Mochizuki-Ono C, Taketomi Y, Irie A
… +19 more, Kano K, Nagasaki Y, Miki Y, Ono T, Nishito Y, Nakajima T, Tomabechi Y, Hanada K, Shirouzu M, Watanabe T, Hata K, Izumi Y, Bamba T, Chun J, Kudo K, Kotani A, Endo Y, Aoki J, Murakami M
Extracellular vesicles (EVs) contribute to intercellular communication, yet how EV-driven lipid signaling is regulated in immune responses remains incompletely understood. Here, we show that genetic deficiency or antibod...Extracellular vesicles (EVs) contribute to intercellular communication, yet how EV-driven lipid signaling is regulated in immune responses remains incompletely understood. Here, we show that genetic deficiency or antibody-mediated neutralization of PLA2G12A, a secreted phospholipase A (sPLA) transiently upregulated in activated CD4 T cells, prevents pathogenic Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on T cell-derived EVs to produce lysophospholipids including the RORγt activator 1-oleoyl-lysophosphatidylethanolamine. Furthermore, these lysophospholipids are converted by autotaxin to lysophosphatidic acid, which amplifies Th17 differentiation via the LPA receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is restored by supplementation with PLA2G12A-modified EVs. These results provide a rationale for the sPLA-EV-lysophospholipid axis as a general mode of sPLA action and suggest that targeting PLA2G12A may be useful for the treatment of Th17-related or possibly other inflammatory diseases.
Cullin-RING ligase 4 (CRL4) complexes achieve substrate specificity through DDB1-CUL4-associated factors (DCAFs), yet how individual DCAFs engage the adaptor protein DDB1 remains incompletely understood. Here, we report...Cullin-RING ligase 4 (CRL4) complexes achieve substrate specificity through DDB1-CUL4-associated factors (DCAFs), yet how individual DCAFs engage the adaptor protein DDB1 remains incompletely understood. Here, we report the cryo-electron microscopy structure of DCAF8 in complex with DDB1 (2.53 Å) and define the molecular determinants underlying CRL4 assembly and cell cycle progression. Our structure reveals that DCAF8 associates with DDB1 primarily through an N-terminal helix-loop-helix (HLH) motif that inserts into a conserved pocket formed by the BPA and BPC domains of DDB1. Disruption of this interface impairs complex assembly, attenuates CDC25A ubiquitination, and results in cell cycle defects. In contrast, residues within the conserved double DxR box of DCAF8 are positioned away from the DDB1 interface and are dispensable for adaptor binding. Together, these findings define a DCAF8-specific recruitment mechanism within CRL4 ubiquitin ligase assemblies.
R-loop remodeling dynamically regulates chromatin states and gene expression; however, its exploitation by cancer to sustain self-renewal and malignancy remains poorly understood. Here, we find that glioblastoma (GBM) st...R-loop remodeling dynamically regulates chromatin states and gene expression; however, its exploitation by cancer to sustain self-renewal and malignancy remains poorly understood. Here, we find that glioblastoma (GBM) stem cells (GSCs) display highly active R-loops compared to differentiated progeny and neural stem cells. Genome-wide mapping reveals cell-specific enrichment and spatial accumulation of R-loops at promoter-proximal regions in GSCs, correlating with active transcription and open chromatin. We identify N-acetyltransferase 10 (NAT10) as a high-affinity R-loop-binding protein in GSCs, where it is overexpressed downstream of OLIG1. NAT10 catalyzes widespread N-acetylcytidine (acC) deposition on the RNA strand of R-loops, stabilizing promoter-associated R-loops and facilitating open chromatin to sustain self-renewal through core stemness regulators, including EGR1. NAT10 knockdown suppresses GSC proliferation and maintenance in vitro and attenuates tumor growth in vivo. Pharmacological inhibition of NAT10/acC-modified R-loops using remodelin phenocopies NAT10 genetic targeting, demonstrating therapeutic promise for targeting cancer.
