The proteasome maintains the integrity of eukaryotic proteomes by selectively degrading ubiquitinated protein substrates. Ubiquitination targets a wide range of substrates for degradation, including translationally stall...The proteasome maintains the integrity of eukaryotic proteomes by selectively degrading ubiquitinated protein substrates. Ubiquitination targets a wide range of substrates for degradation, including translationally stalled nascent chains, misfolded proteins, and properly folded but short-lived proteins destined for regulatory degradation. Distinct structural features and ubiquitination patterns across these classes of substrates remain largely undefined. In this study, we combine structural proteomics and time-resolved isotopic labeling to profile the modification sites, dynamics, and conformational properties of the human ubiquitinome. We show that proteins undergoing rapid proteasomal degradation are ubiquitinated at lysine residues that are normally buried within structured regions of their native conformations. We provide proteome-wide evidence that this high-flux subset of the ubiquitinome is enriched in newly synthesized proteins that have non-native conformations. Together, our findings demonstrate how the lack of structural integrity of misfolded nascent proteins influences their ubiquitination patterns and leads to rapid proteasomal degradation.
The Saccharomyces cerevisiae silent information regulator (SIR) complex performs all core heterochromatin functions. It first drives histone deacetylation in an iterative, spreading manner, then stably incorporates with...The Saccharomyces cerevisiae silent information regulator (SIR) complex performs all core heterochromatin functions. It first drives histone deacetylation in an iterative, spreading manner, then stably incorporates with nucleosomes to compact and epigenetically repress the chromatin. How this switch between dynamic spreading and stable compaction occurs has remained poorly understood, partly due to limited structural data on the intact complex. Using crosslinking mass spectrometry, we identified an uncharacterized inter-subunit interaction connecting these two states: the Sir2 deacetylase interacts with the scaffolding subunit Sir4 through its coiled-coil domain, which also contacts the Sir3 compaction subunit. This interaction hub contains conserved Sir2 residues that can adopt multiple conformations, including orientation toward the active site, alongside co-evolved residues that enable species-specific Sir4 interactions. Mutations disrupting this hub disrupt heterochromatic repression in vivo and affect the deacetylation activity, directly linking catalysis to compaction. Our findings reveal how a multifunctional complex stages a stepwise transition to achieve epigenetic gene repression.
The glioblastoma microenvironment is highly immunosuppressive and enriched with macrophages, which critically promote tumor progression and confer resistance to current therapies. However, the molecular mechanisms by whi...The glioblastoma microenvironment is highly immunosuppressive and enriched with macrophages, which critically promote tumor progression and confer resistance to current therapies. However, the molecular mechanisms by which glioblastoma cells orchestrate macrophage recruitment through post-transcriptional regulation remain poorly understood. Here, we identify a glioblastoma-intrinsic pathway whereby the m6A RNA modification reader IGF2BP3 couples RNA metabolism with metabolic competition and immune remodeling. Specifically, IGF2BP3 selectively binds and stabilizes m6A-modified transcripts encoding the methionine transporter SLC38A2, thereby enhancing methionine uptake by glioblastoma cells. This metabolic advantage creates a methionine-deprived state in neighboring macrophages, leading to reduced global m6A methylation and increased expression of the chemokine CCL5. Elevated CCL5 subsequently drives further macrophage infiltration, establishing a feedforward loop that amplifies nutrient competition and sustains immunosuppression within the tumor microenvironment. Together, our findings delineate an m6A-dependent metabolic circuit that governs macrophage recruitment in glioblastoma and suggest a therapeutic avenue to reprogram the tumor immune microenvironment.
