A stress-induced neuroimmune axis promotes the recruitment of tumor-permissive macrophages to glioma.
The Gram-positive opportunistic pathogen Clostridioides difficile colonizes the gut by adapting to an array of threats from the host and the microbiome. A potential threat from the microbiome is the antimicrobial peptide bacitracin, which is produced by the commensal Bacillus licheniformis and prevents dephosphorylation and recycling of undecaprenyl phosphate, a lipid carrier essential in cell wall biogenesis. Gram-positive bacteria typically use multiprotein membrane complexes called Bce modules to sense and respond to bacitracin. We identified a Bce module in C. difficile that incorporates the undecaprenyl pyrophosphatase BacA2 into the bacterium’s response to bacitracin. This Bce module was essential for C. difficile survival in the presence of B. licheniformis or bacitracin in vitro and in mice. Expression of the genes encoding the Bce module was inhibited by Fur, a transcriptional repressor that controls the expression of genes critical for responding to host-mediated restriction of iron availability in the gut. Low iron reduced undecaprenyl phosphate biosynthesis and sensitized C. difficile to bacitracin. Our findings support a model in which C. difficile is affected independently and collectively by low iron and bacitracin, leading to the inhibition of undecaprenyl phosphate recycling. In response, C. difficile increases the transcription of undecaprenyl recycling and bacitracin efflux genes that are controlled both by Fur and a Bce module. This distinct Bce module forms a system that responds to nutritional immunity and antimicrobial molecules produced by microbiome members to maintain envelope integrity and support host colonization.
Wild-type RAS family members determine the signaling and therapeutic response in cancers driven by mutant HRAS and KRAS because they activate alternate RAS effector pathways. Here, we found that the requirement for wild-type RAS to support mutant NRAS-driven transformation correlated with codon-specific differences in GTP hydrolysis. NRAS with mutations at either Gly12 (G12X) or Gly13 (G13X), which retained the GDP-GTP cycling function, had modest autonomous transforming potential. In contrast, NRAS with GTP-locking mutations at Gln61 (Q61X mutants) was uncoupled from receptor tyrosine kinase (RTK) input, rendering wild-type RAS an obligate partner for RTK-stimulated signaling and oncogenesis. In RASless cells expressing mutant NRAS, reintroduction of wild-type HRAS was sufficient to restore signaling and transformation. Global dependency mapping in human cancer cells revealed functional partitioning, wherein mutant NRAS promoted MAPK signaling and wild-type HRAS promoted PI3K-AKT survival signaling. Consequently, allele-specific or pan-RAS(ON) inhibitors synergized with inhibitors of proximal RTK signaling or of wild-type HRAS or KRAS to overcome this signaling plasticity. Pan-RAS(ON) and HRAS inhibition was synergistic for all NRAS mutants tested, with Q61X mutants showing greater sensitivity. These findings define the signaling partnership between mutant NRAS and wild-type HRAS as a targetable vulnerability and provide a biochemical blueprint for dual RAS inhibition in NRAS-mutated malignancies.
Adhesion G protein-coupled receptors (aGPCRs) are a unique GPCR family defined by large, modular extracellular regions (ECRs) and a conserved seven-transmembrane (7TM) domain. These domains enable aGPCRs to mediate cell-cell and cell-matrix communication, integrate mechanical and chemical signals, and regulate diverse biological processes including neurodevelopment, angiogenesis, and immune response. Traditionally, models of aGPCR activation have focused on the canonical tethered agonist (TA) mechanism, in which autoproteolytic cleavage reveals an intrinsic peptide (TA) that activates the 7TM domain. However, mounting structural, functional, and biophysical evidence indicates that this TA-dependent mechanism is insufficient to explain all aGPCR functions, especially in cleavage-deficient receptors or under physiological forces. A distinct ECR-dependent mechanism has emerged in which allosteric regulation or structural changes in the ECR directly modulate receptor signaling independent of TA exposure. This ECR-dependent model offers reversible, tunable signaling that can accommodate both pulling and compressive forces, expanding traditional views of aGPCR activation. Here, we critically examine current evidence for TA- and ECR-dependent mechanisms, discuss their structural underpinnings, and propose a unified, biological context-dependent framework integrating all known activation modes. This Review advances understanding of aGPCR signaling and provides a basis for therapeutic approaches targeting aGPCRs.