Resolving T cell clonality at single-cell spatial resolution remains a major challenge. Here, we developed an in situ hybridization panel comprising probes for immune and tissue cell types alongside comprehensive coverag...Resolving T cell clonality at single-cell spatial resolution remains a major challenge. Here, we developed an in situ hybridization panel comprising probes for immune and tissue cell types alongside comprehensive coverage of TRAV, TRBV, TRGV, and TRDV gene segments, enabling unbiased spatial mapping of T cell clones in situ. As a proof of principle, we applied this approach to human kidney biopsies from patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and controls. Combinatorial T cell receptor variable (TRV) gene expression allowed identification of T cell clones and their spatial organization. Confined clusters of clonally related αβ T cells were found in proximity to increased numbers of antigen-presenting cells, consistent with local immune activation, while γδ T cells occupied distinct peripheral niches. Although paired αβ chain detection is currently limited, this method establishes a scalable framework for spatially resolved clonotype analysis and provides a broadly applicable tool to investigate tissue-level immune organization across diseases.
Mizuno N, Abraham J, Jimenez-Perez K
… +15 more, Rose I, Springgay L, Boehm D, Ando T, Streblow D, Ward J, Miller S, Pandey U, Junaid A, Joyner D, Muir R, Haddad EK, Burkhart D, Rasheed O, DeFilippis VR
Stimulator of interferon genes (STING) is a pattern recognition receptor that activates type I interferon and proinflammatory responses following cytosolic DNA exposure. Its pharmacologic stimulation enhances vaccine pot...Stimulator of interferon genes (STING) is a pattern recognition receptor that activates type I interferon and proinflammatory responses following cytosolic DNA exposure. Its pharmacologic stimulation enhances vaccine potency and generates anti-tumor responses, but clinical trials evaluating STING agonists have not supported human use. STING activation can occur through the engagement of cytosolic or transmembrane protein domains, processes to which distinct phenotypes are attributed. However, transmembrane agonists are human selective, and conventional in vivo testing is not feasible. We synthesized human-selective STING agonists and describe genetically humanized STING mice as an in vivo model useful for examining these agonists. Experiments suggest that the lead molecule functions through binding to the STING transmembrane region, and its comparison with conventional agonists reveals differences in molecular and immune effects. This work both represents a thorough in vivo immune characterization of the effects of transmembrane STING agonism and demonstrates the efficacy of a potential vaccine adjuvant and oncological therapeutic.
Kövér B, Willis TL, Sherwin O
… +13 more, Kaufman-Cook J, Kemkem Y, Vazquez Segoviano M, Lodge EJ, Zamojski M, Mendelev N, Zhang Z, Smith GR, Bernard DJ, Lu HC, Sealfon SC, Ruf-Zamojski F, Andoniadou CL
Previous single-cell profiling studies of the pituitary gland have yielded minimally reproducible insights due to their low statistical power and methodological inconsistencies. To address this, we generate the uniformly...Previous single-cell profiling studies of the pituitary gland have yielded minimally reproducible insights due to their low statistical power and methodological inconsistencies. To address this, we generate the uniformly pre-processed Consensus Pituitary Atlas (CPA) using all 283 existing mouse pituitary single-cell datasets (∼1.3 million high-quality cells). The CPA reveals cell typing and lineage markers, including low-expression transcripts that previous analyses could not detect. Leveraging the scale of the CPA, we develop machine learning models to automate and standardize cell type annotation and doublet identification for future studies. Utilizing the curated metadata, we identify sex-biased and age-dependent gene expression patterns at cell type resolution. To uncover drivers of cell fates, first we determine consensus cell communication patterns. Second, we use RNA sequencing and chromatin accessibility data to identify transcription factors associated with cell fates across modalities. The epitome platform provides a user-friendly interface with the CPA and allows streamlined analyses.
