarkers. Moreover, PCT combined with SAA is more suitable for diagnosing NS-PIs, and PCT combined with IL-6 is more suitable for diagnosing NS-TIs.

Apoptosis is indispensable for a variety of physio-pathological processes. RNA is one of essential macromolecules for life. Extensive RNA decay is a characteristic feature of apoptosis. However, it is unknown whether there is de novo RNA synthesis in apoptotic cells and metabolites. In this study, we show that apoptotic mesenchymal stem cells (MSCs) and their apoptotic vesicles (apoVs) synthesize de novo RNAs. Nascent RNA-seq showed apoptotic MSCs and apoVs produced numerous nascent RNAs that were different from those in living MSCs, including protein-coding and non-coding RNAs. Mechanistically, apoptotic de novo RNA synthesis was related to the caspase-3/Sp1/RNA polymerase axis. Additionally, we found the LINE-2a (L2a) and LINE-2b (L2b) RNAs were specifically transcribed in apoptotic MSCs and transferred into apoVs to prevent virus infection. Altogether, this study reveals a previously unknown phenomenon that apoptotic cells synthesize various de novo RNAs and identifies that apoptotic LINE-2 RNAs can regulate innate immunity to prevent virus infection.

Proteotoxicity induced by misfolded or aggregated proteins causes progressive neuronal damage. The endoplasmic reticulum (ER) protein quality control (ERQC) pathways are responsible for mitigating the accumulation of these misfolded or aggregated proteins, thus reducing proteotoxicity. Enhancing ERQC pathways is a promising strategy for treating neurodegenerative diseases. However, the mechanisms governing the initiation and degradation of misfolded or aggregated proteins in neurons remain largely unknown in vivo. In studying the maturation of proAVP in mouse AVP neurons, this study discovers that autophagy and ER-associated degradation (ERAD) ERQC pathways collaborate to maintain proAVP maturation and protect AVP neuron survival against proteotoxicity. Autophagy deficiency in mouse AVP neurons leads to the late-onset of diabetes insipidus. Mechanistically, autophagy selectively degrades mutant proAVP aggregates and endogenous HRD1 of the SEL1L-HRD1 ERAD complex through FAM134B mediated ER-phagy. HRD1 induction is responsible for reducing proAVP aggregation and maintaining AVP neuron function and survival under autophagy deficiency. Thus, autophagy and ERAD form a dual-protection system that orchestrates prohormone maturation and endocrine neuron survival, providing new insights in the complexity of neuroendocrinology and the intrinsic mechanism of neurodegenerative diseases, with therapeutic potential in protein folding diseases.

Missense mutations could affect the Liquid-Liquid Phase Separation (LLPS) propensity of proteins and lead to aberrant phase-separating behaviours, which are recently found to be associated with many diseases including Alzheimer's and cancer. However, the regulatory role of mutations in LLPS remains unclear due to challenges in accurately characterizing the LLPS ability of mutants, including the high similarity in features, lack of labeled data, and vast amounts of data involved. To bridge this gap and facilitate the discovery of therapeutic strategies, we propose the first machine learning-based guider for protein phase-separating behaviour alteration, PScalpel. PScalpel leverages both structural information and an auxiliary tasks-based graph contrastive learning framework to distinguish the mutants’ LLPS ability, and incorporates a genetic algorithms-based recommendation method to identify mutants with desired LLPS properties. Comprehensive computational and biological experiments validate the effectiveness of PScalpel as a versatile tool for guiding alterations in protein phase separation behavior.

N6-methyladenosine (m6A) is the most prevalent modification of mRNA which controls diverse physiological processes. Although m6A modification has been reported to regulate type I interferon (IFN) responses by targeting the mRNA of IFN-β and the interferon-stimulated genes (ISGs), the detailed mechanism of how m6A methyltransferase complex (MTC) rapidly responds to conduct the modification on nascent mRNA during IFN-β stimulation remains largely unclear. Here, we demonstrate that WTAP, the adaptor protein of m6A MTC, undergoes dephosphorylation-regulated phase transition from aggregates to liquid-like condensates under IFN-β stimulation, thereby mediating m6A modification of a subset of ISGs to restrict their expression. The phase transition of WTAP promotes the interaction with nucleus-translocated transcription factor STAT1, recruits MTC to the promoter regions of ISGs and directs the co-transcriptional m6A modification on ISG mRNAs. Collectively, our findings reveal a novel regulatory role of WTAP phase transition in manipulating signaling pathways and fine-tuning immune response by orchestrating dynamic m6A modification through the cooperation of transcription factors and MTC. Our findings unveil a novel mechanism by which WTAP phase transition controls immune homeostasis via transcription factor-MTC-driven dynamic m6A modification, thereby proposing a potential therapeutic target for alleviating immune dysregulation.

