A bacterial effector inhibits translation of host mRNAs by chemically modifying them.

Autosomal-dominant hyper-IgE syndrome (HIES) is characterized by chronic mucocutaneous candidiasis, recurrent sinopulmonary complications, noninflammatory atopic dermatitis, high amounts of serum IgE, and nonimmunological manifestations affecting the musculoskeletal and vascular systems. Most cases are caused by heterozygous loss-of-function, dominant-negative (LOF/DN) variants that affect the DNA binding, SH2, or transactivation domains of STAT3. Here, we report a patient with a unique STAT3 allele affecting the STAT3 N-terminal domain (R31Q), who presented with disseminated invasive aspergillosis affecting the brain and lung, bronchiectasis, and high serum IgE but lacked nonimmune disease manifestations. Biochemical analyses in cell lines demonstrated that R31Q reduced the abundance and transcriptional activity of STAT3 and caused it to exhibit negative dominance on wild-type (WT) STAT3. This was due to preferential STAT3[R31Q] dimerization with STAT3[WT], sequestration of STAT3[WT]:STAT3[R31Q] dimers in the cytosol, and a reduction in STAT3 nuclear translocation and transcriptional activity. Lymphocytes from the patient exhibited some typical defects observed in canonical STAT3[LOF/DN] HIES, such as reduced memory B cells, MAIT cells, and TH17 cytokines. However, naïve B cells from this patient had only a partial impairment in immunoglobulin secretion induced by interleukin-21 (IL-21) in vitro, and the patient had normal numbers of TH2 cells and circulating TFH cells. Our findings reveal that the N-terminal domain regulates STAT3 dimer formation and function and provide a previously unknown biochemical mechanism of disease pathogenesis in STAT3[LOF/DN] HIES.

Transfer RNA (tRNA) modifications are increasingly implicated as posttranscriptional regulators of basic neuronal functions. Characterizing tRNA modification profiles in specific neural circuits that control well-defined behaviors is challenging because of the complexity of conventional neurobiological models. Here, we leveraged the numerically simple central nervous system (CNS) of the marine mollusk Aplysia californica to investigate tRNA modification dynamics during habituation of a defensive reflex. We identified and quantified dozens of tRNA modifications and characterized the distribution of select small RNA modifications across different neuronal and nonneuronal tissues. Upon behavioral habituation of the siphon-elicited siphon withdrawal reflex (SSWR), we found that tRNA modification profiles in the major ganglion that controls the SSWR were repatterned with predictable, learning-related changes. We found a family of anticodon loop modifications that changed with habituation learning, including N[6]-isopentenyladenosine (i[6]A), which increased in abundance, and additional modifications N[1]-methyladenosine (m[1]A) and N[7]-methylguanosine (m[7]G), which decreased. These tRNA modification dynamics occurred independently of changes in the expression of their parent tRNAs, illustrating that behavioral habituation altered the activity of tRNA-modifying enzymes. Overall, our work reveals a link between tRNA modifications and behavioral habituation and provides insights toward understanding the posttranscriptional mechanisms of learning and memory.

Stimulation of G protein-coupled receptors (GPCRs) that couple to Gi/o leads to the activation of G protein-activated inward rectifier K[+] (GIRK) channels and membrane hyperpolarization in excitable cells. GIRK channel availability can determine the success of optogenetic silencing strategies based on Gi/o-coupled GPCRs. To selectively enhance Gi/o-mediated GIRK currents, we created expression cassettes that coupled the activity of a GIRK subunit to the light-activated Gi/o-coupled GPCRs Opn4L (an opsin also known as melanopsin), longwave-sensitive opsin Opn1, parapinopsin, or Opn7b. We used the GIRK1 F137S mutant because of its fast activation, generation of large currents, and formation of homomeric channels, which reduced the size of the expression cassette. We demonstrated that light-mediated activation of GIRK1[F137S] through the opsins induced robust GIRK currents in HEK293 cells, cardiomyocytes, and cerebellar Purkinje neurons. Moreover, coupling GIRK1[F137S] activation to the promiscuous Opn4L changed the net effect of G protein signaling from Gq/11-mediated excitation to Gi/o-mediated inhibition in cerebellar Purkinje neurons and cardiomyocytes. Thus, our tools optimize the use of GIRK activity in the optogenetic control of the Gi/o pathway in excitable cells.

Brain vasculature is essential for central nervous system function. Here, we found a cerebrovascular signaling axis in which the arginine methyltransferase PRMT5 in endothelial cells promoted proper inhibitory synaptogenesis and motor coordination in male mice. Cerebrovascular endothelial cell-specific deletion of Prmt5 in mice led to excessive inhibitory synaptic input onto Purkinje cells and progressive motor deficits. Mechanistically, Prmt5 deficiency epigenetically increased the expression of the gene encoding the secreted angiocrine ANGPTL4 through reduced symmetric dimethylation of Arg[8] in histone H3 and Arg[3] in histone H4, which coincided with increased acetylation of Lys[9] in histone H3, at the Angptl4 promoter. Angptl4 deletion in brain endothelium normalized inhibitory synaptic input onto Purkinje cells and partially restored motor coordination in mice with a conditional Prmt5 deficiency. Together, these findings define the endothelial PRMT5-ANGPTL4 axis as a key regulator of cerebellar inhibitory circuitry and motor function, highlighting cerebrovascular mechanisms as potential therapeutic targets for motor coordination disorders.