Cuproptosis, a copper-dependent cell death process induced by excessive copper, represents an emerging therapeutic strategy in oncology. However, tumor-specific molecular pathways regulating this process remain poorly de...Cuproptosis, a copper-dependent cell death process induced by excessive copper, represents an emerging therapeutic strategy in oncology. However, tumor-specific molecular pathways regulating this process remain poorly defined. Here, we demonstrate that copper levels are elevated in lung adenocarcinoma (LUAD), and LUAD cell lines exhibit increased resistance to cuproptosis. Mechanistically, elevated copper stress promotes the expression of the desuccinylase SIRT5 while reducing global succinylation in LUAD cells. Furthermore, we found that SIRT5 is a critical mediator of cuproptosis through the desuccinylation modification on ferredoxin1 (FDX1) protein at Lys84. This modification triggers TRIM8-mediated ubiquitination, leading to FDX1 proteasomal degradation and enhanced cuproptosis resistance. These results reveal the important role of SIRT5 in LUAD cuproptosis resistance. Notably, combining the SIRT5 inhibitor MC3482 with the cuproptosis inducer Elesclomol-Cu synergistically suppresses tumor growth in vivo, suggesting a promising therapeutic strategy. These findings elucidate mechanisms underlying cuproptosis resistance and propose a novel treatment approach for LUAD.
Circular RNAs (circRNAs) are highly enriched in the brain, yet their functional contributions to synaptic plasticity remain unclear. Here, we uncover a plasticity-induced circRNA that links mitochondrial regulation to ac...Circular RNAs (circRNAs) are highly enriched in the brain, yet their functional contributions to synaptic plasticity remain unclear. Here, we uncover a plasticity-induced circRNA that links mitochondrial regulation to activity-dependent spine remodeling. Chemical long-term potentiation in primary hippocampal neurons identified circSamm50, derived from Samm50, which encodes a mitochondrial outer membrane protein, as robustly upregulated. circSamm50 sequesters miR-186-5p to sustain Samm50 mRNA levels, thereby coupling circRNA signaling to mitochondrial gene regulation. Acute CRISPR-Cas13-mediated depletion of circSamm50 disrupted mitochondrial morphology, transport, and bioenergetics across neuronal compartments. Functionally, circSamm50 loss impaired excitatory synaptic transmission, reduced spine density, and compromised two-photon glutamate uncaging-induced structural plasticity. Together, our findings uncover circSamm50 as a plasticity-modulated circRNA that coordinates mitochondrial function with activity-dependent spine remodeling, revealing a mechanism by which circRNAs couple metabolic control to synapse function and structural plasticity.
Human action selection under reinforcement is believed to rely on two distinct strategies: model-free and model-based reinforcement learning. While behavior in sequential decision-making tasks often reflects a mixture of...Human action selection under reinforcement is believed to rely on two distinct strategies: model-free and model-based reinforcement learning. While behavior in sequential decision-making tasks often reflects a mixture of both, the neural basis of individual differences in their expression remains unclear. Here, we conduct a large-scale fMRI study with 179 participants performing a variant of the two-step task. Using both cluster-defined subgroups and computational parameter estimates, we find that in the ventromedial prefrontal cortex, model-based value signals are strongly linked to the degree of model-based behavioral reliance, whereas model-free signals are ubiquitous across individuals regardless of model-free behavioral influence. Individuals lacking model-based behaviors and model-based neural signals, exhibit impaired state prediction errors (a key signal for learning a model of the environment) in the dorsolateral prefrontal cortex and intraparietal sulcus, suggesting that reduced model-based control may depend in part on underlying difficulties in forming accurate model-based predictions.
Neutrophil-involved neuroinflammation in dorsal root ganglion (DRG) plays double-sword roles in chronic pain. How DRG neuron-neutrophil interaction contributes to chronic pain remains unclear. Here, we report that MLKL,...Neutrophil-involved neuroinflammation in dorsal root ganglion (DRG) plays double-sword roles in chronic pain. How DRG neuron-neutrophil interaction contributes to chronic pain remains unclear. Here, we report that MLKL, a key molecule in necroptosis, is constitutively expressed in the nucleus of nociceptive neurons and binds to histone H3. Periphery inflammation disrupted MLKL/H3 interaction, leading to cytoplasmic translocation of MLKL and release of histone H3. Extracellular histone H3 induces neuronal hyperactivity, neutrophil extracellular trap (NET), and hyperalgesia, possibly through P2X7 receptor and Toll-like receptor 4. Nociceptive-specific depletion of Mlkl significantly decreased pain threshold and exacerbated NET formation independent of cell death. Neutralizing extracellular histone, clearing extracellular DNA or restoring nuclear localization of MLKL can reduce both NET formation and hyperalgesia in Mlklmice. These data demonstrated that the nociceptive neuron-neutrophil interaction mediated by this MLKL-histone-NET cascade may serve as a potential therapeutic target for chronic inflammatory pain.