Brown adipose tissue (BAT) regulates whole-body energy balance through uncoupling protein 1 (UCP1)-dependent thermogenesis and secretion of metabolic factors. Recent studies suggest UCP1-independent mechanisms contribute...Brown adipose tissue (BAT) regulates whole-body energy balance through uncoupling protein 1 (UCP1)-dependent thermogenesis and secretion of metabolic factors. Recent studies suggest UCP1-independent mechanisms contribute to energy balance, with UCP1 being conditionally dispensable. However, how adaptation to UCP1 deficiency is regulated remains unclear. Our single-nucleus RNA sequencing of BAT from cold-exposed Ucp1 knockout mice reveals a distinct brown adipocyte subpopulation (U2). U2 adipocytes exhibit a secretory profile enriched in batokines like growth differentiation factor 15 (GDF15), suggesting a shift toward an endocrine role. Functional analyses reveal that GDF15-GFRAL signaling is required to sustain energy expenditure in adipose tissue (AT). The Ucp1/Gfral knockout increased food intake to compensate for decreased energy expenditure in AT. Additionally, a conserved UCP1-GDF15 regulatory axis in human AT is observed. These findings identify a regulatory brown adipocyte subpopulation emerging in response to UCP1 deficiency, representing a compensatory mechanism for maintaining energy homeostasis in mammals.
Lujano-Olazaba O, Robinson M, Jordan GJ
… +13 more, Howe S, Platen A, Lucht C, Vandra D, Gallo C, Shammas M, Shelby K, Wong N, Niesman IR, Schlaepfer DD, Cheresh DA, Annunziata CM, House CD
High-grade serous ovarian cancer (HGSOC) often relapses after chemotherapy due to chemoresistant cancer stem-like cells (CSCs). NF-κB signaling, previously shown to enhance stemness traits, was investigated for its role...High-grade serous ovarian cancer (HGSOC) often relapses after chemotherapy due to chemoresistant cancer stem-like cells (CSCs). NF-κB signaling, previously shown to enhance stemness traits, was investigated for its role in post-treatment tumor regrowth. Here, we found that NF-κB subunits RelA and RelB jointly regulate extracellular matrix genes but differentially control integrin subunits: RelA regulates ITGAV (αV) and RelB regulates ITGB3 (β3). Integrin αVβ3 expression is upregulated on CSCs, and this is partially driven by NF-κB activation. In vivo models demonstrate that αVβ3 cells have higher tumor-initiating capacity and comprise over 90% of cells from relapsed tumors. Cells expressing RelB and αVβ3 preferentially grow on mesentery at relapse. Targeting this pathway, combined RelB knockdown and inhibition of αVβ3 eliminated stress-tolerant CSCs, reduced tumor burden, and extended survival. These findings highlight integrins as promising therapeutic targets and reveal distinct roles for NF-κB subunits in regulating metastasis and relapse in HGSOC.
Obesity is associated with profound immune dysregulation, driving chronic inflammation while compromising host defense against tumors. While trained immunity can enhance innate effector functions, it has thus far require...Obesity is associated with profound immune dysregulation, driving chronic inflammation while compromising host defense against tumors. While trained immunity can enhance innate effector functions, it has thus far required parenteral administration of microbial ligands. Here, we show that incorporating a yeast-derived β-glucan supplement in mouse diets induces trained immunity via reprogramming of hematopoietic stem and progenitor cells. This dietary intervention leads to sustained production of metabolically enhanced monocytes and macrophages that rescue anti-tumor immunity in high-fat diet-induced obese mice, and corrects immune dysfunction sustained after weight loss. Our work reveals that yeast β-glucans act as functional "immuno-nutrients," which remodel innate immunity and identifies the mucosal/bone-marrow axis as a target for dietary manipulations to restore immune resilience without impacting metabolism.
RAG1/2 catalyzes V(D)J recombination to assemble antigen receptor genes, but the RAG1 N-terminal zinc-coordinating domain (NZD) has remained structurally and functionally uncharacterized. Here, we determine the NMR struc...RAG1/2 catalyzes V(D)J recombination to assemble antigen receptor genes, but the RAG1 N-terminal zinc-coordinating domain (NZD) has remained structurally and functionally uncharacterized. Here, we determine the NMR structure of mouse RAG1 NZD, revealing a compact, zinc-dependent fold composed of four α-helices and two short β-strands. This architecture is organized into two interdigitated zinc-coordinating modules, ZMa and ZMb. Structural similarity searches identify no close homolog with the same overall architecture, suggesting that NZD represents a previously undescribed zinc-coordinating fold. Comparative analyses show that NZD is broadly conserved across RAG1 and RAG1-like proteins, while also displaying lineage-specific remodeling, including acquisition of the H2 helix in jawed vertebrates. Guided by structure prediction, we further identify putative NZD-like domains in Chapaev transposases, supporting a possible evolutionary link between RAG1/RAG1L NZDs and Chapa domains. Together, these findings provide a structural framework for mechanistic and evolutionary analyses of RAG1.
