Type IV-C CRISPR-Cas systems remain enigmatic compared to other class 1 systems. Here, we expand the type IV-C catalog, identifying two phylogenetically distinct clades primarily found in archaea (IV-C1) or bacteria (IV-C2), distinguishable by the Cas10IVc subunit architecture. We functionally and structurally characterize type IV-C1 systems from Thermococcus onnurineus (Ton) and Pyrococcus abyssi (Pab). Type IV-C complexes assemble with crRNAs derived from distinct CRISPR arrays and recognize a 5'-GGG-3' protospacer adjacent motif (PAM) to bind double-stranded DNA targets. Target recognition activates the HD domain of Cas10IVc, triggering metal-dependent collateral cleavage of single-stranded DNA and RNA. This behavior is explained by allosteric alignment of the HD active site, triggered by PAM-dependent R-loop formation, as revealed by cryo-EM. Together, our findings suggest that type IV-C systems provide immunity via non-specific cleavage of nucleic acids generated during mobile genetic element replication or transcription.
Sex-dimorphic adipose mitochondrial function (lower activity in males) correlates with visceral adiposity and metabolic risk, yet the underlying mechanisms remain elusive. We find that androgen-androgen receptor (AR) signaling suppresses mitochondrial respiration and thermogenesis in visceral adipose tissue (VAT), promoting visceral fat accumulation. Mechanistically, androgen-AR signaling represses transcription of Pdhb, which encodes a pyruvate dehydrogenase (PDH) subunit, thereby reducing PDH activity, acetyl-CoA levels, H3K27 acetylation, and chromatin accessibility at the promoters of mitochondrial respiration-related genes. Notably, Pdhb overexpression largely reverses these alterations and restores mitochondrial function. Furthermore, sodium dichloroacetate, a PDH activator, enhances mitochondrial respiration and reduces visceral fat in male mice. Multi-omics analyses reveal that the androgen-PDH axis orchestrates a male-specific chromatin-based transcriptional landscape that encompasses mitochondrial and metabolic pathways in visceral adipocytes. Collectively, this study identifies the androgen-PDH axis as a key regulator of sexual dimorphism in mitochondrial metabolism and adipose homeostasis in VAT.
Autoreactive T cells that destroy beta cells in type 1 diabetes are largely targeting insulin signal peptide fragments. HLA knockout beta-cells have been proposed to create hypo-immune cells, but this strategy poses significant tumorigenic risks. Alternatively, we hypothesized that insulin signal peptide modification would give rise to beta cells evading autoimmune recognition while maintaining insulin functionality. We developed human beta-cell lines lacking endogenous insulin and complemented them with insulin-carrying signal peptides from different hormones including chromogranin-A. We evaluated insulin synthesis, processing, secretion, activity, and immune evasion. Chromogranin-A signal peptide substitution directed insulin expression, maturation, and secretion, maintaining physiological proinsulin/insulin ratios. Secreted insulin remained functional. Crucially, beta cells expressing insulin with chromogranin-A signal peptide evaded recognition and killing by autoreactive T cells without inducing ER stress or compromising cellular identity. This approach may offer a targeted alternative to systemic immunosuppression to be used in stem-cell-derived therapies by engineering endogenous insulin signal peptide.
The tumor microenvironment (TME) domesticates macrophage function by decreasing chromatin accessibility. The activation and nuclear translocation of ATP-citrate lyase (ACLY) convert citrate to acetyl-CoA, providing a substrate necessary for histone acetylation. However, the underlying mechanisms in macrophage remodeling are poorly understood. Here, we found that saturated fatty acids (sFA), especially palmitic acid (PA), were lower in TAMs of patients with hepatocellular carcinoma (HCC). Scd1 knockout promoted PA accumulation, resulting in both primary and metastatic liver cancer retardation and overall survival improvement. Mechanisms indicated that ACLY-C893 palmitoylation via PA maintained tetramer stability against CUL3-mediated degradation, facilitating histone acetylation of M1-related genes. Notably, both dietary PA with Scd1KO macrophage infusion and TAM-targeted in vivo PA/shSCD1 reprogramming improved the TME to repress HCC progression. Collectively, our research highlights the crucial role of ACLY palmitoylation in the connection between macrophage FA metabolism and histone acetylation reprogramming, which sheds light on the strategy of macrophage-based HCC immunotherapy.
