Photorespiration is vital for C plant carbon (C) and nitrogen (N) metabolism, yet most engineering ignores N-related constraints. Here, we engineered a chloroplast-targeted bypass in tomato (GCMG), comprising glycolate o...Photorespiration is vital for C plant carbon (C) and nitrogen (N) metabolism, yet most engineering ignores N-related constraints. Here, we engineered a chloroplast-targeted bypass in tomato (GCMG), comprising glycolate oxidase 1 (SlGLO1), catalase 2 (SlCAT2), malate synthase A (SlMSA), and glutamine synthetase 2 (SlGS2). This bypass integrates carbon concentration with enhanced ammonium reassimilation via the GS2/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. GCMG plants showed synergistic improvements in photosynthesis, biomass, and fruit quality; while total yield significantly increased over wild-type (WT), GCMG maintained a favorable trend beyond C-focused GCM lines. Mechanistically, GCMG partitions glycolate flux into parallel routes, maintaining N assimilation while enhancing chloroplastic CO enrichment. N-labeling confirmed this metabolic synergy, revealing a 146% higher N turnover rate (f). GCMG also sustained robust N assimilation under elevated CO and conferred resilience to N-deficiency, high oxygen, and heat stress. Coordinating C/N metabolism boosts productivity and resilience, offering a blueprint for crop improvement.
Tonic pain engages both sensory and affective-motivational processes, which are partially mediated by the medial thalamus. However, the circuit mechanisms by which distinct medial thalamic subnuclei contribute to pain-re...Tonic pain engages both sensory and affective-motivational processes, which are partially mediated by the medial thalamus. However, the circuit mechanisms by which distinct medial thalamic subnuclei contribute to pain-related behaviors remain poorly understood. Taking advantage of intersectional viral genetics, in vivo imaging, and ex vivo physiology, we find that the neurons in the parafascicular (PF) nuclei of the medial thalamus receive monosynaptic inputs from the lateral parabrachial nuclei (LPBN) in mice. The PF neurons downstream LPBN (PF) are nociceptive, sensitized by peripheral neuropathy, and acutely aversive; when activated, they drive both sensory and affective-motivational responses to cold allodynia. In contrast, the LPBN target neurons in the intralaminar centromedian thalamus (CM), another nociceptive nucleus of the medial thalamus, are primarily involved in the affective-motivational aspects of pain. Together, these findings show that LPBN projections to two closely related medial thalamic nuclei differentially shape behavioral responses to cold hypersensitivity associated with peripheral neuropathy.
Lymphovascular invasion (LVI) is a critical early step in metastasis, yet the mechanisms remain elusive. We identify an intragenic antagonistic loop between TNRC6B and circTNRC6B that determines LVI and metastasis in eso...Lymphovascular invasion (LVI) is a critical early step in metastasis, yet the mechanisms remain elusive. We identify an intragenic antagonistic loop between TNRC6B and circTNRC6B that determines LVI and metastasis in esophageal squamous cell carcinoma. This antagonism is conserved across upper gastrointestinal tumors, where TNRC6B expression correlates positively with LVI and poor prognosis, while circTNRC6B shows the opposite associations. TNRC6B protein degrades circTNRC6B via its middle domain, which otherwise functions as a potent tumor suppressor. Mechanistically, circTNRC6B recruits HNRNPA2B1 in an m6A-dependent manner to orchestrate the nuclear-to-cytoplasmic translocation of KLRC3 mRNA. Concurrently, circTNRC6B acts as a molecular decoy, competing with IGF2BP2 for USP4 binding to impair IGF2BP2 deubiquitination, further destabilizing KLRC3 mRNA. Notably, HNRNPA2B1 mediates the exosomal packaging of m6A-modified circTNRC6B to counteract TNRC6B driven pro-tumorigenic effects. This study highlights the TNRC6B/circTNRC6B axis as a key determinant for LVI, providing potential diagnostic biomarkers and therapeutic targets for upper gastrointestinal tumors.
