Cell Research

Bacteria have evolved multiple immune systems to resist phage invasion, however, only a small part of the defensive mechanisms have been clearly uncovered. In this study, we report a type III Druantia two-component defense system, DruH-DruE, identified from Mycobacterium smegmatis. The DruH-DruE prevents phage DNA cyclization and replication.DruE can be replaced from the defense system by either homolog in M. tuberculosis or M. smegmatis. The physical interaction between this two components is essential for fighting against phage infection. Mutations in the interaction sites led to the loss of phage-defending function of the system. The broad-spectrum antiphage ability of the defense system could be activated by the small tail protein Gp25 of phage A10ZJ24. This study fills a major gap in current knowledge of antiphage mechanism of type III Druantia defense system, expanding our understanding of the immune mechanisms in prokaryotic cells.

Thymic colonization by T cell progenitors (TCPs) is essential for adaptive immunity, yet the guiding tissues remain elusive. Here, we unveiled a tunnel microtract (TMT) as an organ indispensable in TCPs homing in both zebrafish and mouse. Disruption of TMT leads to compromised T cell development. Specifically, the zebrafish TMT were positioned bilaterally beneath the fifth branchial levator muscle, connecting the thymus and kidney. They are semi-coiled, non-vascular, non-lymphatic tubes of epithelial signatures. Impressively, Sox10 activates Cdc42 to promote F-actin remodeling in neural crest cells (NCCs), leading to precise elongation and tight packaging of a low-permeability TMT. Remarkably, a homologous CD34 TMT was observed in mice, which bilaterally enveloped the embryonic thymus and extended into the thyroid cartilage. Sox10-Cdc42 signalling functioned recapitulatively in NCC morphogenesis during its construction. These findings establish TMT as an unappreciated NCC-derived organ in TCPs homing, with implications in T cell development and immune disorders.

Single-cell expression quantitative trait loci (sc-eQTL) analyses are powerful in identifying context-specific susceptibility genes from genome-wide association studies (GWAS) loci. However, few studies have comprehensively investigated cells of lung cancer origin in non-European populations. Here, we built a lung sc-eQTL dataset from 129 Korean women never-smokers with epithelial cell enrichment. eQTL mapping identified 2,229 genes with an eQTL in 33 cell types, including East Asian-specific findings when compared to predominantly European datasets. Integration with single-cell chromatin accessibility data demonstrated an enrichment of cell-type specific eQTLs in cell-type matched candidate enhancers, while shared eQTLs were more frequently found near promoters. Colocalization and transcriptome-wide association study unveiled 36 susceptibility genes from 22 cell types in 22 lung cancer loci, including 10 loci not achieving genome-wide significance in prior GWAS. Around 47% of these genes were from cells of the alveoli, underscoring their importance, especially in lung adenocarcinoma (LUAD) susceptibility. Focusing on the trajectory of alveolar epithelial cell regeneration, we detected 785 cell-state-interacting QTLs, which overlapped with 28% (10) of the identified susceptibility genes. Finally, we experimentally validated East Asian-and alveolar type 2 cell-specific eQTL of TCF7L2 underlying East Asian LUAD locus, 10q25.2. Consistent with its role as a Wnt/{beta}-catenin effector, TCF7L2 displayed significant effect on lung adenocarcinoma cell growth. Our data highlighted context-specific susceptibility genes, especially from alveolar cells of lung, contributing to lung cancer etiology.

Iron overload is increasingly recognized as a critical contributor to coronavirus pathogenesis1, yet the underlying induction mechanisms remain unclear. Here, we uncover a fundamental pathway by which coronavirus drives IRP1 RNA-binding activity to induce iron accumulation2 via targeting the TAp73-FDXR axis. Specifically, coronavirus infection represses transcription of FDXR (encoding the key rate-limiting enzyme in host iron-sulfur cluster synthesis3), thereby impairing host iron-sulfur cluster generation to trigger the functional conversion of the cytosolic aconitase 1 (ACO1) into iron-regulatory protein 1 (IRP1)4, ultimately leading to the hosts persistently false perception of iron deficiency. We identify TAp73 as the primary transcription factor governing FDXR expression, and demonstrate that the coronavirus envelope protein (CoV-E) orchestrates TAp73 nuclear export. Subsequently, CoV-E binds TAp73 through a critical valine residue within its C-terminal PBM domain, inducing the K48-linked ubiquitination and proteasomal degradation of TAp73. Furthermore, we developed a CoV-E-targeting molecule, DPTP-FC, which blocks CoV-E-TAp73 interaction via forming steric hindrance and effectively alleviates iron accumulation and tissue damage caused by PEDV, PDCoV, and SARS-CoV-2 infection. Our study reveals the central role of the TAp73-FDXR axis in CoV-induced iron accumulation, highlighting CoV-E as an attractive antiviral target and DPTP-FC as a promising therapeutic candidate.

