Tardigrades, known for their extraordinary resilience to extreme environmental conditions, employ specialized stress-response proteins to maintain cellular integrity under stress. These proteins, including SAHS, CAHS, MAHS, and Dsup, play critical roles in protecting cells from dehydration, radiation, and other environmental stressors. Previous work has characterized many of these proteins in Ramazzottius varieornatus, but the taxonomic breadth, copy-number variation, and structural diversity of these families across other tardigrade lineages remain poorly understood. The recent expansion of genomic resources for additional species motivated a comprehensive multi-species analysis to map ortholog diversity and structural features across tardigrade taxa. We investigated the genetic, structural, and evolutionary features of these stress-response proteins using an integrative bioinformatics approach, analyzing gene structure, orthologous clustering, molecular phylogeny, conserved motifs, and 3D protein structures across four tardigrade species—R. varieornatus, Hypsibius exemplaris, Paramacrobiotus metropolitanus, and H. henanensis. Our results reveal significant variation in predicted gene copy numbers and sequence conservation that reflect species-specific adaptations to environmental stress. Orthologous clustering showed shared evolutionary patterns across species, and conserved motifs were identified within the SAHS, CAHS, and MAHS families. Notably, we identified a new Dsup protein (H.Henanensis.Chr5.66), a potential ortholog in H. henanensis, thereby expanding the known diversity of Dsup proteins. The Dsup protein family exhibited notable sequence diversity across species, yet structural analyses revealed conserved α-helix regions and hydrophobicity patterns, suggesting a flexible conformation that aids DNA protection during extreme stress. Phylogenetic analyses revealed patterns consistent with parallel clustering patterns among stress-response proteins. This study advances our understanding of the molecular adaptations that underpin tardigrades' resilience and offers insights into potential applications for enhancing stress tolerance in other organisms.

Granulomas form to contain Mycobacterium tuberculosis (Mtb) infection in the lung. What constitutes protective or detrimental responses is poorly understood. Here, using spatial transcriptomics and immunofluorescence microscopy of human lung samples and mouse models, we characterize the spatial structure and transcriptome of macrophage and T cell populations in tuberculosis granulomas. We identify signatures of reduced major histocompatibility complex (MHC) class II levels on macrophages and reduced CD4+ T cell activation, particularly in necrotic granulomas, suggesting a compromised interaction between innate and adaptive responses. Further analyses in mouse models and human cells reveal that infection of macrophages, or exposure to mycolic acids, disrupt cholesterol trafficking, leading to cholesterol accumulation and MHC class II sequestration in lysosomes. This inhibits antigen presentation and impairs anti-Mtb CD4+ T cell responses. Pharmacological restoration of cholesterol homeostasis during late-stage infection improves control of Mtb in mice. This study reveals an infection-driven mechanism of cholesterol overload, which impairs control of tuberculosis and could be targeted therapeutically.

Integrin activation is an indispensable step for various integrin-mediated biological functions. Kindlin-2 is known to coactivate integrins with Talin; however, molecules that restrict integrin activation are elusive. Here, we demonstrate that the E3 ubiquitin ligase Smurf1 controls the amount of Kindlin-2 protein in cells and hinders integrin activation. Smurf1 interacts with and promotes Kindlin-2 ubiquitination and degradation. Smurf1 selectively mediates degradation of Kindlin-2 but not Talin, leading to inhibition of αIIbβ3 integrin activation in Chinese hamster ovary cells and β1 integrin activation in fibroblasts. Enhanced activation of β1 integrin was found in Smurf1-knockout mouse embryonic fibroblasts, which correlates with an increase in Kindlin-2 protein levels. Similarly, a reciprocal relationship between Smurf1 and Kindlin-2 protein levels is found in tissues from colon cancer patients, suggesting that Smurf1 mediates Kindlin-2 degradation in vivo. Collectively, we demonstrate that Smurf1 acts as a brake for integrin activation by controlling Kindlin-2 protein levels, a new mechanism that permits precise modulation of integrin-mediated cellular functions.