Ma H, Zhang E, Cai J
… +21 more, Hang D, Ma X, Yang Y, Zhu M, Sun Q, Huang J, Zhang J, Huang G, Zhang C, Jin C, Fu Y, Gong L, Shen C, Lu X, Jiang Y, Dai J, Jin G, Gu D, Qian X, Shen H, Ma H
Metabolic dysregulation has been established as a key driver in tumorigenesis, but its underlying mechanisms in lung cancer remain poorly characterized. In this study, we performed nested case-control analyses in two pro...Metabolic dysregulation has been established as a key driver in tumorigenesis, but its underlying mechanisms in lung cancer remain poorly characterized. In this study, we performed nested case-control analyses in two prospective cohorts (208 and 144 matched pairs) to examine associations between plasma metabolites and lung cancer risk. Untargeted metabolomics identified circulating metabolites of the branched-chain amino acid (BCAA) pathway as significantly associated with lung cancer risk. An animal study demonstrated that a high-BCAA diet accelerated lung cancer progression in the Kras mice model. Among the three BCAAs, leucine contributed much more to promoting lung cancer growth. Mechanistically, AUH-mediated acetyl-CoA production from leucine metabolism fuels cholesterol synthesis, promoting lipid raft formation and EGFR redistribution and activation, thereby driving lung tumorigenesis. Moreover, atorvastatin blocked leucine-induced tumor progression in mice. Overall, our findings provide experimental evidence that leucine-driven BCAA metabolic reprogramming promotes lung tumorigenesis via cholesterol metabolism, revealing a potential therapeutic target.
The glandular stomach is an important digestive organ, and its proper epithelial development is essential for its normal structure and function. However, the molecular mechanisms underlying its epithelial development rem...The glandular stomach is an important digestive organ, and its proper epithelial development is essential for its normal structure and function. However, the molecular mechanisms underlying its epithelial development remain largely unclear. Here, we employ mouse genetics to reveal that Hippo signaling regulates stomach epithelial development by balancing epithelial proliferation and differentiation. Deletion of Yap reduces epithelial growth in the developing glandular stomach, whereas overexpression of constitutively active Yap promotes its expansion. Single-cell RNA sequencing (scRNA-seq) and histological analyses further show that Yap enhances gastric epithelial proliferation while suppressing differentiation. Using 3D organoids, we find that Yap directly governs gastric epithelial proliferation and differentiation. Further genetic studies reveal that Mst1/2 deficiency regulates glandular stomach development partially through Yap. Finally, we demonstrate that Yap controls gastric epithelial proliferation and differentiation by modulating cell cycle activity. Overall, our studies demonstrate that Hippo signaling plays an important role in fine-tuning stomach development.
Benbahouche N, Aze A, Siegfried A
… +12 more, Stefancikova L, Kermi C, Pascussi JM, Pannequin J, Moreaux J, Egger T, Chavanieu A, Hugnot JP, Delisle MB, Moyal E, Uro-Coste E, Maiorano D
The RAD18 (E3) ubiquitin ligase, a key DNA damage tolerance regulator that also functions in DNA double-strand break repair, is overexpressed in the brain cancer glioblastoma. Here, we show that RAD18 promotes glioblasto...The RAD18 (E3) ubiquitin ligase, a key DNA damage tolerance regulator that also functions in DNA double-strand break repair, is overexpressed in the brain cancer glioblastoma. Here, we show that RAD18 promotes glioblastoma cell proliferation in the absence of exogenous damage, independently of its catalytic activity. RAD18 downregulation arrests glioblastoma cells in the G1 phase of the cell cycle, leading to senescence onset, with no apparent increase in DNA damage. We also show that RAD18 sustains glioblastoma stem cells' self-renewal and the growth of tumor orthotropic xenografts in mice. Further, we show that increased RAD18 expression enhances the growth of non-transformed cells and induces the features of oncogenic transformation. Mechanistically, RAD18 interacts and negatively regulates the NF2 tumor suppressor through its SAP domain, thereby facilitating stabilization and nuclear retention of the YAP1 transcription factor. Altogether, these data propose RAD18 as a key target to sensitize glioblastoma to therapy.