Metabolic alterations are increasingly recognized during influenza virus infection, but how local lactate accumulation shapes antiviral immunity remains poorly characterized. By integrating time-series targeted energy metabolomics, single-cell RNA sequencing, flow cytometry, and functional perturbation, we show that influenza virus infection preferentially increases lactate within the lung microenvironment, where it restrains pulmonary CD8+ T cell response. Mechanistically, extracellular lactate enters dendritic cells through monocarboxylate transporter (MCT)-dependent transport and induces a tolerogenic-like state marked by impaired maturation, reduced costimulation, and diminished CD8+ T cell-priming capacity. Direct experimental evidence identifies H3K18la as a prominent lactate-responsive histone lactylation mark, while multi-omics integration links it to enhancer accessibility and NRF2 pathway activation. Functional studies further show that NRF2 promotes dendritic cell suppression by reinforcing tolerogenic programs and limiting mtROS-dependent XBP1 splicing. Together, these findings reveal a lactate-driven histone lactylation-NRF2 pathway that modulates antiviral immunity during influenza infection.
Loss of major histocompatibility complex class I (MHC-I) molecules from the tumor cell surface is a common mechanism of immune evasion; yet, the receptor intrinsic events that initiate their endocytic removal remain unclear. Here, using HLA-A∗11:01, a human MHC-I heavy chain allotype as a model, we identify a membrane responsive regulatory module within its cytoplasmic tail. The conserved 360-365 segment engages phospholipid membranes and undergoes Cys363-dependent self-association under membrane mimetic conditions, while cooperating with the transmembrane region to shape the molecular proximity of full-length HLA-A. Disruption of this segment weakens association with the trafficking GTPase ARF6, slows receptor internalization, and prolongs HLA-A retention at the cell surface. Consistently, a cell penetrating peptide containing this sequence limits endogenous HLA-A internalization across several tumor cell lines. These findings identify the HLA-A cytoplasmic tail as an active regulator of endocytic trafficking and suggest a strategy for preserving surface HLA-A availability in tumor cells.
The molecular basis of neutrophil-driven immunopathology in severe coronavirus disease 2019 (COVID-19) remains poorly defined. Here, we identify the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) SARS-unique domain (SUD)2core domain as a direct binding partner of the neutrophil-associated protein heme binding protein 2 (HEBP2). We show that HEBP2 normally suppresses azurophilic granule exocytosis, whereas SUD2core recruits the E3 ligase LTN1 to ubiquitinate and degrade HEBP2, thereby activating the Rab27a-Synaptotagmin Like 1 (SYTL1) axis to promote granule release, NETosis, and pro-inflammatory cytokine secretion. In a human immuno-epithelial organoid co-culture model, SUD2core amplifies epithelial damage in an HEBP2-dependent manner. Importantly, two small-molecule compounds that disrupt the SUD2core-HEBP2 interaction effectively attenuate neutrophil-mediated inflammation. These findings reveal a viral strategy that dismantles host restraint on neutrophil effector functions and highlight the SUD2core-HEBP2 interface as a promising therapeutic target for COVID-19.
Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer; however, its prognosis remains poor, and the underlying molecular mechanisms have yet to be fully elucidated. In this study, a prognostic model was constructed based on manganese metabolism-related genes, which effectively stratifies patients with LUAD with different prognoses and drug sensitivity. Mendelian randomization analysis identified glutathione peroxidase 3 (GPX3) as a key prognostic gene, which was downregulated in LUAD and significantly correlated with favorable outcomes. Functionally, GPX3 was found to inhibit LUAD cell proliferation, invasion, and metastasis while promoting apoptosis. Mechanistically, GPX3 binds to heat shock protein β-1 (HSPB1), induces its ubiquitination, and facilitates its degradation, thereby activating the Hippo signaling pathway and suppressing malignant phenotypes in LUAD cells. In summary, this study not only established a robust prognostic model based on manganese metabolism-related genes but also uncovered the critical regulatory role of the GPX3/HSPB1/yes-associated protein (YAP) axis in LUAD progression, underscoring the therapeutic potential of targeting GPX3.