TRPC3 is a calcium-permeable, non-selective cation channel that is activated by DAG. It is expressed in several tissues, especially in the cerebellum, and has been implicated in various human diseases. Despite recent progress in understanding the structural mechanism of TRPC3, how the channel opens remains elusive. Here, we present structures of hTRPC3 in an agonist-free resting state, determined using a DAG-binding site mutant. We also present the structure of hTRPC3 in a DAG-bound open state, determined using a constitutively active "moonwalker" (T561A) mutant. These structures, together with electrophysiological results, reveal that the T561A mutation activates hTRPC3 by disrupting a polar interaction with N652. A newly formed {pi}-bulge in S6 leads to rotation and outward tilting of the lower half of S6, resulting in dilation of the pore and thus channel opening. Agonist DAG stabilizes hTRPC3 in the open conformation. BTDM exerts its inhibitory effect by pushing S5 and S6 back to the center to close the pore, while preserving the {pi}-bulge. These results shed light on the opening mechanism of hTRPC3.

The maintenance of protein homeostasis is vital for all cells. Alteration in protein handling underlies several diseases. The small molecule sephin1 is a promising clinical candidate against proteostasis disruption, but its mechanism of action is still uncertain. Our experimental evidence shows that sephin1 binds G-actin and drives actin cytoskeleton misfolding, and eventually, Golgi disintegration. At first, sephin1 impairs the autophagic flux and elicits the phosphorylation of the subunit of eIF2 and the ER-stress independent expression of CHOP via GCN2 kinase. Sephin1 also inhibits the mammalian target of rapamycin (mTORC1), activates the transcription Factor EB (TFEB), drives the expression of TFEB-direct target genes, and eventually stimulates the autophagy lysosomal pathway. Our results reveal that the actin cytoskeleton may regulate autophagy via mTORC1-TFEB complemented with the GCN2-eIF2-CHOP signaling pathway.

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, X-ray absorption spectroscopy (XANES), quantum mechanical calculations, molecular dynamics, and functional assays to provide an integrated characterization of hRec. The 2Ca2+-bound hRec structure was solved at 1.60 [A], showing that, unlike bRec, hRec interacts with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a species-specific charge distribution that might influence membrane interactions. Both recoverins form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray crystallography (1.85 [A]) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1 binding, as the latter can complement the Zn2+ coordination. These data refine our understanding of recoverin function in humans and highlight its role as a key link between calcium and zinc signaling in mammalian photoreceptors under normal and pathological conditions.

The delta-type ionotropic glutamate receptors (iGluRs) GluD1 and GluD2 are ligand-gated ion channels that are fundamental for regulating both excitatory and inhibitory synapses. Rising evidence points to the role of GluD1 in the development of neurological diseases. However, the ultrastructure of human GluD1 (hGluD1) and the molecular basis for its ligand-gating remain unclear. Here, we define the structure of hGluD1 and resolve its ligand-gating mechanism using cryo-electron microscopy (cryoEM) and single channel bilayer recording. While hGluD1 exhibits a non-swapped architecture, it contains conserved iGluR moieties that enable ligand-gating, such as a ligand-binding domain (LBD) tethered to a transmembrane ion channel. Binding of the neurotransmitter {gamma}-aminobutyric acid (GABA) or D-serine to the LBD enables cation influx through the hGluD1 ion channel. Our findings delineate the molecular architecture and function of hGluD1, provide foundations for understanding patient mutations in hGluD1, and will invigorate therapeutic development against hGluD1.

Protein S-palmitoylation is a critical and reversible lipid modification that governs protein localization, trafficking, and signaling. Its dysregulation is increasingly implicated in cancer and therapeutic resistance, highlighting an urgent need for high-throughput computational prediction tools. Palmitoylation is regulated by a complex interplay of sequence motifs, structural conformations, and physicochemical properties. To comprehensively capture these determinants, we developed Deep-Palm: a deep learning framework that integrates multi-view features, including amino acid sequences, spatial constraints from predicted structures, physicochemical descriptors, and protein language model embeddings, for accurate prediction of S-palmitoylation sites. In independent testing, Deep-Palm achieved an area under the curve (AUC) of 0.931, substantially outperforming state-of-the-art tools such as pCysMod, MusiteDeep, and GPS-Palm. Furthermore, Deep-Palm demonstrated robust performance across diverse eukaryotic species. Notably, its predictive accuracy remained consistent regardless of protein functional categories or subcellular localization, indicating that the model captures fundamental, context-invariant determinants of palmitoylation. By embedding amino acid sequences with structural and protein property awareness, Deep-Palm not only delivers stable and high-precision predictions but also provides a framework for uncovering novel regulatory mechanisms and therapeutic targets in protein post-translational modification (PTM).