Qian L, Su R, Wang W
… +20 more, Yan B, Qiu L, Zhao Z, Yang X, Li Y, Ding C, Hu R, Yu X, Yan Q, Niu M, Wu K, He Q, Yang Y, Johnson DR, Lawson CE, Aharoni A, Nikoloski Z, Zhou J, He J, He Z
Coastal ecosystems play critical roles in biogeochemical cycling, food webs, climate regulation, and aquaculture. Their functions are shaped by multitrophic interactions across microbe-microbe, microbe-plant, and microbe...Coastal ecosystems play critical roles in biogeochemical cycling, food webs, climate regulation, and aquaculture. Their functions are shaped by multitrophic interactions across microbe-microbe, microbe-plant, and microbe-animal interfaces, yet our understanding of the underlying mechanisms remains limited. Synthetic ecology offers a promising approach to disentangle such interactions using simplified and controllable synthetic communities (SynComs). Here, we review microbe-plant-animal interactions toward ecosystem function improvements and provide a biological foundation for SynCom design. We further propose a framework for coastal synthetic ecology, including SynCom design and construction, experimental validation of SynCom functions, and laboratory scaling-up and field applications with a focus on greenhouse gas reduction, carbon sequestration, and pollutant degradation. Finally, we discuss future directions for coastal synthetic ecology, with a focus on biogeochemical cycling, food web structure and function, and biological stoichiometry. Overall, this review highlights the potential of SynComs to address environmental and ecological challenges in coastal ecosystems.
Long non-coding RNAs (lncRNAs) are key regulators across eukaryotes, yet their functions in non-Dikarya fungal pathogens remain largely unexplored. Here, we provide a comprehensive identification and characterization of...Long non-coding RNAs (lncRNAs) are key regulators across eukaryotes, yet their functions in non-Dikarya fungal pathogens remain largely unexplored. Here, we provide a comprehensive identification and characterization of lncRNAs in the non-Dikarya fungal order Mucorales, a WHO high-priority group of opportunistic human pathogens. Using Mucor lusitanicus and the clinically relevant pathogen Rhizopus microsporus as models, we show that NDF lncRNAs exhibit conserved features, dynamic regulation during host interactions, and integration into gene regulatory networks. Although preferentially encoded in inactive chromatin compartments, lncRNA expression in R. microsporus is associated with N6-methyladenine (6mA). We further show that lncRNAs are targeted by canonical and non-canonical RNA interference (RNAi) pathways. Comparative genomics analyses identified conserved lncRNAs, including two essentials for viability (lncRNA2 and lncRNA4). Disruption of lncRNA4, even in heterokaryosis, severely impairs growth and filamentation. These results establish lncRNAs as important regulators of fungal physiology and pathogenicity, highlighting their potential as novel antifungal targets.
In many cancers, stably elevated MYC levels drive sustained activation of anabolic programs and the cell cycle, creating opportunities for the synthetic-lethal targeting of MYC tumors. Enhanced mitochondrial respiration...In many cancers, stably elevated MYC levels drive sustained activation of anabolic programs and the cell cycle, creating opportunities for the synthetic-lethal targeting of MYC tumors. Enhanced mitochondrial respiration is a hallmark of MYC overexpressing cancer cells. Mitochondrial respiration sustains the TCA cycle by regenerating NAD through complex I-mediated oxidation of NADH, supporting the anabolic demand of MYC-driven cells. Metabolic carbon tracing reveals that MYC shifts the TCA cycle carbon source from glucose to glutamine. Inhibition of the glutamine-fueled TCA cycle using NAD-depleting complex I inhibitors promotes MYC-dependent synthetic lethality in breast cancer cells. In mouse models of MYC tumors, combined inhibition of complex I and glutaminolysis produces persistent suppression of tumor growth. Altogether, the elevated respiration of MYC cells supports a glutamine carbon-enriched TCA cycle that meets anabolic demand, rendering MYC tumors selectively vulnerable to mitochondrial respiration and glutaminolysis inhibitors.