Neuroinflammation often disrupts cerebral cholesterol homeostasis and leads to white matter damage and demyelination. However, the molecular mechanisms underlying these defects have remained enigmatic. In this study, we report that inflammation is associated with aberrant activation of Wnt signaling in astrocytes. While Wnt signaling increases astrocytic expression of SREBF2 protein and intracellular cholesterol level, it simultaneously inhibits the expression of APOE and reduces cholesterol efflux from astrocytes. The extracellular cholesterol supply is essential for the activation of the expression of MYRF transcription factor and myelin-associated genes in oligodendrocytes. Moreover, the defective oligodendrocyte differentiation and myelin gene expression caused by astrocytic Wnt activation in transgenic mice can partially be rescued by chemical-enhanced ApoE expression. Our findings suggest a regulatory pathway of “inflammation → Wnt signaling → cholesterol metabolism → myelin integrity” and provide important insights into the molecular mechanisms underlying inflammation-induced demyelinating diseases and future therapeutic strategies.
Diabetic complications, including retinopathy, are driven by hyperglycemic metabolic reprogramming and aberrant angiogenic transcription. Recent work highlights lactate accumulation and protein lactylation as key regulators, yet their underlying mechanisms in diabetic retinopathy remain unknown. Here, we report that hyperglycemia triggers aminoacyl-tRNA synthetase 1 (AARS1)-mediated lactylation of histone deacetylase 1 (HDAC1) at lysine 412. This modification sequesters HDAC1 in the cytoplasm, preventing its nuclear translocation and abrogating deacetylation of H3K56. The consequent H3K56ac increase activates transcription of the angiogenic gene vascular endothelial growth factor A (VEGFA). Disruption of the HDAC1 K412 lactylation impairs vascular morphogenesis and causes embryonic lethality in zebrafish, whereas Hdac1-K412A knockin mice are protected from pathological retinal angiogenesis in an STZ-induced diabetic model. Pharmacologically, the small molecule exifone inhibited HDAC1 lactylation, suppressing aberrant angiogenesis in diabetic mice and impairing vascular development in chick embryos. Our findings establish HDAC1 lactylation as a critical metabolic-epigenetic switch and a promising therapeutic target.
Many HIV-1 broadly neutralizing antibodies (bnAbs) account for envelope (Env) glycan shielding by supplementing antibody-protein interactions with antibody-glycan interactions. Further, bnAbs that interact predominantly via glycans can augment their binding through antigen-binding fragment (Fab) dimerization, generally utilizing non-variable region interactions. Here, we examined a donor whose serum identified glycan-reactivity (antibody 2G12-like) and CD4 binding-site (CD4bs) reactivity and isolated both glycan-reactive and CD4bs-reactive antibodies. The CD4bs antibodies were members of the VRC01-antibody class and neutralized nearly 70% of HIV-1 (208-strain panel). The glycan-reactive antibody had ∼30% breadth, and cryo-EM analysis revealed it to be a Fab-dimerized glycan (FDG)-reactive antibody with a distinct architecture wherein the Fab arms dimerized through a disulfide bond at the tips of the complementarity-determining loops that wedged between two glycans. Overall, we identified an FDG antibody that recognized through a “dual-glycan clamp” epitope and is the second FDG antibody to be isolated from an HIV-infected donor since antibody 2G12.
