β-1,3-Glucan synthase FKS1 is essential for fungal cell wall biogenesis and serves as a validated target of widely prescribed antifungal drugs. However, the molecular mechanism of pathogenic FKS1, its modes of inhibition, and the associated resistance mechanisms remain elusive, hindering the antifungal development. Here, we focus on Candida glabrata FKS1, a clinically relevant target frequently linked to antifungal resistance. We present cryo-EM structures of C. glabrata FKS1 in multiple detergent environments and in complex with the triterpenoid antifungal enfumafungin. Integrated functional studies revealed a key catalytic residue within the conserved ED motif and uncovered structural adaptations to different membrane-mimetic environments, rationalizing FKS1's sensitivity to its surrounding membrane context. Enfumafungin binds at the extracellular membrane leaflet of cgFKS1 transmembrane domain, engaging a convex surface separated from the cytosolic active site, consistent with its role as a non-competitive inhibitor. Further analyses identified key enfumafungin-binding residues from TM5-TM6 and elucidated the molecular basis of drug resistance in C. glabrata. Collectively, these findings establish a mechanistic framework for pathogenic FKS1 function and inhibition and provide a molecular basis for the rational design of next-generation antifungal therapeutics. ### Competing Interest Statement The authors have declared no competing interest.

Loss of immune homeostasis in Type 1 diabetes (T1D) leads to a dysregulated and autoreactive immune response that destroys pancreatic β cells, causing absolute insulin deficiency. Nevertheless, current strategies for restoring immune homeostasis remain limited. Inspired by our earlier research, we leveraged the single-cell RNA sequencing data from T1D patients, and unexpectedly found that STAT1 overrepresentation is much more prominent in dendritic cells (DCs) rather in CD4 T cells. Subsequently, we repurposed the clinically-applied STAT1 inhibitor fludarabine in T1D setting. To resolve the underlying mechanism, we employed a multipronged approach in animal studies, incorporating FACS, RNA-seq, ChIP-qPCR, Co-IP/MS, CESTA, TEM, Seahorse assay and Conditional gene knockout model. Furthermore, we investigated the significance of our data in human autoimmune diabetes. Other than directly targeting CD4+ effector T cells, fludarabine also elevated regulatory T cell (Treg) frequency, and therefore, its administration markedly alleviated T1D pathogenesis. Interestingly, fludarabine did not show a direct effect on Treg cells but indirectly fosters Treg program via inducing tolerogenic DCs (tolDCs). The fludarabine-reprogrammed tolDCs are featured by the metabolic shift towards mitochondrial oxidative respiration and exert protective effects on the adoptive transfer studies. Since the emergence of tolDCs could not be fully explained by STAT1 itself, we further explored whether fludarabine alters the STAT1 interactome. Notably, fludarabine binds to STAT1 and disrupts its interaction with the aryl hydrocarbon receptor (AhR), thereby facilitating AhR nuclear translocation. Activated AhR transcriptionally upregulated the expression of anti-inflammatory, anti-ferroptotic, and mitochondrial respiration genes to uphold the tolerogenic DC phenotype. Our findings identified that fludarabine could be a promising immunometabolic therapeutic candidate to restore immune tolerance, which may be a viable approach against T1D in clinical settings.

Fungal inositol phosphorylceramide (IPC) synthase is an essential enzyme complex that catalyzes a critical step in sphingolipid biosynthesis. It is the molecular target of potent antifungal aureobasidin A (AbA). Despite its therapeutic relevance, the lack of structural and mechanistic insights into IPC synthase function and inhibition has impeded rational antifungal drug development. Here, we present cryo-EM structures of Saccharomyces cerevisiae IPC synthase in two distinct functional states: a ceramide-bound form and an AbA-inhibited complex. Our study reveals a conserved heterodimeric architecture formed by Aur1 and Kei1, stabilized through extensive protein-protein and lipid-mediated interactions. Within catalytic Aur1, we identify a membrane-embedded reaction chamber harboring a conserved H-H-D catalytic triad (H255, H294, and D298) essential for IPC synthesis. Structural comparisons illuminate the mechanism of ceramide recognition and reveal how AbA acts as a competitive inhibitor by occupying the substrate-binding pocket. Further analyses identify key residues involved in AbA binding and explain the molecular basis of drug resistance. Together, these findings advance the mechanistic understanding of fungal IPC biosynthesis and inhibition, and establish a foundation for developing new antifungal drugs targeting IPC synthase.