Host restriction factors mediate intrinsic immunity against infections, thus serving as promising targets for host-directed therapy (HDT) against drug-resistant pathogens. While restriction factors counteracting viruses have been extensively studied, those targeting bacteria, particularly those with broad-spectrum activity, remain largely unexplored. Here, through screening for host factors promoting lysosomal acidification, a crucial process clearing pathogens, we identify the host small GTPase Rab14 as a restriction factor with broad-spectrum activity against multiple bacteria and viruses. Mechanistically, upon pathogen infections, GTP-bound Rab14 increases and binds to the calcium/calmodulin-dependent protein kinase type 2 delta (CAMK2D), suppressing CAMK2D-mediated phosphorylation of V0a1, the critical subunit determining V-ATPase localization, thus promoting V0a1 binding to the COPⅡ complex to facilitate V-ATPase trafficking from the endoplasmic reticulum to lysosomes, resulting in lysosomal acidification and pathogen clearance. Taken together, our data demonstrate an unrecognized intrinsic immune mechanism mediated by Rab14-CAMK2D-V-ATPase axis, which might be a promising target for infectious diseases.

Glioblastoma (GBM) remains a significant therapeutic challenge. While GBM-derived extracellular vesicles (EVs) are known to remodel the normal blood-brain barrier (BBB) into a blood-tumour barrier (BTB), the underlying mechanism is largely not understood. Here, we reveal that nucleolin (NCL) is transferred via GBM-derived EVs to the surface of brain endothelial cells, where it promotes BTB formation. Furthermore, the NCL-specific aptamer AS1411 exploits this pathway, crossing the BTB through receptor-mediated transcytosis and selectively entering GBM cells in an NCL-dependent manner. Proteolysis-targeting chimeras (PROTACs), heterobifunctional molecules that recruit E3 ligases to degrade target proteins, show therapeutic potential but are hindered by inefficient brain penetration in GBM. Capitalizing on the BTB-penetrating capability of AS1411 and our prior finding that AS1411 can intracellularly recruit the E3 ligase MDM2 via employing NCL as a molecular bridge, we engineered AS1411-based PROTACs against VEGFR2 and EGFR. These PROTACs induced NCL- and MDM2-dependent ubiquitination and degradation of VEGFR2 or EGFR in GBM cells, demonstrating potent anti-tumour activity. Collectively, our findings identify EV-transferred NCL as a key mediator of BTB formation and a functional transcytosis receptor for AS1411, providing a promising strategy for developing BTB-permeable, targeted therapy for GBM.

BACKGROUND/AIMS:Dysregulated cholesterol metabolism is a hallmark of hepatocellular carcinoma (HCC) that drives tumor initiation and progression. However, clinical targeting of cholesterol metabolism has yielded limited benefits due to stringent feedback in tumor cells. Identifying a central mediator capable of restoring cholesterol homeostasis within the cell's intrinsically fine-tuned regulatory framework is urgently needed. METHODS:We integrated a proteomic dataset from patients with cholesterol-dysregulated HCC into a global cholesterol metabolic regulatory network to identify potential therapeutic targets for disrupted cholesterol homeostasis. The prognostic significance of the candidate targets was further validated in an independent cohort through immunohistochemistry. Functional and mechanistic studies were conducted in vitro using HCC cell lines and in vivo using mouse models. The pharmacological efficacy of the candidate agent was evaluated in both subcutaneous and orthotopic HCC mouse models. RESULTS:ER lipid raft-associated 1 (ERLIN1), a pivotal regulator of cholesterol metabolism reprogramming, was identified as an independent favorable prognostic indicator in HCC. ERLIN1 constrains HCC progression both in vitro and in vivo by stabilizing the INSIG1-SCAP-SREBP2 axis and maintaining the metabolic balance of intracellular cholesterol. Under hypoxia, impaired factor-inhibiting hypoxia-1-dependent hydroxylation of ASB11 at asparagine residues 90 and 92 enhances ASB11-mediated ERLIN1 degradation. Pharmacological targeting of this axis using zoledronic acid (ZoA) attenuated HCC progression by weakening the ASB11-ERLIN1 interaction and restoring cholesterol homeostasis. CONCLUSIONS:ERLIN1 represents a druggable metabolic vulnerability in cholesterol-dysregulated HCC. Targeting the ASB11-ERLIN1 axis with the clinically approved ZoA reestablishes cholesterol homeostasis and offers a promising therapeutic strategy to overcome the current limitations of cholesterol-targeted HCC therapies.