Xenophagy is a selective autophagy process crucial for eliminating intracellular pathogens, yet its regulatory mechanisms remain poorly defined. In this study, acetylome profiling identifies dynamic acetylation of the xenophagy receptor NDP52 at K202, and its deacetylation enhances during Salmonella Typhimurium infection. Acetyltransferase CREBBP/KAT3A and deacetylase HDAC3 reciprocally regulate NDP52 K202 acetylation. Deacetylated NDP52 binds more strongly to ATG8 family proteins (MAP1LC3A/B and GABARAPL2), promotes autophagosome-lysosome fusion and pathogen degradation. Innate immune kinase TBK1 phosphorylates HDAC3 at S424, stabilizes it by inhibiting ubiquitination-dependent degradation, and this regulatory cascade links innate immunity to xenophagy. Liver-specific overexpression of deacetylation-mimetic NDP52K202R in mice reduces hepatic Salmonella Typhimurium burden, attenuates liver necrosis, and suppresses proinflammatory cytokines. This study uncovers a post-translational modification paradigm in xenophagy, where NDP52 acetylation dynamics fine-tune receptor function in antibacterial responses, highlighting the HDAC3-NDP52 axis as a potential therapeutic target for infectious diseases.
Long-chain fatty acids (LCFAs) are common in lipid-rich wastewater, yet the dose-dependent effects on anaerobic ammonium oxidation (anammox) remain unclear. In this work, the effect of oleate on anammox was evaluated in short- and long-term experiments at different C/N ratios using multi-omics approaches. Oleate showed a dose-dependent effect. Low oleate dosages, particularly at a C/N ratio of 0.1, improved nitrogen removal performance, whereas high dosages shifted nitrite consumption from anammox toward heterotrophic reduction. Multi-omics analysis showed that appropriate oleate dosages also stimulated central carbon metabolism, while the Wood-Ljungdahl pathway remained comparatively stable. 13C-isotope tracing results demonstrated that oleate-derived carbon entered acetate, central-carbon intermediates, amino acids, peptides, and vitamin-related metabolites, supporting a cross-feeding framework involving LCFA-degrading heterotrophs and anammox bacteria. In contrast, excessive oleate caused incomplete oleate degradation, membrane damage, and community restructuring. These findings provide a basis for controlling LCFA dosage to improve anammox-based treatment of organic-rich side streams.
Astrocytes and neuronal synapses function as bidirectional partners in the modulation of synaptic transmission, and remodeling of perisynaptic astrocytic processes (PAPs) in the hippocampus gates the strength of recent fear memory. Whether cortical PAP remodeling contributes to remote memory consolidation, and whether this is different between synapses of engram versus non-engram neurons, is elusive. Here, we present Astro-GRASP, a genetically targeted approach to investigate astrocyte-synapse interactions. In the prelimbic cortex (PL), fear conditioning (FC) globally increases astrocyte-synapse coverage during recent memory consolidation, whereas astrocytes selectively retract from non-engram synapses at a remote time point after learning. Ezrin depletion from astrocytes reduces local astrocyte-synapse coverage and enhances remote memory expression, confirming the functional relevance of astroglial interaction with cortical synapses. Together, our findings indicate that astrocyte-synapse coverage on PL non-engram, rather than engram neurons, shapes remote memory strength, highlighting a previously unrecognized role for astrocytic remodeling of memory consolidation in cortical circuitry.
Influenza A virus protein PA-X mediates host shutoff, yet its molecular mechanism and the role of its C-terminal region remain unclear. When ectopically expressed via plasmids, PA-X cleaves its own mRNAs, leading to remarkably low intracellular expression, which makes its characterization challenging. In this study, we utilize an endonuclease inhibitor to suppress PA-X shutoff activity, enabling its robust expression and purification. Our biochemical analyses reveal that PA-X directly binds RNA via its unique C-terminal basic-rich region and induces mRNA degradation with minimal sequence specificity. Furthermore, we find that while PA-X targets a broad range of RNAs, viral RNAs are protected from its endonuclease activity through their assembly into vRNP complexes. These findings provide critical insights into the PA-X-mediated shutoff strategy, revealing how PA-X modulates the cellular environment while sparing the viral genome to favor viral replication.
Mother-to-offspring microbial transmission is a foundational process for seeding the infant gut microbiome, yet the relative contributions of maternal body sites and the influence of birth delivery mode remain incompletely understood. We use shotgun metagenomic sequencing in 68 mother-infant dyads to investigate species- and strain-level sharing of the maternal gut and vaginal microbiomes with the infant gut during the first year of life. At 2-4 months of age, infants share an average of 35% of species with their mother's microbiomes, with markedly greater sharing from the maternal gut than the vagina. Vaginally delivered infants exhibit higher levels of sharing than those born by cesarean section (C-section). Strain-level analyses reveal persistent mother-infant transmission across multiple Bacteroides and Bifidobacterium species genome bins, with strain-sharing frequencies varying by species and birth mode. C-section reduces the extent of mother-infant species- and strain-level sharing.