The p24 transmembrane emp24 domain family of cargo receptors is central to secretory trafficking, yet its molecular organization and regulatory principles have remained elusive. Here, we present cryo-EM structures of human p24 complex in three pH states, integrated with systematic functional analyses focused on glycosylphosphatidylinositol-anchored protein (GPI-AP) cargo. We identify a tightly interlocked p24α2-p24β1-p24δ1-p24γ2 heterotetramer as the minimal unit for GPI-AP engagement, resolving long-standing uncertainties regarding p24 stoichiometry and higher-order assembly. We reveal functional redundancy between p24α2 and p24α3, alongside selective incorporation of p24γ2 required for cargo binding. Systematic screening identifies two stable subassemblies: p24β1-p24δ1 and p24α2-p24β1-p24δ1, suggesting a stepwise assembly pathway. Comparative structural analysis across multiple pH states uncovers a conserved proton-sensing network centered on Coiled-coil layers 0 and +1 that drives coordinated transmembrane rearrangements, underlying pH-dependent cargo release. Our findings establish an integrated framework linking p24 architecture, isoform diversity, and pH regulation, offering a basis for understanding p24 trafficking complexity.

GLT25D1 O-galactosylates hydroxylysine residues in collagen and is essential for collagen maturation and function. Dysfunctions of GLT25D1 cause various tissue disorders. Despite its biological significance, the action mechanism of GLT25D1 remains enigmatic. Here we report the cryo-EM structures of human GLT25D1 and its ternary complex with UDP and hydroxylated acceptor substrates, revealing a bi-lobe architecture for the GLT25D1 monomer that organizes into dimeric and hexameric oligomers. The N-lobe of GLT25D1 contains a high-affinity UDP-galactose binding site, and the C-lobe is the catalytic domain of the enzyme. The structures together with biochemical analyses unravel the key recognition of the consensus "Hyl-Gly" motif from collagen acceptor substrates and associated catalytic mechanism. We further demonstrate that GLT25D1 mutations linked to cerebral small vessel disease and musculoskeletal defects adversely affect its function via distinct mechanisms. Our findings elucidate the molecular mechanism underlying collagen glycosylation and provide a molecular framework for understanding GLT25D1-related diseases.

SUMOylation is an evolutionary conserved regulatory mechanism, in which Ubc9 is the only E2 conjugating enzyme. Previous studies demonstrated that SUMOylation is involved in multiple biological processes, but its role in dendritic cells (DCs) remains to be fully addressed. Herein in this report, we found that DCs deficient in Ubc9 protected mice from dextran sulfate sodium (DSS)-induced colitis, as evidenced by the ameliorated weight loss, colon length, and disrupted colon structure. Mechanistically, Ubc9 mediated SUMOylation of RBPJ, by which it stabilized RBPJ from ubiquitin-mediated degradation to enhance its transcriptional activity, while Ciita, a critical transcription factor, is a direct target downstream of RBPJ, which forms an enhanceosome complex to transcribe the expression of MHC II genes. Therefore, loss of Ubc9 abolished RBPJ SUMOylation, which was coupled with reduced Ciita transcription, thereby attenuating the expression of MHC class II genes. As a consequence of defective MHC II expression, Ubc9-/- DCs were featured by the impaired capability to process antigen and to prime effector CD4+ T cells, thereby protecting mice from DSS-induced colitis. Together, our results shed novel insight into the understanding of SUMOylation in the regulation of DC functions in pathological conditions.