Neurovascular coupling ensures that active neuronal circuits receive the blood supply needed to sustain brain function. In this issue of Science Signaling, Lavanderos et al. identify STIM1-ORAI-mediated Ca2+ entry as a mechanism that reinforces capillary-to-arteriole communication, sustaining functional hyperemia during repetitive neuronal stimulation.
Neurovascular coupling (NVC), which is initiated by the brain's dense capillary network, matches blood flow to neuronal activity. We found that ORAI1 channels and their regulator STIM1, the main drivers of store-operated Ca2+ entry, were essential for communication from capillaries, which detect neuronal metabolic need, to upstream arterioles, which dilate to increase regional flow. Endothelial cell-specific knockout of either Stim1 or Orai1 disrupted capillary Ca2+ signals, impaired sustained capillary-driven arteriole dilation, and reduced increases in blood flow in the somatosensory cortex evoked by whisker stimulation, indicating that ORAI1 and STIM1 sustain cerebral blood flow during prolonged neuronal stimulation. Moreover, mice with endothelial cell-specific deficiency of Stim1 or Orai1 showed cognitive impairment, whereas mice with endothelial cell-specific deficiency of Orai3 showed anxiety-like behaviors. These in vivo results link impaired capillary-to-arteriole signaling to isoform-specific behavioral aberrations. These findings demonstrate that intravascular communication mediated by ORAI channels and STIM1 is fundamental for NVC and brain health.
Although reperfusion therapy effectively restores blood flow to occluded brain arteries after ischemic stroke, many patients develop persistent white matter injury, a major contributor to long-term neurological disability. Currently, there are few approved clinical therapies that specifically target white matter repair to enhance functional recovery after stroke. We demonstrated that microglial adenosine 2A receptor (A2AR) is essential for spontaneous white matter regeneration after ischemic injury in mice. Deletion of A2AR in microglia specifically impaired chronic-phase repair without altering the severity of acute ischemic injury. Pharmacological activation of A2AR signaling with blood-brain barrier-permeable agonistic micelles during the early reperfusion phase enhanced white matter structural repair and led to sustained improvements in cognitive and sensorimotor function in mice. Mechanistically, A2AR activation promoted microglial efferocytosis of apoptotic cells and myelin debris in the lesioned white matter, thereby limiting secondary necrosis-induced inflammation, enhancing neurotrophic factor release, and establishing a reparative microenvironment conducive to oligodendrocyte precursor cell differentiation and remyelination. Moreover, A2AR signaling engaged HIF1α-dependent metabolic reprogramming to increase glycolysis, thereby providing the energetic support required for efficient and sustained efferocytosis. Together, these findings identify A2AR as a critical regulator of microglia-mediated white matter repair and a promising therapeutic target for enhancing regeneration after stroke.
Dopamine neurons in the ventral tegmental area (VTA) have roles in motivation, learning, and psychiatric disorders. We found that genetically defined VTA dopamine neuron subtypes had distinct electrophysiological properties, neuronal signaling dynamics in response to reward- and aversion-related stimuli, and roles in somatic optogenetically induced reward dependent on the neurotransmitters they released. Nonglutamate-dopamine neurons increased activity after reward-related stimuli and decreased activity after the omission of an expected reward (negative reward prediction error) and aversion-related stimuli. Glutamate-dopamine and glutamate-only (nonGABAergic and nondopaminergic) neurons were activated by both rewarding and aversive events, but only glutamate-dopamine neurons had sustained cue-induced reward signaling. Recordings of all dopamine neurons without considering glutamate cotransmission showed mixed population responses during prediction error and aversive stimuli that obscured the distinct signaling patterns of its constituent subpopulations. Although the examined cell types largely differ in mediolateral location, cell-type identity better accounted for functional differences than mediolateral location. Glutamate-dopamine neurons were more excitable than nonglutamate-dopamine neurons. Glutamate-dopamine and nonglutamate-dopamine axons had similar but not identical dopamine release dynamics in the nucleus accumbens. Only nonglutamate-dopamine neurons supported somatic optogenetically induced reward and reinforcement. In glutamate-dopamine neurons, the dopamine synthetic enzyme TH contributed to associative learning of aversive or less-beneficial outcomes, whereas the vesicular glutamate transporter VGLUT2 contributed to reward- and exploration-related vigor. Our results suggest that glutamate cotransmission is a distinguishing feature of VTA dopamine neuron signaling patterns and roles in natural reward- or aversion-motivated behavior.