A central goal in neuroscience is to develop cross-species frameworks that reveal conserved principles of movement control. Recent work has suggested a potential species difference in the role of primary motor cortex (M1...A central goal in neuroscience is to develop cross-species frameworks that reveal conserved principles of movement control. Recent work has suggested a potential species difference in the role of primary motor cortex (M1) during skilled forelimb tasks: in rodents, extensive practice appears to shift control to subcortical circuits, leading to "disengagement" of M1 from task execution. Importantly, there is no evidence of such disengagement in macaques. We hypothesized that these differences instead reflect task demands, particularly the need for fine (i.e., grasping) versus gross motor control. Strikingly, M1 lesions in macaques produced recovery patterns similar to rodents: gross motor control, including an exclusively gross motor task, recovered rapidly, whereas fine motor control exhibited persistent deficits. Detailed kinematic analyses in both macaques and mice performing a single-pellet reach-to-grasp task (RGT) revealed analogous disruptions in the temporal structure of sub-movements. These findings underscore the importance of task design and the translational value of rodent models for understanding motor control and recovery.
In temperate and boreal perennial trees, precise timing of growth cessation under short photoperiods is essential for survival and geographic adaptation. In Populus, the circadian evening complex (EC) component EARLY FLO...In temperate and boreal perennial trees, precise timing of growth cessation under short photoperiods is essential for survival and geographic adaptation. In Populus, the circadian evening complex (EC) component EARLY FLOWERING 3 (ELF3) integrates temperature and photoperiod signals to regulate growth cessation, yet the underlying mechanism remains incompletely understood. Here, we demonstrate that the tree LUX ARRHYTHMO (LUX) homologs LUX1 and LUX2 interact with ELF3.1 to form functional EC. Mutant analyses show that elf3.1 single and lux1/2 double mutants fail to cease growth under short days, correlating with sustained FT2 expression. Mechanistically, the EC directly binds the promoters of FT2, GI/GIL, and FKF1a/b and represses their expression. Conversely, the GI-FKF1 complex physically interacts with EC components to counteract their repression on FT2. These findings reveal a reciprocal transcriptional-translational feedback circuit between the EC and GI-FKF1 modules that fine-tunes FT2 expression, ensuring precise photoperiodic control of seasonal growth in trees.
The spindle assembly checkpoint (SAC) delays anaphase onset until all kinetochores are stably attached to microtubules, thus promoting error-free chromosome segregation. Multiple molecular events are implicated in SAC si...The spindle assembly checkpoint (SAC) delays anaphase onset until all kinetochores are stably attached to microtubules, thus promoting error-free chromosome segregation. Multiple molecular events are implicated in SAC silencing, including removal of phospho-marks, protein (un)binding, and structural reorganization of the kinetochore-but we currently lack a quantitative map of how these events unfold through time. Here, we use the levels of the checkpoint protein MAD2 to create a pseudo-timeline of SAC silencing at single kinetochores. We demonstrate how silencing proceeds through an ordered series of molecular events where MAD2-Spindly unbinds first and then the KNL1 catalytic platform disassembles, with a pool of MPS1 retained. Coincidently, the NDC80 ensemble reconfigures, an event dependent on microtubule binding. Kinetochores next transition into a mature attachment state, marked by a switch-like recruitment of Astrin-SKAP. This then undergoes gradual further stabilization through NDC80 tail dephosphorylation. By preventing biorientation, we also define otherwise hidden kinetochore states involved in error correction cycles. These results provide a critical temporal framework for understanding the mechanisms of SAC silencing and error correction at single human kinetochores.