BACKGROUND:Endogenous retroviruses (ERVs) occupy >8% of the human genome. Aberrant resurgence of ERVs has been implicated recently in several critical pathologies. However, the possible incidence and role of ERV resurgence in heart failure (HF), a leading cause of global morbidity and mortality, remain unexplored. METHODS:We established a total RNA sequencing analyzing pipeline to assess the ERV occurrence in human and murine HF models. We generated 2 myocardium-specific mouse lines by crossing Myh6-MerCreMer (Myosin heavy chain 6 promoter driving MerCreMer recombinase) with TRIM28f/f and SETDB1f/f mice to identify the molecular regulators of ERV resurgence and the downstream pathways in the heart. We evaluated ERV expression by total RNA sequencing, reverse transcription-quantitative polymerase chain reaction and RNA fluorescence in situ hybridization. We restrained ERV activation by overexpressing TRIM28 (tripartite motif-containing 28) using adeno-associated virus serotype 9. The therapeutic potential of the ERV-mediated inflammatory pathway was tested in a myocardial ischemia/reperfusion model. RESULTS:ERVs, particularly class I ERVs, were prominently activated in multiple cross-species models of HF. Depletion of TRIM28, an epigenetic repressor, attenuated the epigenetic surveillance of trimethylation at lysine 9 of histone H3 and N6-methyladenosine, leading to the activation of ERVs in the failing heart. This ERV activation stimulated the antiviral innate immune pathways of TLR7/9 (Toll-like receptor 7/9) and NF-κB and lead to myocarditis and acute HF. Furthermore, restraining ERV activation and ERV-mediated innate immune responses by either adeno-associated virus serotype 9-mediated TRIM28 expression or a small-molecule TLR7/9 inhibitor improved heart function and alleviated HF in an ischemia/reperfusion model. CONCLUSIONS:ERV resurgence is a specific molecular trait of HF, driven by TRIM28 depletion in cardiomyocytes. ERV resurgence activates the innate immune TLR7/9-NF-κB pathway and induces myocarditis and HF. Inception of ERVs and the ERV-mediated immune pathway confers cardiac protection. These results identify TRIM28-ERV-TLR7/9-NF-κB as a target for therapeutic management of myocarditis and HF.

The glycosylphosphatidylinositol (GPI) biosynthesis pathway is critical for antifungal drug development. As a key component of this pathway, GPI transamidase (GPIT) catalyzes the attachment of GPI anchors to proteins, a process essential for fungal cell wall integrity and virulence. Despite its biological significance, structural and mechanistic insights into fungal GPIT remain limited. Here, a series of cryo-electron microscopy structures capturing distinct functional states of Saccharomyces cerevisiae GPIT is reported, including GPIT complexed with a GPI anchor, GPIT bound to a substrate-mimetic peptide, and an unprecedented dimeric GPIT assembly. These structures reveal the conserved GPI anchor binding site formed by Gab1 and Gpi16, as well as a key protein substrate recognition site, Gpi16 Y550. Comparative structural analyses uncover fungal-specific adaptations and the dynamic accommodation of catalytic subunit Gpi8. The dimeric GPIT structure exhibits a unique T-shaped organization unexpectedly mediated by transmembrane helices of Gab1 and Gaa1, a configuration unlikely to form in the human counterpart. This study provides a molecular framework for understanding GPIT function and species-specific divergences, providing a molecular basis for antifungal drug development.

Introduction and Objective: This study analyzes trends in Type 1 diabetes mellitus (T1DM) incidence, prevalence, mortality, and disability-adjusted life years (DALYs) in China from 1990 to 2021, with projections through 2036. Methods: Data from the GBD 2021 study were used to calculate annual changes in age-standardized rates (ASRs) for T1DM via joinpoint regression. Age-period-cohort analysis assessed age, period, and cohort effects, while decomposition analysis examined demographic and epidemiological factors. Bayesian modeling projected trends to 2036. Results: From 1990 to 2021, new T1DM cases in China rose from 22,722 to 32,058, with age-standardized incidence increasing by 1.16% annually. Prevalence doubled to 1,442,775, with a 1.15% annual rise. Mortality fell from 5,732 to 3,960, and DALYs dropped from 328,727 to 248,596. The highest incidence was in children aged 5-9, while prevalence peaked in older adults. Males had a higher disease burden, with a narrowing gender gap in mortality and DALYs. Decomposition analysis showed that epidemiological factors were key drivers. Projections indicate continued increases in incidence and prevalence, especially among males, through 2036. Conclusion: T1DM burden has increased over the past three decades, especially among older adults and males, emphasizing the need for targeted interventions and improved healthcare infrastructure. Z. Li: None. R. Duan: None. X. Chen: None. J. Li: None. W. Ren: None. X. Li: None. X. Fan: None. P. Yang: None. D. Yan: None. C. Wang: None. S. Liu: None. Key Laboratory Construction Plan Project of Shanxi Province (202404010920011); Fundamental Research Program of Shanxi Province (202303021212330); Science and Technology Achievements Transformation and Guidance Special Program Project of Shanxi Province (202304021301066); Scientific Research Funding Project for Returned Overseas Scholars of Shanxi Province (2024-143); Special Program for Science and Technology Innovation Talent Teams of Shanxi Province (202204251002029); Metabolic Disease (Type 1 Diabetes) Clinical Medical Research Center Construction Project of Shanxi Province (20240410501001); National Key Technology Research and Development Program on Prevention and Treatment of Cancer, Cardiovascular and Cerebrovascular Diseases, Respiratory Diseases, and Metabolic Diseases (2023ZD0507302); Research and Innovation Team Project for Scientific Breakthroughs at Shanxi Bethune Hospital (2024AOXIANG03)