Öberg M, Myers C, Saffarzadeh N
… +19 more, Maric I, Murillo-León M, Strömberg A, Fabrikova D, Fabrik I, Rivas-Galvez L, Deshmukh MV, Enriquez J, Ali KX, Crescitelli R, Angeletti D, Sayin VI, Jin T, Rafie K, Skibicka KP, Kurzawa-Akanbi M, Paul G, Gekara NO, Härtlova A
All animals age. However, aging is a heterogeneous process, and individual organisms age differently. Moreover, within the same organism, cells or organs do not age at the same speed. For instance, neurodegeneration, a h...All animals age. However, aging is a heterogeneous process, and individual organisms age differently. Moreover, within the same organism, cells or organs do not age at the same speed. For instance, neurodegeneration, a hallmark of aging, generally manifests later than other peripheral aging signs. The genetic determinants of aging are not completely understood. Gain-of-function (GoF) mutations in leucine-rich repeat kinase 2 (LRRK2) are major genetic risk factors for Parkinson's disease (PD). By analyzing PD patients and LRRK2 mice, we show that PD represents an accelerated aging disorder driven by STING-dependent inflammation. This inflammation begins peripherally, disrupts the blood-brain barrier, and causes dopaminergic neurodegeneration. Mechanistically, aging or LRRK2 causes endolysosomal decline, resulting in cytosolic self-DNA accumulation and the release of DNA-containing extracellular vesicles (EVs) that activate the cGAS-STING pathway within and between cells. Our findings identify LRRK2 as a key driver of accelerated aging and systemic inflammaging through DNA-containing EVs, highlighting potential therapeutic targets to counteract inflammaging and neurodegeneration.
Assaf S, Bozek DA, Heemskerk K
… +11 more, Banks A, MacLeod G, Bahia RK, Cutts E, Johnston MJ, Cseh O, Angers S, Nikolic A, Gallo M, Luchman HA, Weiss S
Harboring a low mutational burden, glioblastoma relies on various epigenetic regulators to fuel its development and progression, several of which remain mechanistically enigmatic. Here, we show that the histone methyltra...Harboring a low mutational burden, glioblastoma relies on various epigenetic regulators to fuel its development and progression, several of which remain mechanistically enigmatic. Here, we show that the histone methyltransferase DOT1L shapes chromatin accessibility of glioblastoma stem cell enhancer elements to reversibly regulate fate- and growth-related transcriptional programs. A genome-wide chemogenomic knockout screen reveals that the mediator complex tail module subunit, MED23, is essential for glioblastoma stem cell growth arrest following DOT1L inhibition, critically relying on CCND2 repression. MED23 knockout (KO) glioblastoma stem cells do not display the chromatin accessibility changes at enhancer elements following DOT1L inhibition. Consequently, MED23-KO rescues glioblastoma stem cell growth by enabling CCND2 transcription in the context of DOT1L inhibition. Our results uncover valuable mechanistic insights involving DOT1L cooperation with the MED23-driven mediator complex to regulate chromatin accessibility and coordinate transcriptional programs governing glioblastoma stem cell growth.
Chromatin compartments reorganize dramatically during cell-cycle transitions, shifting from a compartmentalized interphase to a condensed mitotic configuration in which large-scale compartment signals are attenuated. Whi...Chromatin compartments reorganize dramatically during cell-cycle transitions, shifting from a compartmentalized interphase to a condensed mitotic configuration in which large-scale compartment signals are attenuated. While histone modifications are linked to compartment identity, their contribution to compartment organization remains unclear. Using meiotic (mouse spermatogenesis) and mitotic (mouse neuronal progenitor cell [mNPC]) models, we mapped histone landscapes alongside compartment dynamics. We found that histone H4 lysine 16 acetylation (H4K16ac) strongly tracked compartment reorganization, remaining enriched in A compartments during compartmentalization but redistributing upon chromatin compaction. In spermatogenesis, H4K16ac loss on the X chromosome coincided with condensation and H3K9me3 spreading. Machine learning highlighted H4K16ac as a stronger predictor of compartment transitions than H3K27ac. Acute degradation of MOF, the principal H4K16ac acetyltransferase, disrupted both the spatial distribution and global abundance of H4K16ac and progressively impaired compartmentalization in mNPCs. These results position H4K16ac as a histone modification selectively coupled to chromatin compartment transitions.