Mengozzi A, Costantino S, Mongelli A
… +20 more, Duranti E, Mohammed SA, Gorica E, Cappelli F, Telesca M, Armenia S, Hornemann T, Hulsmeier A, de Ciuceis C, Agabiti-Rosei C, Ippolito C, Bellini R, Pugliese NR, Matter CM, Dzemali O, Masi S, Taddei S, Ruschitzka F, Virdis A, Paneni F
Epigenetic reading of histone marks by BET proteins is emerging as a pivotal mechanism governing gene transcription and may be implicated in cardiometabolic disease (CMD). Using vessels from mice and patients with CMD, w...Epigenetic reading of histone marks by BET proteins is emerging as a pivotal mechanism governing gene transcription and may be implicated in cardiometabolic disease (CMD). Using vessels from mice and patients with CMD, we show that pharmacological inhibition of BET proteins by RVX-208 prevents the maladaptive crosstalk between perivascular adipose tissue (PVAT) and blood vessels. The effect of the BET inhibitor on endothelial function was more pronounced in the presence of PVAT. In mouse and human PVAT, RVX-208 rescued maladaptive transcriptional programs with a marked downregulation of hexokinase-2 (HK2). This was associated with reduced glycolysis, lipid accumulation, and secretion of pro-inflammatory cytokines. In human adipocytes, HK2 overexpression induced a pro-inflammatory phenotype fostering endothelial damage. Pharmacological inhibition of HK2 in vessels from cardiometabolic patients restored endothelial-dependent vasorelaxation. This unveiling of a BET/HK2-dependent crosstalk between PVAT and blood vessels highlights the potential of therapeutic strategies to prevent vascular damage in cardiometabolic patients.
The enhancer lysine acetyltransferases CBP/p300 are compelling targets for multiple myeloma therapy. Chemical inhibition of these multidomain factors, either through the bromodomain or the catalytic acetyltransferase dom...The enhancer lysine acetyltransferases CBP/p300 are compelling targets for multiple myeloma therapy. Chemical inhibition of these multidomain factors, either through the bromodomain or the catalytic acetyltransferase domain, show promising activity in pre-clinical models. Chemical degradation is the only modality that can completely disrupt all functional domains. Our previous attempts to induce CBP/p300 targeted degradation led to a potent tool compound, dCBP-1. Here we comprehensively demonstrate across a large panel of cell lines how CBP/p300 degradation compares to inhibition, with pronounced selective antiproliferative activity toward multiple myeloma. We use chemical linker optimization strategies to create a compound with better pharmacokinetic properties. Through these we define an advanced analog of dCBP-1, dCBP-30, that has improved potency and improved in vivo properties including oral bioavailability. dCBP-30 led to potent and sustained loss of CBP and p300, potent inhibition of several myeloma-specific dependency programs, and elicits tumor reduction in xenograft models.
Antibiotics (Abx) are essential in medicine but can disrupt gut microbiota, potentially contributing to inflammatory bowel diseases (IBDs). This study employed fecal metagenomics and metaproteomics to evaluate the effect...Antibiotics (Abx) are essential in medicine but can disrupt gut microbiota, potentially contributing to inflammatory bowel diseases (IBDs). This study employed fecal metagenomics and metaproteomics to evaluate the effects of Abx in patients with pouchitis, ulcerative colitis (UC), and non-IBD controls. Each group displayed distinct microbiome profiles, with metaproteomes more affected by Abx than metagenomes. Proteomic analysis revealed increased pancreatic protease activity and fecal proteolytic activity in all groups, except in patients without IBD before Abx, consistent with impaired epithelial barrier integrity. Abx also decreased bacterial protease inhibitors, which may control proteolysis and help maintain gut balance. These findings emphasize the importance of understanding Abx-induced proteolytic shifts in IBD and highlight metaproteomics as a valuable tool for studying host-microbiome interactions. Future research should explore the molecular mechanisms that regulate bacterial protease inhibitor levels and their effects on intestinal health.