Ferroptosis is closely linked with various pathophysiological processes, including aging, neurodegeneration, ischemia-reperfusion injury, viral infection and, notably, cancer progression; however, its post-translational regulatory mechanisms remain incompletely understood. Here we revealed a crucial role of S-palmitoylation in regulating ferroptosis through glutathione peroxidase 4 (GPX4), a pivotal enzyme that mitigates lipid peroxidation. We identified that zinc finger DHHC-domain containing protein 8 (zDHHC8), an S-acyltransferase that is highly expressed in multiple tumors, palmitoylates GPX4 at Cys75. Through small-molecule drug screening, we identified PF-670462, a zDHHC8-specific inhibitor that promotes the degradation of zDHHC8, consequently attenuating GPX4 palmitoylation and enhancing ferroptosis sensitivity. PF-670462 inhibition of zDHHC8 facilitates the CD8+ cytotoxic T cell-induced ferroptosis of tumor cells, thereby improving the efficacy of cancer immunotherapy in a B16-F10 xenograft model. Our findings reveal the prominent role of the zDHHC8–GPX4 axis in regulating ferroptosis and highlight the potential application of zDHHC8 inhibitors in anticancer therapy. Zhou et al. show that palmitoylation of GPX4 via ZDHHC8 sustains ferroptosis resistance in melanoma and other cancer cells and propose targeting ZDHHC8 with a small molecule to boost CD8+ T cell-induced ferroptosis, thereby improving immunotherapy.

ABBREVIATION:17-ODYA: 17-octadecynoic acid; 293T: HEK293T; 2-BP: 2-bromopalmitate; 2CS: Cys155Ser and Cys156Ser; ABE: acyl-biotin exchange; AP: adaptor protein; APEX2: ascorbate peroxidase 2; ATG: autophagy related; baf A1: bafilomycin A1; CRISPR: clustered regularly interspaced short palindromic repeats; CTD: C-terminal domain; Cys: cysteine; DAB: 3,3'-diaminobenzidine; EV: empty vector; H2O2: hydrogen peroxide; IF: immunofluorescence; IP: immunoprecipitation; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NTD: N-terminal domain; PAS: phagophore assembly site; PBS: phosphate-buffered saline; PtdIns3K-CI: class III phosphatidylinositol 3-kinase complex I; PM: plasma membrane; PTM: post-translational modifications; Ser: serine; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TGN: trans-Golgi network; ULK1: unc-51 like autophagy activating kinase 1; WCL, whole cell lysates; WDR45/WIPI4: WD repeat domain 45; WT: wild-type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Sepsis is a leading cause of death, with the liver being particularly vulnerable to sepsis-related injuries. This damage significantly contributes to disease progression, underscoring the need for new treatments. Brown adipose tissue (BAT) secretes various cytokines, including neuregulin 4 (Nrg4), which plays a protective role in hepatic glucose and lipid metabolism. Ferroptosis, a key type of cell death in sepsis-induced liver injury, has recently gained attention. This study aimed to investigate how BAT-secreted cytokines alleviate liver ferroptosis in sepsis. Septic liver injury was induced in the control and BAT group using cecal ligation and puncture (CLP) and lipopolysaccharide injections. BAT removal worsened ferroptosis; in contrast, CL316243 activation reduced it. These findings suggest that Nrg4 secretion following BAT activation protects the liver during sepsis by inhibiting ferroptosis. Future therapies targeting BAT activation and Nrg4 could potentially mitigate sepsis-induced liver damage, offering new insights into treatment strategies.

Innate immune cells can acquire a memory phenotype, termed trained immunity, but the mechanism underlying the regulation of trained immunity remains largely elusive. Here, we demonstrate that inhibition of Aurora kinase A (AurA) dampens trained immunity induced by β-glucan. ATAC-seq and RNA-seq analysis reveal that AurA inhibition restricts chromatin accessibility of genes associated with inflammatory pathways such as JAK-STAT, TNF, and NF-κB pathways. Specifically, AurA inhibition promotes nuclear localization of FOXO3 and the expression of glycine N-methyltransferase (GNMT), a key enzyme responsible for S-adenosylmethionine (SAM) consumption. Metabolomic analysis confirms a reduction in SAM level upon AurA inhibition. As a result of SAM deficiency, trained mouse macrophages exhibit decreased H3K4me3 and H3K36me3 enrichment on gene regions of Il6 and Tnf . Additionally, the tumor inhibition effect of β-glucan is notably abolished by AurA inhibition. Together, our findings identify an essential role of AurA in regulating trained immunity via a methylation-dependent manner by maintaining endogenous SAM levels through the mTOR-FOXO3-GNMT axis.