Many signals contribute to the selective assembly of neural circuits alongside the developing brain vasculature. In this issue of Science Signaling, Lu et al. identify endothelial PRMT5 as an epigenetic brake that restrains Angptl4 expression to limit inhibitory synapse formation in the cerebellum, linking angiocrine signaling to synaptic specificity and circuit function.

The Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a cytosolic multiprotein immune sensor that is restrained from assembling at steady state. Mutations in NLRP3 associated with cryopyrin-associated periodic syndrome (CAPS) enhance inflammasome formation and activation, resulting in neutrophil-predominant systemic and tissue inflammation. Here, we investigated how gasdermin D (GSDMD) and GSDME drive interleukin-1β (IL-1β) release, pyroptosis, and autoinflammation during constitutive and canonical NLRP3 activation in neutrophils. Mass cytometry analysis of neutrophils from patients with CAPS revealed heterogeneous subtypes with differing amounts of intracellular IL-1β. In mice, neutrophil-specific expression of activating Nlrp3 mutations orthologous to those in patients with CAPS uncovered nonredundant roles for GSDMD and GSDME downstream of inflammasome activation. Combined deletion of Gsdmd and Gsdme in these CAPS models reduced IL-1β secretion, prevented pyroptosis, and mitigated autoinflammation. Loss of both GSDMD and GSDME reduced caspase-1 cleavage and pro-IL-1β processing downstream of NLRP3 activation, whereas GSDMD deficiency alone increased cleavage of GSDME and caspase-8. Clusters of GSDMD and GSDME were closely associated with one another at the plasma membrane of activated neutrophils from CAPS mice. These findings indicate that the coordinated actions of membrane-associated GSDMD and GSDME reinforce cytosolic NLRP3 inflammasome activation to promote IL-1β secretion, lytic cell death, and autoinflammation.

Bifunctional antibodies linking a GPCR with the transferrin receptor lead to GPCR down-regulation.

Chemokine receptors are G protein-coupled receptors (GPCRs) that drive cell migration through interactions with chemokines. Even in the absence of ligand, chemokine receptors spend a portion of their time in active conformational states, rendering them constitutively active. Here, we identified and characterized two mutations in the third intracellular loop of atypical chemokine receptor 3 (ACKR3) that inhibited or promoted its constitutive activity as assessed by ligand-independent β-arrestin recruitment and receptor internalization. Single-molecule fluorescence resonance energy transfer experiments showed that these effects on activity corresponded to changes in the conformational equilibrium of the receptor, which also affected the kinetics of chemokine binding. The potencies of chemokines and small-molecule agonists for β-arrestin recruitment were differentially affected by the mutations, showing that the effects of an altered receptor conformational equilibrium on receptor activation were ligand dependent. Together, our findings reveal an inside-out allosteric mechanism for ACKR3 in which intracellular, conformation-altering mutants affect the ability of extracellular ligands to bind to and activate the receptor.

Regulator of G protein signaling 4 (RGS4) is a Gα subunit-interacting protein that modulates the functional responses of various G protein-coupled receptors (GPCRs) and that is present in several components of the nociceptive circuitry. Here, we found that RGS4 in lumbar dorsal root ganglia (DRGs) is required for the maintenance of chronic pain states in mice. Rgs4 knockout in Mrgprd[+] nonpeptidergic C-fibers was sufficient to attenuate sensory hypersensitivity in several acute and chronic injury models without affecting acute nociceptive responses. Single-nucleus RNA sequencing in ipsilateral lumbar DRGs from mice with prolonged peripheral nerve injury identified cell subset-specific gene expression changes in Rgs4 knockout mice after recovery from mechanical hypersensitivity, including Gabbr2, which encodes the GABAB receptor, in Mrgprd[+] fibers. RGS4 competed with Gαi-interacting protein (GINIP) to modulate GABAB receptor signaling, which suppresses neuronal excitability. Analysis of genetically modified mice suggested that imbalances in the interplay between RGS4 and GINIP contribute to the maintenance of sensory hypersensitivity and chronic pain states. Overall, our study reveals critical G protein-modulatory pathways in the DRG that promote the maintenance of sensory hypersensitivity in chronic pain models.