How a local infection triggers systemic humoral immunity remains unclear. Here we identify farnesyl pyrophosphate (FPP), a mevalonate pathway metabolic intermediate1, as an endogenous alarmin that enhances IgG antibody responses through keratinocyte-derived IL-6 and CCL20. This signalling axis potentiates the differentiation of T follicular helper cells and migratory dendritic cells2,3. FPP accumulates within keratinocytes after infection or ultraviolet irradiation through the activation of the mevalonate pathway mediated by the unfolded protein response-SREBF pathway, amplifying germinal centre (GC) responses in draining lymph nodes. Mechanistically, accumulated FPP in the cytosol engages transient receptor potential vanilloid 3 (TRPV3) by binding to its intracellular domains, inducing Ca2+ influx that subsequently activates the calmodulin-calcineurin-NFAT and PYK2-RAS-ERK pathways to enhance IL-6 and CCL20 production. This FPP-TRPV3-IL-6/CCL20-GC axis potentiates pathogen-specific antibody production, conferring protection in wild-type but not TRPV3-deficient mice. Single-cell RNA-sequencing analyses of systemic lupus erythematosus (SLE) skin lesions and pathogen-infected mouse skin demonstrate hyperactivation of this signalling axis, particularly in the TRPV3high keratinocyte subset. In mouse models of SLE, the activation of this axis correlates with exacerbated disease pathology. Thus, FPP potentiates systemic humoral immunity through the TRPV3-IL-6/CCL20-GC signalling axis, providing insights for the development of vaccine adjuvants and potential therapeutics for SLE.

The integrated stress response (ISR) is activated in response to intrinsic and extrinsic stimuli, playing a role in tumor progression and drug resistance. The regulatory role and mechanism of ISR in liver cancer, however, remain largely unexplored. Here, we demonstrate that OTU domain-containing protein 3 (OTUD3) is a deubiquitylase of eukaryotic initiation factor 2α (eIF2α), antagonizing ISR and suppressing liver cancer. OTUD3 decreases interactions between eIF2α and the kinase EIF2ΑK3 by removing K27-linked polyubiquitylation on eIF2α. OTUD3 deficiency in mice leads to enhanced ISR and accelerated progression of N-nitrosodiethylamine-induced hepatocellular carcinoma. Additionally, decreased OTUD3 expression associated with elevated eIF2α phosphorylation correlates with the progression of human liver cancer. Moreover, ISR activation due to decreased OTUD3 expression renders liver cancer cells resistant to sorafenib, while the combined use of the ISR inhibitor ISRIB significantly improves their sensitivity to sorafenib. Collectively, these findings illuminate the regulatory mechanism of ISR in liver cancer and provide a potential strategy to counteract sorafenib resistance.

E3 ubiquitin ligases recognize substrates through specific interfaces. Accurate delineation of these interfaces is essential, as mutations disrupting them impair protein ubiquitination and drive cancer progression. However, available E3-substrate interface data are sparse and systematic prediction methods remain lacking. Here, we propose MetaESI, a deep learning framework that simultaneously predicts E3-substrate interactions and leverages its interpretable architecture to infer binding interfaces de novo. With a two-stage meta-learning strategy, MetaESI generalizes across diverse E3s and achieves state-of-the-art performance in both interaction and interface prediction. We applied MetaESI at the proteome scale to generate MetaESI-Atlas, which comprises 68,056 annotated interactions across eight species. Integrating multi-omics data, we identified mutations at MetaESI-predicted interfaces that disrupt E3-substrate binding, and experimentally validated representative examples including JunB Q244E and SPOP F102C as oncogenic drivers. By combining interpretable AI with mechanistic insight, MetaESI establishes a methodological paradigm for interpretable model design and a foundational resource for precision oncology and targeted protein degradation.