The predictability of evolution across lineages remains unclear. We examine repeated adaptation in the globally distributed sibling species, Drosophila melanogaster and D. simulans. We assemble a high-quality reference g...The predictability of evolution across lineages remains unclear. We examine repeated adaptation in the globally distributed sibling species, Drosophila melanogaster and D. simulans. We assemble a high-quality reference genome for D. simulans, and integrate whole-genome data from approximately 2,000 strains sampled across major continents. Population genomic analyses indicate more recent global colonization of D. simulans than D. melanogaster. Using complementary selection scans, we quantify signatures of positive selection across evolutionary timescales and genomic contexts. Despite substantial divergence, 12%-17% of adaptively evolving genes are shared between species across methods, indicating repeated selection of the same genes and biological pathways. Convergence is particularly pronounced for insecticide resistance genes. Gene-level repeatability is further supported by oxidative stress experiments. Our study provides a quantitative, multiscale framework for dissecting hierarchical convergence and clarifies how genomic architecture, environmental change, and genetic background shape the repeatability of adaptation.
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovium infiltration of immune cells, leading to inflammatory activation of fibroblast-like synoviocyte (FLS). Neutrophils infiltrate the synovi...Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovium infiltration of immune cells, leading to inflammatory activation of fibroblast-like synoviocyte (FLS). Neutrophils infiltrate the synovium early in RA and exacerbate disease by releasing neutrophil extracellular traps (NETs). CCDC25 has been identified as a sensor for NETs-DNA, but its role in FLS activation remains to be investigated. Here, CCDC25 was found upregulated in RA-FLS, with reduced dimeric and increased monomeric forms relative to controls, and only monomeric CCDC25 bound to NETs-DNA. NETs-DNA binding to monomeric CCDC25 enhanced Rac1-Sar1 interaction, disrupted COPII-dependent ER-to-Golgi trafficking, induced endoplasmic reticulum stress (ERS), and promoted inflammatory activation of FLS. In vivo, CCDC25 deficiency alleviated arthritis severity in adjuvant-induced, collagen-induced and serum-transfer models, reduced synovitis, and weakened the neutrophil-FLS feedback loop. These findings support monomeric CCDC25 as a NETs-DNA sensor that induces inflammatory activation of FLS, with potential as a therapeutic target in RA.
Ruple et al. explores the potential of a set of quantitative gut microbiome parameters as diagnostic and therapeutic readouts beyond gut microbiome taxonomic composition or gene content. The authors profile QMPs' changes...Ruple et al. explores the potential of a set of quantitative gut microbiome parameters as diagnostic and therapeutic readouts beyond gut microbiome taxonomic composition or gene content. The authors profile QMPs' changes across diets and murine host genetic backgrounds.
Gut microbiome composition has been frequently but inconsistently associated with human disease. Using a food dye-based gastrointestinal (GI) passage assay to measure GI transit time, stool production (fecal mass), fecal...Gut microbiome composition has been frequently but inconsistently associated with human disease. Using a food dye-based gastrointestinal (GI) passage assay to measure GI transit time, stool production (fecal mass), fecal microbiota density (microbial load), and absolute microbiota proliferation (fecal microbiota excretion) in mice, we show quantitative microbiome parameters (QMPs) to be controlled by diet, feeding pattern, and IL-10 deficiency, with food intake and GI transit time acting as separate modulators. High-fiber diet (HFiD) and time-restricted feeding (TRF) reduce GI transit time and induce convergent QMP reductions, whereas high-fat diet (HFaD) produces contrary effects. Intestinal gene expression signatures are consistent with the described shared anti-inflammatory effects of HFiD and TRF, which sharply contrast differences in fecal taxonomic microbiota profiles that are explained by ecological and experimental confounders. Similar QMP and gene expression modulations by distinct diet and feeding interventions warrant exploration of a diagnostic and therapeutic QMP potential for microbiome-mediated disease.