Type I interferons (IFNs) are induced by pattern recognition receptors (PRRs) of the innate immune system to protect against foreign pathogens and malignant transformation, and their production must be tightly regulated to balance antiviral defense with tissue homeostasis. To define the genetic network that governs IFN regulation, we conducted genome-wide CRISPR screens for genes that positively or negatively regulated IFNB1 gene expression induced by the PRR cGAS and its downstream effector STING, in both unprimed THP-1 cells and cells primed with IFN-α to mimic an ongoing inflammatory response. Distinct subsets of genes affected IFNB1 induction in the unprimed and primed states, and many regulators had not previously been associated with IFN responses, thereby linking IFNB1 regulation to various cellular pathways. For example, we found that the NCoR/SMRT corepressor complex components TBL1XR1 and HDAC3 cooperated to support IFNB1 expression, with HDAC3 promoting activation of the kinase TBK1, an essential driver of type I IFN responses. These datasets are a resource for identifying genes associated with type I IFN-related inborn errors of immunity and cancer and for developing therapies to modulate STING signaling in interferonopathies and other conditions of dysregulated type I IFN.
Polyunsaturated fatty acids (PUFAs) play a crucial role in tumor development by influencing not only tumor cells but also immune cells within the tumor microenvironment. Here, we explored the mechanisms by which PUFAs are transported and function within immune cells to regulate tumor growth. We found that PUFA transport through LDL receptor-related protein 5 (LRP5) into natural killer (NK) cells played an essential role in modulating the cells' antitumor function. LRP5 deficiency or expression of LRP5 lacking the LDLa domain enhanced the cytotoxicity and antitumor activity of NK cells both in vivo and in culture. However, wild-type NK cells cultured in the absence of PUFAs and NK cells from mice fed a PUFA-free diet also exhibited enhanced cytotoxicity, eliminating the functional difference between wild-type and NK cells expressing LDLa domain-deficient LRP5. Mechanistically, LRP5-mediated PUFA transport suppressed mTORC1 signaling and glycolysis in NK cells, a metabolic pathway essential for NK cell cytotoxicity. Thus, our study identified LRP5 as an immune checkpoint that restrains NK cell activity through PUFA transport-dependent suppression of mTORC1 signaling.
Mapping mutations to cellular outcomes reveals the molecular mechanisms underlying STING activity.
Astrocytes, oligodendrocyte lineage cells, and microglia dynamically shape the neural extracellular matrix (ECM) across brain development and aging. Recent findings highlight the potential of glial regulation of the ECM as a framework for understanding circuit formation and identify previously unrecognized targets in neurological diseases.
Recognition of the bacterial product lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) initiates inflammatory responses. The unfolded protein response (UPR) elicited by endoplasmic reticulum (ER) stress can strengthen TLR4-dependent inflammatory responses. Here, we report that the ER-localized E3 ubiquitin ligase TRIM13 restrained LPS-induced inflammatory responses in macrophages and in mice by protecting the cells from ER stress. TRIM13 mediated Lys33-linked polyubiquitylation of the ER-localized Ca2+ sensor STIM1, promoting its degradation. TRIM13 deficiency caused STIM1 accumulation and activated store-operated Ca2+ entry (SOCE) and the inositol-requiring enzyme 1 α (IRE1α) branch of the UPR. Suppressing SOCE, chelating extracellular Ca2+, relieving ER stress, or blocking IRE1α activation inhibited the amplification of inflammatory responses caused by the loss of TRIM13 in macrophages. Pharmacological inhibition of IRE1α ameliorated chemically induced colitis in TRIM13-deficient mice. Our study suggests that TRIM13 restrains inflammation by limiting LPS-induced activation of SOCE and the IRE1α branch of the UPR.
A molecular glue degrader tackles mutant BRAF in drug-resistant colorectal cancer by disrupting mRNA splicing.