IRAK4 plays a pivotal role in autoimmune diseases by exerting both kinase and scaffolding functions. Conventional inhibitors of IRAK4 target its kinase activity while leaving the scaffolding function intact. PROTACs, which induce the complete degradation of target proteins, offer a promising strategy to overcome the constraints of traditional inhibition. Here, we designed and synthesized a series of nucleolin (NCL)-bridged, MDM2-recruiting PROTAC degraders by conjugating oridonin (Ori) with Zimlovisertib (Zim). Structure-activity relationship studies identified Ori-Zim-6 as the most potent degrader. Mechanistic investigations revealed that Ori-Zim-6 triggered the proteasomal degradation of IRAK4. Ori-Zim-6 effectively inhibited pro-inflammatory response across multiple cell types in vitro. In a mouse model of psoriasis, oral administration of Ori-Zim-6 resulted in robust therapeutic efficacy and a favorable safety profile. Notably, Ori-Zim-6 exhibited superior anti-inflammatory activity compared to the reference degrader KT-474. These findings establish Ori-Zim-6 as an orally available IRAK4 degrader for the treatment of autoimmune diseases.

Liver fibrosis is the initial stage of most liver diseases, and it is also a pathological process involving the liver in the late stages of many metabolic diseases. Therefore, it is important to systematically understand the pathological mechanism of liver fibrosis and seek therapeutic approaches for intervention and treatment of liver fibrosis. Disordered proteins and their post-translational modifications, such as phosphorylation, play vital roles in the occurrence and development of liver fibrosis. However, the regulatory mechanisms that govern this process remain poorly understood. In this study, we analyzed and quantified the liver proteome and phosphoproteome of carbon tetrachloride-induced early liver fibrosis model in mice. Proteomic analysis revealed that the pathways involved in extracellular matrix recombination, collagen formation, metabolism and other related disorders, and protein phosphorylation modification pathways were also significantly enriched. In addition, Western blotting and phosphoproteomics demonstrated that phosphorylation levels were elevated in the context of liver fibrosis. A total of 13,152 phosphosites were identified, with 952 sites increased, whereas only 156 sites decreased. Furthermore, the upregulated phosphorylation sites, which exhibited no change at the proteome level, mainly shared a common [xxxSPxxx] motif. Consequently, the kinase-substrate analysis ascertained the overactive kinases of these upregulated substrates, which ultimately led to the identification of 13 significantly altered kinases within this dataset. These kinases were mainly cataloged into the STE, CMGC, and CAMK kinase families. Among them, STK4 (serine/threonine-protein kinase 4), GSK3α (glycogen synthase kinase 3α), and CDK11B (cyclin-dependent kinase 11B) were subsequently validated though cellular and animal experiments, and the results demonstrated that their inhibitors could effectively reduce the activation of hepatic stellate cells and extracellular matrix production. These kinases may represent potential therapeutic targets for liver fibrosis, and their inhibitors may serve as promising antihepatic fibrosis drugs.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene protein (STING) signaling plays a critical role in innate immunity and must be tightly regulated to maintain immune homeostasis, but the mechanism underlying the spatiotemporal regulation of this pathway remains largely elusive. Here, it is shown that during DNA viral infection, the linear ubiquitin chain assembly complex (LUBAC) and ovarian tumor deubiquitinase with linear linkage specificity (OTULIN) reversibly catalyze the linear ubiquitination of STING. At the early stage of the infection, LUBAC promotes STING linear ubiquitination to drive its trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus through binding to the Sec24b subunit of the coat protein complex II (COPII) complex. Later on, OTULIN is recruited to TANK1 binding kinase 1 (TBK1)-phosphorylated STING and removes its linear ubiquitin chains, thus preventing excessive antiviral immune responses. Together, the study uncovers a linear ubiquitination-governed spatiotemporal regulatory mechanism that fine-tunes STING-driven antiviral immunity.

Membrane-less organelles (MLOs), formed via liquid-liquid phase separation, are crucial for organization of biomacromolecules and cellular processes, yet their dynamic nature challenges compositional analysis. Conventional proximity labeling methods lack the specificity to overcome high background and systematic biases inherent in studying these condensates. Here we introduce Phase-APEX2, a three-input AND-gate strategy that dramatically enhances labeling specificity. By integrating a split-APEX2 system with a light-controlled H 2 O 2 generator, we developed Phase-APEX2 platforms for two application contexts: Phase-APEX2-MS for proteomics and Phase-APEX2-seq for genomics. Our analyses revealed not only the core interactomes of the nucleolus and stress granules with high accuracy but also uncovered distinct “molecular grammars” governing their assembly and a surprising link between nucleolar dynamics and the transcriptional regulation of mitosis-related genes. This highly specific and adaptable platform opens new avenues for exploring the composition and function of diverse biomolecular condensates.