Salmonella enterica serovar Typhimurium (STM) employs various strategies to endure antibiotic stress, but the contribution of central metabolites to resistance remains insufficiently characterized. In this work, we ident...Salmonella enterica serovar Typhimurium (STM) employs various strategies to endure antibiotic stress, but the contribution of central metabolites to resistance remains insufficiently characterized. In this work, we identify intracellular formate, produced by pyruvate-formate lyase (PflB), as a key determinant of STM susceptibility to meropenem and ciprofloxacin. Deletion of pflB disrupts cellular pH homeostasis and is associated with impaired efflux pump function, increased reactive oxygen species, and membrane depolarization, collectively correlating with heightened antibiotic sensitivity. Supplementing extracellular formate reverses these phenotypes. Mechanistically, pH imbalance activates the RpoE-csrB regulatory pathway, leading to reduced expression of efflux pump genes acrB and tolC, while extracellular formate engages the BarA/SirA two-component system to further influence the CsrA-csrB network. Overall, our results position formate as a metabolic driver of antibiotic outcomes in STM and highlight it as a potential target for mitigating resistance.
Frizzled4 (FZD4) is a receptor for Norrin and WNT7A/B ligands, is expressed in endothelial cells (ECs), is required for endothelial blood-central nervous system (CNS) barrier function as well as CNS angiogenesis, and tra...Frizzled4 (FZD4) is a receptor for Norrin and WNT7A/B ligands, is expressed in endothelial cells (ECs), is required for endothelial blood-central nervous system (CNS) barrier function as well as CNS angiogenesis, and transduces β-catenin-dependent signaling. Despite its fundamental importance in neurovascular biology, including as a drug target, the molecular mechanisms governing FZD4 regulation remain poorly understood. Here, we employed proximity biotinylation to identify proteins that regulate FZD4. We identified ATPase plasma membrane Ca transporting 1 (ATP2B1, also known as PMCA1) as a FZD4 proximity interactor. Functional analyses revealed that ATP2B1 depletion increased EC Ca, activated NFAT, and significantly attenuated Norrin/Frizzled4 signaling. Endothelial-specific Atp2b1 deletion caused CNS vascular phenotypes consistent with compromised Norrin/Frizzled4 signaling. This study identifies ATP2B1 as a regulator of Norrin- and WNT7A/B-induced FZD4 signaling and suggests that in pathological contexts with elevated EC Ca-levels, EC function may be modulated by suppression of β-catenin-dependent signaling.
Developmental gene expression is tightly regulated by tissue-specific enhancers. Understanding this process in the context of the three-dimensional (3D) genome requires dedicated interrogation on a per gene and per tissu...Developmental gene expression is tightly regulated by tissue-specific enhancers. Understanding this process in the context of the three-dimensional (3D) genome requires dedicated interrogation on a per gene and per tissue basis. Here, we functionally dissect the mechanisms that regulate Wnt4/WNT4 gene expression. We focus our efforts on the mouse mammary gland and human breast, where Wnt4/WNT4 expression is restricted to hormone-responsive luminal epithelial cells and generally assumed to lie downstream of progesterone. Using freshly isolated cells and cell lines, we map the Wnt4/WNT4 enhancer network, reveal Grainyhead-like (GRHL) proteins as novel co-regulators of Wnt4/WNT4 expression, and identify a conserved distal enhancer that physically loops to Wnt4/WNT4. We integrate our findings to propose a working model in which a conserved distal chromatin hub, bound by CTCF and GRHL, recruits other species-specific enhancers, to facilitate PGR-dependent and -independent Wnt4/WNT4 gene regulation via 3D chromatin looping.