Triglycerides can be formed by fatty acid esterification of glycerol 3-phosphate (G3P) and by de novo lipogenesis (DNL). We identified G3P as a stimulus that activated mTORC1, a nutrient-sensing protein complex that promotes DNL in the liver. We found that the major source of G3P in primary mouse hepatocytes was glycerol kinase (GK), which generates G3P from glycerol. Mice with a liver-specific GK deficiency showed reductions not only in hepatic triglyceride production and storage but also in mTORC1-dependent DNL. Sequentially blocking hepatic pathways for G3P metabolism and analysis of hepatocytes and mice deficient in glycerol phosphate dehydrogenases, alternative enzymatic sources for G3P, showed that mTORC1 activation positively correlated with G3P amounts and was not mediated by a G3P precursor or other glycerol metabolites. G3P generated by wild-type GK in glycerol-treated cells induced mTORC1 activation through GATOR2, a complex that also activates mTORC1 in response to amino acids. In contrast, GK with inactivating mutations found in GK deficiency did not induce activation of mTORC1 in response to glycerol. These results show that by coordinating the production of substrates needed for esterification, G3P stimulates hepatic DNL through mTORC1 activation. In obesity, higher glycerol levels and enhanced GK-mediated metabolism drive hepatic TG accumulation, contributing to metabolic dysfunction-associated fatty liver disease.
Individual G protein-coupled receptors (GPCRs) are present on many cell types and regulate multiple physiological responses, making it necessary to design ligands that are selective for the targeted GPCR in a tissue-selective manner. Here, we found ligand-selective effects of the GPCR free fatty acid receptor 2 (FFAR2) mediated through differential activation of Gq and Gi family proteins in various tissues. These effects were elucidated with ago-allosteric regulators of FFAR2 that bind to overlapping sites (compound 187 and AZ1729) and to a distinct site (4-CMTB) in tissues from mice expressing a hemagglutinin-tagged human FFAR2 DREADD (designer receptor exclusively activated by designer drugs). The Gq-activating ligand compound 187 stimulated the release of the gut hormones GLP-1 and PYY from colonic crypts and the secretion of insulin from pancreatic islets, effects that were prevented by the Gi-biased ligand AZ1729. Both compound 187 and AZ1729 promoted Gi-mediated neutrophil migration in bone marrow and antilipolytic effects in adipose tissue synergistically with an orthosteric DREADD agonist, whereas 4-CMTB was ineffective in both of these tissues. In contrast, 4-CMTB, alone or with the orthosteric agonist, produced Gq signaling-dependent effects in colonic crypts and pancreatic islets. These insights into the functional selectivity of allosteric ligands acting on the same GPCR may enable the development of physiologically specific therapeutics.
The WNT signaling pathway regulates cell proliferation and stem cell maintenance. Its sustained and inappropriate activation results in excessive cell division and cancer. WNT signaling is activated upon interaction of the soluble WNT ligand with the Frizzled (FZD) receptor. The RING E3 ubiquitin ligases RNF43 and ZNRF3 promote the ubiquitylation and internalization of FZD, thereby turning off WNT signaling, and their inactivation causes cancer. Here, we identified the determinants of ubiquitin transfer by ZNRF3 and RNF43 and report the structure of the RING domain from ZNRF3. We found that the RING domain was monomeric and that RING dimerization was not required for its ubiquitin ligase activity. However, the ectodomain of ZNRF3 dimerizes, and our data supported a model in which the cytoplasmic domains are in close proximity in cells and interact, even though RING dimerization was not required for ubiquitin transfer. Our studies provide a framework for understanding how the E3 ubiquitin ligase activity of ZNRF3 and RNF43 is regulated.
To mount a robust T cell-dependent immune response, antigen-specific B lymphocytes require the stimulation of the transmembrane receptor CD40 through immune synapse formation with CD4+ T follicular helper cells. CD40 stimulates the activation of mammalian target of rapamycin complex 1 (mTORC1) and remodels mitochondria to meet the increased bioenergetic and anabolic demands of activated B cells. Here, we found that diacylglycerol kinase ζ (DGKζ) supported mTORC1 activation downstream of CD40 stimulation in mouse B cells. We showed that DGKζ was required for organellar translocation to the CD40-mediated immune synapse and for the recruitment of mTORC1 to lysosomes, the latter of which was necessary for mTORC1 activation and function. The production of phosphatidic acid by DGKζ was crucial for these processes. DGKζ-/- B cells exhibited defects in protein biosynthesis, metabolite transporter expression, and cell cycle progression, together with dysregulation of the transcriptional network that determines B cell fate. To sustain their bioenergetic and metabolic demands, DGKζ-/- B cells enhanced their mitochondrial function. Together, these effects of DGKζ loss led to decreases in germinal center responses and in the generation of long-lived plasma cells and memory B cells in mice. Thus, our data identify DGKζ as an essential mediator of CD40 functions in the B cell immune response.
Oxytocin-induced lipolysis in adipocytes in the lactating mammary gland ensures a high lipid content in milk.