Trained immunity confers innate immune memory via metabolic and epigenetic reprogramming, yet the intercellular mediators regulating this process in host defense remain largely elusive. Here, through plasma exosomal profiling of tuberculosis (TB)-resistant individuals, we identify a trained immunity-inducing long non-coding RNA (lncRNA), termed tuberculosis resister-derived CLOCK regulator 1 (TRCR1). Mechanistically, exosome-derived TRCR1 collaborates with the RNA-binding protein FXR2 to stabilize CLOCK mRNA by forming lncRNA-protein-mRNA complexes in monocytes, thus enhancing circadian regulator CLOCK expression and promoting CLOCK-mediated histone H3 acetylation (K9/K14) at immune gene promoters, ultimately establishing epigenetic memory-mediated antimicrobial activity. We further reveal that Mycobacterium tuberculosis (Mtb)-secreted protein MPT53 induces lung epithelial cells to release TRCR1-enriched exosomes. In mice, TRCR1 training strengthens host anti-Mtb immunity and improves Bacille Calmette-Guérin (BCG) vaccine efficacy. Collectively, our findings unveil an intercellular TRCR1-FXR2-CLOCK axis driving trained immunity at the lung-systemic immune interface, providing a strategy for refining BCG vaccination and preventing infectious diseases.

The human oral microbiome has been associated with multiple inflammatory conditions including inflammatory bowel disease (IBD). Identifying functional changes in oral microbiome by metaproteomics helps understanding the factors driving dysbiosis related to intestinal diseases. However, enriching bacterial cells from oral samples (such as saliva and mouth rinse) rich in host proteins is challenging. Here, we present an Optimized Salivary MetaProteomic sample analysis workflow (OSaMPle) to enrich salivary bacteria and reduce host-derived interferences for in-depth analysis of the oral metaproteome. Compared to a conventional approach, OSaMPle improved the identification of bacterial peptides and proteins by 3.2 folds and 1.7 folds, respectively. Furthermore, applying OSaMPle to analyze mouth rinse samples from IBD patients revealed significant alterations in bacterial protein expressions under disease conditions. Specifically, proteins involved in the fatty acid elongation pathway in Peptostreptococcus were significantly less abundant in IBD patients, whereas proteins associated with the TCA cycle in Neisseria were significantly more abundant. The OSaMPle workflow is capable of processing small-volume oral samples and adaptable to high-throughput automation. It holds promise as a strategy for investigating the functional responses of oral microbiomes under disease conditions and identifying disease-associated microbes with their proteins, providing critical insights for detecting disease-related biomarkers within the oral microbiome.