Natural killer (NK) cells are known for their cytotoxic and regulatory functions in immune responses. Here, we identify a subset of insulin-like growth factor binding protein 2 (IGFBP2) NK cells enriched in liver cancer,...Natural killer (NK) cells are known for their cytotoxic and regulatory functions in immune responses. Here, we identify a subset of insulin-like growth factor binding protein 2 (IGFBP2) NK cells enriched in liver cancer, exhibiting impaired cytotoxicity and correlating with poor prognosis in patients with liver cancer. The hypoxic tumor microenvironment induces IGFBP2 expression in NK cells. Mechanistically, hypoxia drives lactate accumulation in intratumoral NK cells, promoting histone H3 lysine 18 lactylation (H3K18La) at the IGFBP2 promoter and enhancing IGFBP2 transcription. NK cell-secreted IGFBP2 inhibits the expression of stress-related activating immune ligands (MICA/B and ecto-CRT) on neighboring tumor cells, thereby reducing their sensitivity to cytotoxic immune cells and promoting immune evasion. Antibody-mediated IGFBP2 blockade increases tumor cell sensitivity to cytotoxic immune cells. Additionally, IGFBP2 blockade synergizes with immune checkpoint therapy to enhance anti-tumor efficacy in liver cancer models.
Shin S, Hernandez M, Dauvillier S
… +17 more, Ducoux-Petit M, Mouton-Barbosa E, Estève D, Moutahir M, Rengel D, Delos O, Roumiguié M, Dray C, Valet P, de Medina P, Poirot M, Silvente-Poirot S, Bertrand-Michel J, Burlet-Schiltz O, Reina N, Muller C, Attané C
Bone marrow adipocytes (BMAds) are a major component of the bone marrow (BM) that regulate bone turnover and hematopoiesis. In rodents, two distinct adipocyte populations exist: constitutive BMAds (cBMAds), located in ar...Bone marrow adipocytes (BMAds) are a major component of the bone marrow (BM) that regulate bone turnover and hematopoiesis. In rodents, two distinct adipocyte populations exist: constitutive BMAds (cBMAds), located in areas devoid of hematopoietic cells and resistant to metabolic cues, and regulatory BMAds (rBMAds), interspersed within hematopoietic niches and responsive to energy stress. Despite their potential importance, rBMAds have remained poorly characterized due to their scarcity in rodents. Here, we used a high-yield method to isolate human rBMAds, enabling structural, proteomic, lipidomic, and functional analyses. Remarkably, human rBMAds are anucleate yet retain organelles and maintain lipid and glucose metabolism, but unlike their rodent counterparts, they lack lipolytic activity. They actively secrete factors that support hematopoiesis in vitro, implicating them as functional contributors to the BM niche. Collectively, these findings redefine key aspects of BMAds diversity in humans.
Genomic instability drivers of senescence and carcinogenesis. The ribosomal RNA gene (rDNA) locus in budding yeast provides an excellent model to study these processes. Because of its highly repetitive structure and the...Genomic instability drivers of senescence and carcinogenesis. The ribosomal RNA gene (rDNA) locus in budding yeast provides an excellent model to study these processes. Because of its highly repetitive structure and the gene amplification system that maintains its copy number, rDNA represents one of the most unstable regions in the genome. Here, we demonstrate that the integrity of rDNA transcription is essential for maintaining genomic stability and lifespan. Loss of Paf1, an elongation factor associated with RNA polymerase, reduces rDNA transcription, stability, and lifespan. In paf1 mutants, R-loops accumulate within the rDNA, generating single-stranded regions prone to breakage. This triggers double-strand breaks at replication forks, leading to rDNA copy number variation and DNA fragmentation. Torsional stress generated by R-loops also results in the accumulation of Top1. These abnormalities are partially dependent on canonical amplification recombination pathways, including those regulated by replication fork blocking and non-coding transcription.