Highly pathogenic avian influenza viruses (HPAIVs) continue to cause substantial disease in birds and mammals, with repeated H5N1 spillovers highlighting the need for broadly protective antiviral strategies. Here we develop a programmable RNA-targeting antiviral platform based on RfxCas13d and evaluate its activity in avian cells. Screening of five Cas13 orthologs in chicken DF1 fibroblasts revealed RfxCas13d as the most potent and well tolerated effector. Virus-specific CRISPR RNAs (crRNAs) targeting conserved regions of positive- and negative-sense influenza RNA were tested against A/WSN/033[H1N1] and multiple HPAIV isolates, including a member of clade 2.3.4.4b H5N1. Targeting positive-sense RNA conferred superior influenza inhibitory activity and further enhanced by multiplexed crRNA expression. These findings establish RfxCas13d as a versatile RNA-guided antiviral platform and provide a route for broad-spectrum influenza control through conserved RNA targeting.

Mitochondrial dynamics, involving fission and fusion, are critical for the maintenance, function, distribution, and inheritance of mitochondria, allowing their morphology to adapt to the cells physiological needs. Mitofusins, large GTPase transmembrane proteins, play a role in this process by driving the tethering and fusion of mitochondrial outer membranes. Dysfunction in mitofusins has been associated with neurodegenerative diseases such as Parkinsons, Alzheimers, Huntingtons, and Charcot-Marie-Tooth type 2A, as well as various cancers, where their dysregulated expression influences cell proliferation, invasion, and chemotherapy resistance. Despite their importance, the precise molecular mechanisms underlying mitofusin-mediated fusion remain unclear and require further structural elucidation. In fact, no complete high-resolution structures exist for mitofusins, including their yeast homolog, Fzo1. Here, we generated and analyzed full-length structural models of mitofusins using AlphaFold. Monomeric, dimeric, and tetrameric assemblies were produced, including complexes with fusion partners Ugo1 and SLC25A46. While AlphaFold predicted limited conformational diversity for isolated monomers, structural variability emerged for predicted homo- and hetero-oligomers. Notably, our models reveal a previously undescribed cross-type dimerization mode involving interactions between heptad repeat domains, not reported in current experimental structures. Comparison with recently resolved experimental data further supports the structural relevance of this interface. These full-length models allowed us to propose a new hypothetical mechanism of outer mitochondrial membrane fusion.

Genetic regulation of splicing uniquely contributes to trait-associated genome-wide association studies (GWAS) signals. However, quantitative trait loci (QTL) analysis using short-read sequencing of bulk tissues fails to capture full-length and cell-type-specific isoforms. Here, we present an isoform-level lung cell atlas from 129 never-smoking Korean women using single-cell long-read RNA-sequencing, identifying abundant unannotated and cell-type-specific isoforms. Isoform-level signatures of 37 lung cell types display a larger difference and therefore improve cell-type classification compared to gene-level expression. Notably, isoform-QTLs (isoQTLs) detect unannotated and/or cell-type-specific isoforms with independent genetic regulation from expression-QTL (eQTL), supported by enriched splicing functional elements. IsoQTLs nominate susceptibility isoforms from previously unexplained lung function and cancer GWAS loci, via eQTL-independent signals. We highlight a potentially functional novel variant of PPIL6 in multiciliated cells underlying lung cancer risk through alternative splicing. This isoform-level resource advances our understanding of cell-type-specific isoform regulation and its contribution to lung traits and diseases.

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca{superscript 2}-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.

Dopamine is a neurotransmitter essential for cognition, and its dysregulation is associated with neurological diseases1,2. Historically, dopamine has been understood to signal exclusively through metabotropic receptors3. Delta-type ionotropic glutamate receptors (GluDs), which have recently been established as ligand-gated ion channels4,5, are fundamental for synaptic maintenance, are implicated in neurological disorders, and co-localize with dopaminergic machinery. Here, we report that dopamine is a direct agonist of GluDs, eliciting ionotropic activity, as visualized by cryo-electron microscopy (cryo-EM), bilayer recordings, mutagenesis, and patch clamp recordings. Dopamine binds to the GluD ligand binding domain, inducing clamshell closure and channel activation through a distinct molecular interface. GluD channel activity is tightly regulated by G-proteins, which act as molecular switches to tune GluD activity: free G{beta}{gamma} inhibits ligand-gating, while G or inactive G-protein heterotrimers enable dopamine-induced GluD currents. This tuning of GluD activity by G-proteins is uncoupled in a point mutation associated with neurodegeneration. These findings expand mechanisms of neuronal dopaminergic signaling, uncover how G-proteins tune GluD channel activity, and provide a framework for targeting GluDs in neurological diseases.