The human gut microbiome exhibits characteristics of complex ecosystems, including the ability to resist and compete with exogenous species or communities. Understanding the microbiome response that emerges from such competitive interactions is crucial, particularly for applications like fecal microbiota transplantation (FMT), where the success of treatment largely depends on the outcome of these microbial competitions. During these processes, microbial communities undergo coalescence, a phenomenon where distinct microbial communities combine and interact, leading to complex ecological outcomes that are still being uncovered. In this study, we examined the coalescent dynamics of 10 different pairs of human gut microbiota by co-culturing the plateau-phase communities of individual samples in vitro, and highlighted the critical role of metaproteomics in elucidating the competitive dynamics of co-cultured human fecal samples. Results showed that microbiome changes observed after coalescent co-culture were not straightforwardly an approximate average of the initial taxonomic or functional compositions of the two samples. Instead, both coalescent microbiotas behaved as cohesive structures, influencing the competitive outcome toward one of them. Although co-cultured communities usually exhibited high degrees of taxonomic similarities to one of its parental samples, we found that 23% of the observed proteins still showed differential expression or abundance at the metaproteomic level. Interestingly, and somewhat counterintuitively, no specific microbial ecological characteristic could linearly determine which of the two initial microbiotas would act as the driving microbiota. Instead, we observed that the outcomes of the microbial co-cultures resembled a "rock-paper-scissors"-like dynamic. Through an analysis of co-colonizing species in such "rock-paper-scissors"-like triangle, we discovered that co-colonizing species that contributed to winning each between-community competition differed from one community pair to another. This suggests that no single species or function consistently dominates across all situations; instead, this involves more complex mechanisms, which require further in-depth investigation in future studies. Our findings demonstrate that the complex competitive interactions between microbial communities make predicting success through a single parameter challenging, whereas pre-co-culturing shows promise as an effective method for predicting outcomes in ecological therapies such as FMT. SUMMARY: This study underscores the critical importance of integrating metaproteomics with microbial systems ecology to gain a functional understanding of microbial coalescence. By addressing the ecological question of how two communities compete when they are brought into contact, we investigated the metaproteomic responses of pairs of coalescent co-cultured human gut microbiotas. Our results revealed significant insights: post-co-culture microbiota changes were not merely a simple average of the initial compositions but instead exhibited distinct shifts toward one of the original samples. Notably, due to the observed rock-paper-scissors-like cycle of winning, we argue that no single microbial ecological characteristic could straightforwardly predict which of the two samples would dominate as the driving microbiota. Overall, our findings suggest that during coalescence, microbial communities behave as cohesive structures both taxonomically and functionally, influencing competitive dynamics and ecosystem complexity, indicating that an in vitro coalescence pretest may help predict the success of therapies like FMT.

Osteoclasts derived bone marrow monocytes have been documented to modulate bone quality by directly sensing mechanical forces. However, the mechanisms by which osteoclasts perceive and respond to mechanical disturbances remain unclear. Through integrating multi-omics data of bone tissues from hindlimb unloading (HLU) and control mice, it is revealed that glutamine (Gln) catabolism-induced suppression of apoptosis is critical for monocytes sensing and responding to mechanical unloading. Gln uptake is essential for the survival of monocytes under mechanical unloading. Deprivation of Gln or blockade of Gln transporter solute carrier family 1 member 5 (SLC1A5) inhibits bone resorption by enhancing apoptosis of monocytes. Unloading exposure-induced cell survival is mediated by X-linked inhibitor of apoptosis protein (XIAP)/direct IAP binding protein with low pI (Diablo) axis. Upon mechanical unloading XIAP is upregulated, then interacts with Diablo in mitochondrial and promotes the K63-linkage ubiquitylation of Diablo at the K212 site. This sequesters Diablo within the mitochondrial and inhibits its release into the cytosol, ultimately inhibiting cell apoptosis of osteoclasts and the precursors. Clinically, the serum Gln levels are positively correlated with cross linked C-telopeptide of type I collagen (CTX) levels, indicating that serum Gln levels might serve as a potential biomarker for predicting the risk of osteoporosis. Gln-deficient diet, as well as SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA), effectively preserves bone mass in HLU mice, implicating attractive approaches for combating bone loss induced by weightlessness or disuse.

Host immune cells are equipped with cytosolic sensors to detect invading pathogens and initiate anti-infectious responses. However, how pathogens undermine host intracellular surveillance for persistent infection is not fully understood. Here, we identify that Mycobacterium tuberculosis protein kinase PknG subverts inflammasome sensor NLRP3-mediated cytokine release and pyroptosis by targeting host linear ubiquitin chain assembly complex (LUBAC). Mechanistically, PknG phosphorylates the LUBAC subunit HOIL-1L to prevent it from engaging in LUBAC formation, thereby suppressing linear ubiquitination of inflammasome adaptor ASC to dampen NLRP3 inflammasome assembly. Meanwhile, this phosphorylation stabilizes and activates HOIL-1L, which, in turn, exerts ubiquitin ligase activity to mediate K48-linked ubiquitination of NLRP3 for degradation. Disrupting the kinase activity or HOIL-1L-interacting region of PknG facilitates host NLRP3-dependent anti-Mtb immunity in mice. Thus, the bacterial kinase disrupts host linear ubiquitin machinery and coopts its ubiquitin ligase subunit to constitute an inter-species enzymatic cascade that drives inflammasome sensor degradation for counteracting immune surveillance.