Hippocampal theta oscillations coordinate computations underlying learning and memory. The frequency of theta varies with factors such as locomotion and anxiety, but the effect of an animal's sleep history on theta frequ...Hippocampal theta oscillations coordinate computations underlying learning and memory. The frequency of theta varies with factors such as locomotion and anxiety, but the effect of an animal's sleep history on theta frequency remains unknown. Using long-duration CA1 recordings in rats, we found that awake theta frequency progressively decreases following sleep but remains elevated during sleep deprivation. These changes were not accounted for by movement but were predicted by the proportion of sleep in the preceding 30 min. In contrast, theta frequency remained relatively stable during rapid eye movement sleep. In aged rats, sleep-dependent frequency changes were less pronounced, likely due to sleep fragmentation. Systemic administration of the phosphodiesterase inhibitor rolipram during sleep deprivation reduced theta frequency, mimicking natural sleep. Collectively, these findings reveal a robust influence of sleep history on hippocampal theta frequency, with potential implications for understanding memory function, age-related sleep disruptions, and the potential for pharmacological interventions.
Tanu T, Cox AM, Karlow JA
… +18 more, Sharma P, Sun K, He X, Babu S, Roelofs MK, Jeon E, Chen J, Wu C, Brown J, Barrientos NB, McCallion AS, Brown KM, Chanock SJ, Liu D, Zhang T, Burns KH, Boutz PL, Insco ML
Endogenous retroviral (ERV) RNA is highly expressed in cancer, suggesting a selective advantage. We identify recurrent truncating mutations in ZC3H18 (Z18), a nuclear RNA surveillance factor lost in 30% of all cancers. W...Endogenous retroviral (ERV) RNA is highly expressed in cancer, suggesting a selective advantage. We identify recurrent truncating mutations in ZC3H18 (Z18), a nuclear RNA surveillance factor lost in 30% of all cancers. We show that Z18 truncating mutations are oncogenic and that Z18 plays an evolutionarily conserved role in the nuclear surveillance of oncogenic ERV RNA. In zebrafish, Z18 truncation accelerates melanoma onset and selectively increases ERV RNA. Human cancer cell lines and patient tumors with Z18 mutations also upregulate ERV RNA. In engineered human melanoma cells, Z18 truncation enhances ERV RNA accumulation more than heterozygous Z18 loss, consistent with dominant-negative activity. Truncated Z18 directly stabilizes and relocalizes ERV RNA to the cytoplasm. ERV RNA expression accelerates melanoma and is required for Z18 truncation-mediated melanoma onset in zebrafish. Growth of human melanoma cells similarly depends on Z18-regulated ERV RNA. Together, these findings support Z18-regulated ERV RNA as a driver of oncogenesis.
Whether and how pyrimidine metabolites promote systemic autoimmunity is unknown. Here, metabolomics and N-amide glutamine tracing show enhanced flux through de novo pyrimidine synthesis in systemic lupus erythematosus (S...Whether and how pyrimidine metabolites promote systemic autoimmunity is unknown. Here, metabolomics and N-amide glutamine tracing show enhanced flux through de novo pyrimidine synthesis in systemic lupus erythematosus (SLE)-prone B cells. Temporal inhibition of pyrimidine synthesis dampens SLE-prone but not foreign antigen-specific germinal center (GC), plasma cell (PC), and antibody responses. Uridine monophosphate synthase (UMPS) conditional deletion, however, reveals a B cell-intrinsic requirement of de novo pyrimidine synthesis in foreign antigen-driven and SLE-prone GC, PC, and antibody responses and kidney immune complex deposition. Metabolomics, mitochondrial stress test, metabolic flow cytometry, glycolytic rate assay, and RNA sequencing highlight the importance of pyrimidine synthesis in promoting aerobic glycolysis and oxidative phosphorylation in SLE-prone B cells. De novo pyrimidine synthesis helps SLE-prone B cells maintain heightened metabolic state and expression of metabolic regulator, cMYC. Mechanistically, mTORC1 and S6K1 downstream of TLR7 and CD40 signaling in B cells promote pyrimidine synthesis by activating CAD, a rate-limiting enzyme of this pathway.