CD4[+] effector T helper 1 (TH1) cells drive inflammatory bowel disease (IBD). Here, we investigated how integrated signaling, metabolic, and epigenetic programs sustain their pathogenic persistence. We found that enforcing mitochondrial pyruvate metabolism with methyl pyruvate (MePyr) suppressed TH1 cell effector function and induced the acquisition of a regulatory-like phenotype. MePyr suppressed the activity of glycogen synthase kinase 3β (GSK3β), promoting a metabolic shift from glycolysis toward oxidative phosphorylation (OXPHOS) and limiting the accumulation of intracellular acetyl-CoA. This metabolic reprogramming decreased histone H3 acetylation at genes encoding inflammatory cytokines, thereby decreasing TH1 cell-associated gene expression. Restoration of inflammatory cytokine production by acetate supplementation or histone deacetylase inhibition established acetyl-CoA-dependent histone acetylation as a mechanistic link between metabolic flux and effector function. Conversely, the cytokine interleukin-21 enhanced TH1 cell effector activity by promoting a GSK3β-dependent shift from OXPHOS to glycolysis. Single-cell transcriptomic analysis of Crohn's disease tissue revealed enrichment of glycolytic gene programs in nonregulatory CD4[+] T cells. Consistent with these findings, pharmacologic inhibition of GSK3β attenuated disease severity in a T cell-transfer mouse model of colitis. Together, these findings identify a GSK3β-dependent metabolic-epigenetic axis that sustains TH1 cell pathogenicity and highlight GSK3β as a potential therapeutic target for IBD.

Atypical protein kinase C (aPKC) isozymes play opposing roles in oncogenic and tumor-suppressive signaling pathways. These kinases are activated by phosphoinositide-dependent kinase 1 (PDK1) as well as by direct binding of the lipid second messenger sphingosine 1-phosphate (S1P). Here, we found that S1P and PDK1 provide mutually exclusive activation of aPKC by a common switch mechanism. In silico docking, molecular dynamics, and biochemical approaches revealed that the phosphate on S1P occupied the same position as the phosphate of the PDK1-phosphorylated Thr residue in the activation loop to structure the active site for catalysis. The interaction of either phosphate with the activation loop was coordinated by conserved Arg and Lys residues. When aPKC was phosphorylated, these residues positioned the activation loop in a manner that made the binding of S1P energetically prohibitive. Cellular studies revealed that S1P sustained basal signaling of aPKC, which suppressed apoptosis. In contrast, PDK1-dependent phosphorylation promoted apoptosis. These data identify a regulatory mechanism by which a ligand mimics a posttranslational modification and reveal that aPKC is at the crossroads of lipid second messenger signaling through cytosolic S1P pathways and canonical PDK1 signaling.

Light is an important environmental stimulus that is necessary for vision and circadian entrainment. Here, we present a system-wide analysis of the kinase-mediated light response in Drosophila using optimized data-independent acquisition mass spectrometry (DIA-MS) for quantitative phosphoproteomics analysis. Exposure to standard white light or high-intensity blue light induced a widespread increase in phosphorylation relative to dark-adapted flies. The light-sensitive phosphoproteins were part of multiple pathways, including phototransduction, circadian control, lipid metabolism, and synapse organization, and exhibited distinct phosphorylation signatures under dark adaptation or white or blue light. The Ca[2+]-permeable transient receptor potential (Trp) channel was differentially phosphorylated at multiple sites in a white and blue light-dependent manner, and phosphorylation stoichiometric analysis identified Thr[849] in Trp as a pivotal functional switch. It was entirely unphosphorylated in the dark, but 80% of the Trp molecules were phosphorylated in either light condition. This phosphorylation event was mediated in part by the eye-specific protein kinase C InaC and regulated the eye's electrophysiological response to oscillating light. Thus, our results show that light remodels the Drosophila phosphoproteome, establishing a crucial mechanism for sensory adaptation to a changing light-dark environment.

Immunotherapies affect T cell function by modifying the immune synapse, which forms at the interface between T cells and their targets. Phosphoprotein associated with glycosphingolipid-rich microdomains 1 (PAG) is a T cell adaptor protein within the immune synapse that mediates signaling by the inhibitory co-receptor PD-1. Because PAG undergoes palmitoylation, which positions it within plasma membrane lipid rafts, and contains a carboxyl-terminal PDZ-binding motif that connects to the actin cytoskeleton, PAG is a potential target for modulating immune function. Here, we investigated how PAG functioned with actin to regulate T cell immune synapse organization and function. We found that the dynamics of PAG and actin were closely synchronized during immune synapse maturation. Mutation of the PDZ-binding motif of PAG disrupted the PAG-actin interaction, impairing immune synapse formation, stability, and function. To assess the function of the PDZ-binding motif of PAG in vivo, we used mouse models of type IV hypersensitivity and highly immunogenic cancer. In both cases, mice with T cells expressing mutant PAG lacking this motif showed reduced immune responses, particularly in terms of cytotoxicity. These results highlight the importance of the PAG-actin connection for optimal formation and function of the T cell immune synapse. Our research indicates that targeting PAG may be a promising approach to improving immunotherapy in those patients who do not respond or experience immune-related adverse events.

cMAF drives the identity and signaling of a brain macrophage subset involved in vascular homeostasis.