Background: N 6-methyladenosine (m6A) modification plays a crucial role in various physiological processes by regulating mRNA biology. However, the exact impact of m6A modification on macrophages in adipose tissues under obese settings remains to be further elucidated. Methods: We established macrophage-specific Wtap-deficient mice to explore the effects of Wtap on obesity and metabolic disorders induced by high-fat diet (HFD) in mice. The molecular targets were explored by MeRIP-qPCR, and the metabolomic assays were performed to detect the alteration of relevant metabolites. Results: Wilms tumor 1-associated protein (WTAP), one of the m6A "writers", was downregulated in adipose tissue macrophages (ATMs) from obese individuals and negatively correlated with clinical metabolic traits. Depletion of Wtap in mouse macrophages exacerbated the metabolic consequences of high-fat diet (HFD) induced obesity. Additionally, energy expenditure and adipose beiging were considerably lower in Wtap-deficient mice in response to cold exposure. Mechanistic study revealed that WTAP-mediated m6A modification of isocitrate dehydrogenase 1 (Idh1) transcripts enhanced its stability and translation in macrophages leading to α-ketoglutarate (α-KG) production. Alpha-KG further supported alternative activation of macrophages by metabolic reprogramming. Conclusions: Our data support that Wtap modulates HFD-induced macrophages through interfering with the IDH1-α-KG axis, and highlight the importance of WTAP-mediated m6A modification in maintaining alternative macrophage activation, proposing potential targets for the regulation of obesity and related metabolic diseases.

Although Ubc9-mediated SUMOylation are recognized to regulate the multiple aspects of hepatic biological processes, its impact on hepatic senescence and metabolic dysfunction-associated steatotic liver disease (MASLD), however, is yet to be fully addressed. Herein noted an age-dependent decrease of hepatic Ubc9 expression is first noted along with an escalated decrease of protein SUMOylation, which is coupled with enhanced senescent marker expressions both in humans and mice. Interestingly, Ubc9 is dispensable for liver development at the embryonic stage. However, Ubc9 deficiency in hepatocytes rendered mice with an exacerbated hepatic aging phenotype and more susceptible to fatty liver disease and steatohepatitis following the challenge of a methionine- and choline-deficient (MCD)-diet. Ii is further demonstrated that nuclear ribosomal protein L3 (RPL3) interacts with DExD/H-box (DDX/DHX) helicases (DHX9), which then recruits RNA polymerase II to the p16 promoter to transcribe its expression, thereby exacerbating the hepatocyte aging process. However, Ubc9-mediated SUMOylation prevents RPL3 nuclear translocation, by which it represses the expression of senescent markers such as p16 to attenuate the hepatic aging process. Together, the study highlights that Ubc9-mediated SUMOylation of RPL3 could be an unappreciated mechanism against hepatic aging in clinical settings.

Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.

Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172 V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172 V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172 V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172 V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.

AIMS:We aimed to investigate the association between glycemic variability (GV) and the abnormal differentiation of T-cell subpopulations in patients with type 2 diabetes mellitus (T2DM). METHODS:In total, 108 hospitalized patients with T2DM were enrolled and divided into two subgroups (normal glycemic excursion (NGE) and high glycemic excursion (HGE)) according to their mean amplitude of glycemic excursion (MAGE) level. The MAGE was evaluated via continuous glucose monitoring for 72 h consecutively. Flow cytometry was used to determine the proportions of T cell subpopulations. RESULTS:The T helper (Th) 1 cell/Th2 cell ratio was significantly higher, and the proportion of regulatory T cells (Tregs) was significantly lower in the NGE group than in the HGE group (all P < 0.05). After fully adjusting for confounders, the MAGE was positively associated with the Th1 cell/Th2 cell ratio (β = 0.370; P = 0.009) and negatively associated with the proportion of Tregs (β = -0.554; P = 0.001). CONCLUSION:The MAGE was an independent risk factor for abnormally high Th1 cell/Th2 cell ratio and proportion of Tregs. Abnormal differentiation of T cell subpopulations induced by GV may impair β-cell function, aggravate insulin resistance, and contribute to the development of diabetic complications.