P97/VCP is a protein unfoldase of the AAA+ ATPase family that plays essential roles in numerous processes, including ER-associated degradation and DNA replication. For unfolding of proteins modified with K48-linked ubiqu...P97/VCP is a protein unfoldase of the AAA+ ATPase family that plays essential roles in numerous processes, including ER-associated degradation and DNA replication. For unfolding of proteins modified with K48-linked ubiquitin chains, p97 works with the heterodimeric cofactor Ufd1-Npl4, and the cofactor Faf1 was shown to enhance this activity during replisome disassembly by unknown mechanisms. Here, we employ an in vitro reconstituted system with human components for biochemical experiments, FRET-based assays, and cryo-EM structure determination to reveal that Faf1 generally accelerates ubiquitin-dependent substrate processing by promoting the unfolding of an initiator ubiquitin and its engagement by the ATPase. Faf1 thereby uses its p97-bound C-terminal UBX domain to anchor a long helix that braces Ufd1's UT3 domain and stabilizes Ufd1-Npl4 for ubiquitin unfolding. Our findings demonstrate how p97 works simultaneously with several cofactors to facilitate the unfolding of ubiquitinated proteins, indicating more complex regulatory mechanisms than for the simpler yeast Cdc48.
Kinase-mediated signal transduction regulates most cellular processes, and concentration-dependent autophosphorylation is a common mechanism to promote kinase signaling. Many kinases undergo phase separation to form cond...Kinase-mediated signal transduction regulates most cellular processes, and concentration-dependent autophosphorylation is a common mechanism to promote kinase signaling. Many kinases undergo phase separation to form condensates. Despite the central role of autophosphorylation in regulating kinase activity, how condensates impact kinase autophosphorylation has not been systematically studied. Using biochemical reconstitution and cellular studies, we find that phase separation can concentrate kinases to effectively trigger the trans-autophosphorylation of the tyrosine kinases FAK and Abl, as well as the serine/threonine kinase Mst2. Moreover, kinase condensates can create a chemical environment that enriches ATP, and positively charged intrinsically disordered regions are one feature that enrich ATP into condensates. Thus, kinase phase separation is a general mechanism to activate kinase signaling pathways by locally concentrating both kinases and ATP to trigger autophosphorylation.
Periodontitis is one of the most common human inflammatory diseases, yet the immune mechanism linking oral and systemic immune responses is not well defined. Here, we show that the accumulation of interleukin-17 (IL-17)-...Periodontitis is one of the most common human inflammatory diseases, yet the immune mechanism linking oral and systemic immune responses is not well defined. Here, we show that the accumulation of interleukin-17 (IL-17)-producing γδT (γδT17) cells occurs not only in the gingiva and cervical lymph nodes (CLNs) but also in the peripheral lymph nodes and spleen of ligation-induced periodontitis (LIP) mice. Strikingly, oral γδT17 cells derived from LIP migrate to the inflamed joint and exacerbate rheumatoid arthritis (RA) in a collagen-induced arthritis mice model. Mechanistically, we demonstrate that dysbiosis in LIP increases the production of complement component 3 (C3)-enriched extracellular vesicles (EVs) derived from macrophages. These C3-enriched EVs are taken up by γδT cells and stimulate the intracellular C3a receptor to drive IL-17A production in γδT cells. Our findings reveal a previously unrecognized immunological mechanism linking periodontitis to RA through the accumulation and migration of oral γδT17 cells.
ZYG11B is a substrate specificity factor for the cullin-2-RING ubiquitin ligase (CRL2), which plays a critical role in the recognition and degradation of Gly/N-degrons. Yet, how ZYG11B assembles with CRL2, and how ZYG11B...ZYG11B is a substrate specificity factor for the cullin-2-RING ubiquitin ligase (CRL2), which plays a critical role in the recognition and degradation of Gly/N-degrons. Yet, how ZYG11B assembles with CRL2, and how ZYG11B couples specific substrate recognition to CRL2-mediated ubiquitination, is unknown. We present the cryo-electron microscopy (cryo-EM) structures of the CRL2 holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1. The structures indicate that ZYG11B folds into a leucine-rich repeat followed by two armadillo repeat domains that promote assembly with CRL2 and specific recognition of the NLRP1 Gly/N-degron that is revealed by viral protease cleavage. Our structural and functional data indicate that blocking ZYG11B recognition of the NLRP1 Gly/N-degron inhibits NLRP1 inflammasome activation by a viral protease. Overall, we show how the CRL2 E3 ligase complex recognizes Gly/N-degron substrates, including those that are involved in viral protease-mediated activation of the NLRP1 inflammasome.