Neutrophils are central mediators of innate defense in bone marrow, where infection rapidly reshapes local hematopoietic and immune niches. Here, we identify a subset of Mrgpra2 neutrophils that supports antimicrobial im...Neutrophils are central mediators of innate defense in bone marrow, where infection rapidly reshapes local hematopoietic and immune niches. Here, we identify a subset of Mrgpra2 neutrophils that supports antimicrobial immunity through neutrophil extracellular trap (NET) formation during bone marrow infection. Using a murine Staphylococcus aureus marrow infection model, we show that Mrgpra2 is enriched in neutrophil precursors and supports their survival and effector activation under infectious stress. Single-cell and bulk transcriptomics show that Mrgpra2 neutrophils exhibit a transcriptional program enriched for NET formation and inflammatory signaling. Mechanistically, Mrgpra2 and TNFR signals converge on a PLC-Ca-PKC-NADPH oxidase axis to drive reactive oxygen species (ROS)-dependent NET release while preserving neutrophil viability. In vivo, Mrgpra2 deficiency impairs bacterial clearance, exacerbates tissue injury, and reduces the therapeutic benefit of β-defensin. These findings define a marrow neutrophil pathway that couples infection-derived signals with controlled NET deployment to preserve bone marrow immune homeostasis.
Zhang and colleagues identify a Mrgpra2⁺ neutrophil subset that deploys neutrophil extracellular traps (NETs) in infected bone marrow. NET release requires coincident defensin and TNF-α signaling, a combination that defi...Zhang and colleagues identify a Mrgpra2⁺ neutrophil subset that deploys neutrophil extracellular traps (NETs) in infected bone marrow. NET release requires coincident defensin and TNF-α signaling, a combination that defines a dual-signal mechanism for context-specific antimicrobial defense during osteomyelitis.
Cell cycle-dependent maintenance of centromere protein A (CENP-A) levels and its spatiotemporal assembly are essential for centromere propagation. CENP-A synthesis peaks in late G2, while its assembly occurs during late...Cell cycle-dependent maintenance of centromere protein A (CENP-A) levels and its spatiotemporal assembly are essential for centromere propagation. CENP-A synthesis peaks in late G2, while its assembly occurs during late telophase/early G1. We have previously shown that phosphorylation of CENP-A at Ser68 by CDK1-cyclin B during mitosis impairs its binding to holiday junction recognition protein (HJURP) and facilitates DCAF11-dependent polyubiquitination and degradation. However, the mechanisms governing CENP-A stability remain elusive. Here, we demonstrate that spindlin interactor and repressor of chromatin binding (SPINDOC), as an M-phase-specific maintenance factor for CENP-A assembly, binds and stabilizes CENP-A by antagonizing DCAF11-dependent polyubiquitination. In addition, SPINDOC bridges CENP-A to HJURP, ensuring that CENP-A is poised for subsequent deposition at centromeres. Interestingly, SPINDOC promotes liver cancer development in vitro and in vivo, and alterations in its levels disrupt CENP-A homeostasis, resulting in chromosomal instability. Together, this study identifies SPINDOC as a mitotic molecular counter of newly synthesized CENP-A, coordinating its spatiotemporal assembly by presenting it to HJURP.
Abnormal myocardial fuel utilization contributes to heart failure (HF). Myocardial glucose uptake in response to insulin is suppressed in patients with HF, but the mechanisms of this metabolic inflexibility are not fully...Abnormal myocardial fuel utilization contributes to heart failure (HF). Myocardial glucose uptake in response to insulin is suppressed in patients with HF, but the mechanisms of this metabolic inflexibility are not fully understood. The present studies employ culture surfaces with tunable stiffness, quantitatively mimicking the healthy and diseased heart milieu. We observe that human and rat adult cardiomyocytes cultured on stiff surfaces develop blunted insulin-mediated glucose uptake, associated with intracellular aggregation of the high-affinity glucose transporter GLUT4 within the microtubule network, and with impaired contractility. These effects are prevented by blocking stiffness-induced detyrosination of α-tubulin, and can be partially rescued by metformin, through AMPK activation. Similarly, disabling motor proteins that mediate microtubule-based trafficking of GLUT4 independently alter insulin-mediated glucose uptake and contractility in myocytes. These findings demonstrate a cell-autonomous mechanism of stiffness-induced impairment of GLUT4 trafficking and glucose uptake in adult rat and human cardiomyocytes.