Transmembrane (TM) proteins play essential roles in biology as transporters, ion channels, chaperones, enzymes, and mediators of signal transduction. However, membrane proteins often suffer from inefficient folding and intrinsic instability. Misfolding in cells can cause numerous loss-of-function pathologies. Likewise, denaturation upon purification in the laboratory is a critical barrier to structure determination and characterization of key biochemical mechanisms. Generalizable strategies to stabilize membrane proteins remain limited. Here, we developed an informatics-based de novo design strategy to create synthetic auxiliary subunits that interact with the TM helices of a model pentameric ion channel, thereby bolstering folding while maintaining channel function. Biochemical and structural characterization reveal the synthetic TM subunits can also be used to create larger multi-spanning designer proteins of custom topology. This proof-of-concept motivates the feasibility of computationally designed accessory TM helices as potential pharmacological chaperone "folding correctors" of membrane proteins in disease and as tools in structural biology.

Mainland China experienced multiple waves of COVID-19 pandemic during 2020-2022, driven by emerging variants and changes in public health and social measures (PHSMs). We developed a hypergraph-based Susceptible-Vaccinated-Exposed-Infectious-Recovered-Susceptible (SVEIRS) model to reconstruct epidemic dynamics across 31 provinces, capturing transmission heterogeneity associated with clustered contacts. We assessed key characteristics of transmission at national and provincial levels during four outbreak periods: initial, localized pre-delta, Delta, and widespread Omicron, which accounted for 96.7% of all infections. We found significant diversity in transmission contributions across cluster sizes, with a small fraction of larger clusters responsible for a disproportionate share of infections. Counterfactual analyses showed that reducing cluster-size heterogeneity, while holding overall exposure constant, could have lowered national infections by 11.70-30.79%, with the largest effects during Omicron period. Ascertainment rates increased over time but remained spatially heterogeneous with a range: (14.40, 71.93)%. Population susceptibility declined following mass vaccination (to 42.49% in Aug 2021, nationally) and rebounded (to 89.89% in Nov 2022) due to waning immunity with variations across the provinces. Effective reproduction numbers displayed marked temporal and spatial variability, with higher estimates during Omicron. Overall, these results highlight critical role of group contact heterogeneity in shaping epidemic dynamics.

The developmental program governing meibomian gland (MG) morphogenesis and proliferation remains poorly understood, largely due to the lack of physiologically relevant model systems. Here, we established a novel high-fidelity, three-dimensional organoids model derived from mouse meibomian gland (mMGO) epithelium. Transcriptomic and phenotypic analyses demonstrated that mMGOs faithfully recapitulate postnatal gland development in vivo, including dynamic transcription program, branching morphogenesis, lineage differentiation, and functional lipid accumulation. Leveraging this model, we identified the Hippo-YAP pathway as a pivotal regulator of MG epithelial proliferation and homeostasis for the first time. YAP inhibition severely impaired organoids growth, while pharmacological inhibition of Hippo pathway with XMU-MP-1 enhanced proliferation and progenitor clonogenicity. Crucially, in inflammation-induced atrophic organoids, XMU-MP-1 treatment rescued YAP nuclear localization and stimulated regrowth and functional restoration. Our study provided new mechanistic insights and a robust organoids platform for MG development research, and nominated targeted Hippo pathway inhibition as a promising strategy to reverse glandular atrophy in meibomian gland dysfunction.

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.

Nitric oxide (NO) signaling is pivotal in numerous physiological processes and is implicated in a spectrum of human diseases. Nitric oxide synthases (NOS) initiate NO signaling and govern its magnitude and duration, making them key drug targets. Despite decades of investigation, the structural mechanism by which NOS enzymes transfer electrons from NADPH to haem remains incompletely understood. Here, we report cryo electron microscopy studies of the inducible NOS (iNOS) homodimer in complex with calmodulin captured under the catalytic turnover condition, resolving two important functional states: the electron input state and output state. In the input state, the FMN binding subdomain (FMND) docks onto the FAD/NADPH-binding subdomain (FNR), positioning the FMN cofactor to accept electrons from FAD. The FMND then undergoes a large rotational movement to engage the oxygenase domain of the other protomer, adopting the output state, which enables electron transfer from FMN to the haem center via W366. This dynamic movement of the FMND shuttles electrons from the reductase domain to the oxygenase active site in iNOS. A point mutation (S594E) that disrupts the FMND oxygenase interface markedly reduces catalytic activity of iNOS and traps the enzyme in a non-productive intermediate conformation. Together, these findings elucidate the structural mechanism of FMND mediated electron transfer in the iNOS catalytic cycle.