Abstract Purpose Patients with end stage renal disease (ESRD) lose the capacity of renal potassium excretion and often suffer from persistent hyperkalemia, especially for those requiring maintenance hemodialysis (HD). Sodium zirconium cyclosilicate (SZC) is the most recently approved K + binding agent in China. It is reported SZC is an effective and well-tolerated treatment for pre-dialysis hyperkalemia in patients with ESRD undergoing adequate hemodialysis. We thus conducted a retrospective study to compare the therapeutic efficiency of SZC and sodium polystyrene sulfonate (SPS, another classic K + binding agent) on hyperkalemia in HD patients. Methods: 38 patients with persistent pre-dialysis hyperkalemia were included, and 18 patients were treated by SZC while 20 patients were treated by SPS. The changes of serum potassium level were followed up for 7 months. Results: We observed that the potassium reducing capacity of SZC and SPS were comparable at the first 3 months, but SZC displayed better long-term therapeutic effect. Conclusion: Our results supported that SZC is a good option for treatment of hard-controlled pre-dialysis hyperkalemia.

The regulation of autoimmunity against pancreatic islet β cells for type 1 diabetes (T1D) onset is still unclear. NOD/ShiLtJ (NOD) mice are prone to the onset of autoimmune diabetes, but its congenic strain, ALR/Lt (ALR), is not. Here we show that dendritic cells (DC) in ALR mice have impaired migratory and T-cell priming capability. Genomic comparative analysis maps a 33-bp deletion in the ALR Myosin IXb (Myo9b) gene when compared with NOD genome; meanwhile, data from knock-in models show that this ALR Myo9b allele impairs phenotypic and functional maturation of DCs, and prevents the development and progression of spontaneous autoimmune diabetes in NOD mice. In parallel, while the ALR 33-bp deletion of Myo9b is not conserved in human, we find a MYO9B R133Q polymorphism associating with increased risk of T1D and enhanced DC function in patients with T1D. Our results thus hint that alterations in Myo9b may contribute to altered DC function and autoimmune diabetes onset.

The membrane-integrated synthase FKS is involved in the biosynthesis of β-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane–cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1–membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5–6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal β-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS. Using cryo-electron microscopy, the molecular architecture and catalytic mechanism of action of the fungal β-1,3-glucan synthase FKS1 are determined.

Although DNA methylation has been recognised in the pathogenesis of idiopathic pulmonary fibrosis (IPF), the exact mechanisms are yet to be fully addressed. Herein, we demonstrate that lungs originated from IPF patients and mice after bleomycin (BLM)-induced pulmonary fibrosis are characterised by altered DNA methylation along with overexpression in myofibroblasts of methyl-CpG-binding domain 2 (MBD2), a reader responsible for interpreting DNA methylome-encoded information. Specifically, depletion of Mbd2 in fibroblasts or myofibroblasts protected mice from BLM-induced pulmonary fibrosis coupled with a significant reduction of fibroblast differentiation. Mechanistically, transforming growth factor (TGF)-β1 induced a positive feedback regulatory loop between TGF-β receptor I (TβRI), Smad3 and Mbd2, and erythroid differentiation regulator 1 (Erdr1). TGF-β1 induced fibroblasts to undergo a global DNA hypermethylation along with Mbd2 overexpression in a TβRI/Smad3 dependent manner, and Mbd2 selectively bound to the methylated CpG DNA within the Erdr1 promoter to repress its expression, through which it enhanced TGF-β/Smad signalling to promote differentiation of fibroblast into myofibroblast and exacerbate pulmonary fibrosis. Therefore, enhancing Erdr1 expression strikingly reversed established pulmonary fibrosis. Collectively, our data support that strategies aimed at silencing Mbd2 or increasing Erdr1 could be viable therapeutic approaches for prevention and treatment of pulmonary fibrosis in clinical settings.