Malaria continues to exact a high disease burden worldwide, and improved vaccines and therapeutics are desperately needed. Both licensed vaccines target only the circumsporozoite protein (CSP) expressed during early stag...Malaria continues to exact a high disease burden worldwide, and improved vaccines and therapeutics are desperately needed. Both licensed vaccines target only the circumsporozoite protein (CSP) expressed during early stages of Plasmodium falciparum infection. Vaccines targeting the subsequent blood stage, in particular those based on RH5, are also showing clinical promise. Concrete data demonstrating the utility of a combination of antigens are lacking. Using a humanized liver mouse model, we show that anti-CSP monoclonal antibodies (mAbs) lose protection below ∼30 μg/mL serum concentration and plateau above ∼60 μg/mL. Yet breakthrough infections have multiple logs fewer parasites emerging from the liver. The addition of an anti-RH5 blood stage mAb to a partially protective anti-CSP mAb yields additional benefits by controlling the ensuing blood stage parasitemia to a transient and extremely low-level infection. These results provide the proof-of-concept evidence that multi-stage antibodies may be more protective compared to a single stage approach.
Amyloid-β 1-42 (Aβ42) aggregation is among the earliest pathological signs in Alzheimer's disease (AD). Here, we characterized Aβ42 species at several aggregation stages at the single-molecule level and examined their to...Amyloid-β 1-42 (Aβ42) aggregation is among the earliest pathological signs in Alzheimer's disease (AD). Here, we characterized Aβ42 species at several aggregation stages at the single-molecule level and examined their toxicity in murine organotypic brain slices, where we observed a stage-dependent recapitulation of multiple aspects of the cellular phase of AD. Aggregates formed during the lag phase of the Aβ42 aggregation elevated neuronal baseline Ca levels and impaired long-term potentiation (LTP), while promoting microglial homeostatic exit and transition to disease-associated microglia (DAM) state. In contrast, aggregates enriched during the growth phase downregulated homeostatic microglial markers and induced TLR4-mediated microglial activation, cytokine production, and complement activation, leading to synaptic engulfment and severe disruption of neuronal activity. Together, these findings reveal that structurally distinct Aβ42 aggregate species engage different cellular and molecular pathways. This framework advances mechanistic understanding of amyloid toxicity in neurodegeneration and could inform the design of combination therapeutic strategies.
Kang M, Yoon SH, Kang M
… +15 more, Park SP, Song WS, Kim J, Lee S, Park DH, Song JM, Kim B, Park KH, Joe EH, Woo HG, Kaang BK, Han D, Lee YS, Kim MH, Suh YH
Mitochondrial antiviral signaling protein (MAVS) forms prion-like aggregates to activate innate immunity against RNA viruses, but the metabolic regulation of MAVS remains poorly understood. Here, we show that viral infec...Mitochondrial antiviral signaling protein (MAVS) forms prion-like aggregates to activate innate immunity against RNA viruses, but the metabolic regulation of MAVS remains poorly understood. Here, we show that viral infection induces the formation of lipid droplets (LDs), which physically interact with mitochondria to promote the assembly of MAVS prion-like aggregates. Mechanistically, the LD-resident protein PLIN3 binds to the mitochondrial fusion protein MFN2, thereby relieving MFN2-mediated inhibition of MAVS and enabling its oligomerization. Furthermore, LD-mitochondria contact sites facilitate fatty acid transfer, sustaining mitochondrial membrane potential required for MAVS signaling. Oleic acid (OA)-enriched diets enhance LD formation and boost antiviral immunity in vivo, while myeloid-specific Seipin (an LD biogenesis regulator) deficiency attenuates MAVS activation and exacerbates viral susceptibility. These findings establish LDs as metabolic platforms that bridge cellular lipid metabolism with innate antiviral defense through organelle crosstalk, suggesting LD induction as a novel therapeutic strategy against viruses.