Mitochondria are continuously exposed to damage that contributes to aging and disease. While prolongedly damaged mitochondria are eliminated by mitophagy, how cells respond to transient damage remains unclear. Here, we establish a cell-based system to induce transient mitochondrial stress and resolve its recovery dynamics. We identify the E3 ubiquitin ligase mahogunin ring finger 1 (MGRN1) as a damage-threshold sensor that discriminates between transient and prolonged mitochondrial insults. Under transient stress, MGRN1 shows enhanced association with the outer mitochondrial membrane via MFN1, where it restrains mitophagy, potentially preserving mitochondria for repair. Loss of MGRN1 disrupts this checkpoint, leading to inappropriate mitophagy and impaired recovery. Mechanistically, mitochondrial repair is coordinated by the DELE1-eIF2α-ATF4 axis, Nrf2 signaling, and JUN/FOS activation, which collectively drive an antioxidant program, with TXNRD1 and SLC7A11 as downstream effectors. Together, our findings uncover a damage-sensing checkpoint that gates the decision between recovery and clearance, and reveal active and regulated pathways for mitochondrial repair.
The low efficiency in the production of induced pluripotent stem cell (iPSC) remains a major barrier to the widespread reprogramming applications in regenerative medicine and drug screening. Small molecules that modulate key signaling pathways offer a promising approach to overcome the reprogramming limitation. Here, we demonstrated that Thiazovivin (TZV), a selective Rho-associated coiled-coil kinase inhibitor, significantly increased human fibroblast reprogramming rate by approximately 1.4-fold (*p < 0.01). Treatment with TZV during the early phase of reprogramming enhanced colony formation, improved cell survival, and promoted the mesenchymal-to-epithelial transition (MET). Quantitative Real Time Polymerase Chain Reaction (RT-PCR) analysis revealed the downregulation of mesenchymal markers (N-cadherin, 52%; vimentin, 61%; p < 0.05) and upregulation of epithelial markers (E-cadherin, 165%; EpCAM, 95%; p < 0.05). Flow cytometry confirmed elevated expression of NANOG and TRA-1-60 pluripotency markers. Collectively, these findings suggest that TZV facilitates reprogramming through MET, offering a simple and effective strategy to enhance the efficiency and scalability of human iPSC generation.
Wang et al. identify a gut-brain pathway in which loss of the commensal bacterium Alistipes shahii reduces indole production, prevents hippocampal indole-AhR signaling, and drives anxiety and depression behaviors in patients and mouse models of irritable bowel syndrome.1
Mesenchymal stem cells (MSCs) maintain bone homeostasis through osteogenic differentiation. During aging, MSCs undergo a fate shift toward adipogenesis rather than osteogenesis, but the post-transcriptional mechanisms remain unclear. Here, we identify ovarian tumor domain-containing protein 1 (OTUD1) as an RNA-binding protein that controls MSC fate by stabilizing osteogenic transcripts. OTUD1 directly binds and stabilizes BMP2 mRNA, thereby supporting osteogenic differentiation. In vivo, OTUD1 deficiency does not affect early skeletal development but progressively impairs bone homeostasis during aging and exacerbates bone loss in ovariectomy- and glucocorticoid-induced osteoporosis models. Structure-function analyses reveal that N-terminal intrinsically disordered region of OTUD1 mediates RNA binding and osteogenic activity, whereas its deubiquitinase catalytic domain is dispensable. Multi-omics profiling demonstrates that OTUD1 coordinates the stability of transcripts involved in collagen remodeling and extracellular matrix organization, thereby maintaining MSC stemness and activation. Together, these findings define a mechanism linking RNA stability control to MSC lineage commitment and skeletal homeostasis during aging and disease.
