T cell activation requires cell polarization and changes in gene expression. Target cell stiffness contributes to the activation of immune cells, and tumor cell softening is linked to cancer progression. We investigated...T cell activation requires cell polarization and changes in gene expression. Target cell stiffness contributes to the activation of immune cells, and tumor cell softening is linked to cancer progression. We investigated how substrate stiffness influences T cell activation using functionalized, T cell-activating substrates of varying stiffness and softened target cells. Reorientation of the microtubule-organizing center (MTOC) toward the immunological synapse and nuclear translocation of the transcription factor NFAT1 were impaired on softer hydrogels or upon contact with softer target cells. The increase in intracellular Ca induced by target engagement also depended on stiffness and was reduced on soft substrates. Stiffness-dependent Ca signaling was crucial for both rapid (MTOC reorientation) and long-term (NFAT translocation) responses. Whereas MTOC reorientation depended on the mechanosensitive Ca-permeable channel PIEZO1, NFAT1 translocation depended on the Ca channel ORAI1. Our results demonstrate that target stiffness directly influences MTOC reorientation and NFAT1 translocation in T cells, and these two processes are governed by different plasma membrane Ca channels, indicating that these stiffness-regulated rapid and long-term responses can be decoupled. Our findings imply that tumor cell stiffness regulates T cell functionality and suggest that pathways regulated by PIEZO1 and ORAI1 might differentially control rapid and long-term responses to stiffness in other cell types.
A high-fat diet impairs the growth of a commensal that produces lipids with anti-obesogenic effects in mice.A high-fat diet impairs the growth of a commensal that produces lipids with anti-obesogenic effects in mice.
The chemical reagent pervanadate is widely used to study phosphotyrosine signaling because it is a potent, irreversible inhibitor of protein tyrosine phosphatases that globally increases tyrosine phosphorylation across t...The chemical reagent pervanadate is widely used to study phosphotyrosine signaling because it is a potent, irreversible inhibitor of protein tyrosine phosphatases that globally increases tyrosine phosphorylation across the proteome. In this issue of , Mulholland show that pervanadate also modulates phosphotyrosine signaling by oxidizing specific cysteine residues on the tyrosine kinase SRC, thereby leading to hyperactivation of this ubiquitous signaling enzyme.
Freund PA, Panayotis N, Yuanxiang P
… +13 more, Samer S, Otikovs L, Kawaguchi R, Oses-Prieto J, Zomorrodinia T, Rishal I, Schweizer M, Tsoory M, Grochowska KM, Karpova A, Burlingame AL, Kreutz MR, Fainzilber M
Axonal localization and local translation of mRNA encoding importin β1 are important for retrograde injury signaling and axonal growth. Here, we found that loss of importin β1 in axons through deletion of the mRNA's 3' u...Axonal localization and local translation of mRNA encoding importin β1 are important for retrograde injury signaling and axonal growth. Here, we found that loss of importin β1 in axons through deletion of the mRNA's 3' untranslated region (3'UTR) caused a specific spatial memory deficit in mice. Electrophysiological analyses of hippocampal circuits revealed that mice expressing importin β1 transcripts lacking the 3'UTR (∆3'UTR) had impaired long-term potentiation (LTP) in presynaptic mossy fibers. Furthermore, mass spectrometry revealed an altered synaptic proteome and phosphoproteome, and sequencing of ribosome-associated RNAs showed suppression of locally translated presynaptic mRNAs in dentate gyrus neurons from mice expressing ∆3'UTR importin β1. This dysregulation was reflected in a reduction in the readily releasable pool of synaptic vesicles in mossy fiber synapses. Thus, axonal localization of importin β1-encoding mRNA is required to shape the presynapse, and disruption of this mechanism causes memory deficits.
Dynamic regulation of protein tyrosine phosphorylation (pTyr) by kinases and phosphatases enables cells to sense and respond to environmental changes. The widely used chemical pervanadate induces the accumulation of pTyr...Dynamic regulation of protein tyrosine phosphorylation (pTyr) by kinases and phosphatases enables cells to sense and respond to environmental changes. The widely used chemical pervanadate induces the accumulation of pTyr in mammalian cell lines. This effect is primarily attributed to its inhibition of protein tyrosine phosphatases (PTPs), leading to the assertion that PTPs are master gatekeepers of intracellular pTyr homeostasis. Here, we used several approaches to reveal that pervanadate disrupted cellular redox homeostasis and directly activated tyrosine kinases of the SRC family through the oxidation of specific cysteine residues. Mass spectrometry and biophysical approaches showed that pervanadate-induced oxidation of cysteine-188 and cysteine-280 activated SRC by disrupting autoinhibitory intramolecular interactions between the catalytic domain and the SH2/SH3 domains and by impairing SH2 domain binding to phosphopeptides, including the regulatory carboxyl-terminal tail phosphotyrosine-530. Redox-sensitive cysteine residues were essential for SRC to promote the overgrowth of mouse fibroblasts. Our findings call for a reevaluation of pervanadate-based experiments and demonstrate that SRC cysteines control its oncogenic properties.
T cells achieve precise antigen discrimination by relying on the temporal stability of T cell receptor (TCR) interactions with antigens. Given that time is central to antigen discrimination, we used real-time, single-cel...T cells achieve precise antigen discrimination by relying on the temporal stability of T cell receptor (TCR) interactions with antigens. Given that time is central to antigen discrimination, we used real-time, single-cell imaging in a controlled TCR-antigen system to characterize extracellular signal-regulated kinase (ERK) signaling dynamics as a function of antigen affinity to better understand the temporal patterns of signaling downstream of the TCR. We found that intermediate-affinity antigens elicited pulsatile ERK activity at different frequencies and that T cell activation correlated with the cumulative amount of ERK activity. Mechanistically, we found that the ERK pulsing frequency depended on the rate of activity of the Src family kinase LCK at the plasma membrane, whereas mitogen-activated protein kinase (MAPK) kinase (MEK) modulated the amplitude of ERK signaling. Moreover, we showed that ERK activity dynamics in T cells depended on members of two upstream MAP3K groups: mixed lineage kinases (MLKs) and RAFs, which played distinct roles promoting or sustaining the formation of upstream signaling condensates containing the transmembrane adaptor molecule LAT. Together, our findings reveal insights into the spatiotemporal organization of TCR signaling activities and their roles in T cell activation.
T cell activation critically depends on the calcium ion (Ca)-mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate), which induces the formation of Ca microdomains that initiate global Ca signa...T cell activation critically depends on the calcium ion (Ca)-mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate), which induces the formation of Ca microdomains that initiate global Ca signals. NAADP is produced in immune synapses in T cells by dual NADPH oxidase 2 (DUOX2). Here, we investigated the mechanisms that stimulate DUOX2 activity in T cells. DUOX2 activity was enhanced by a modest increase in intracellular Ca concentration, similar to that induced by Ca microdomains that arise in resting T cells through different T cell receptor (TCR)-independent mechanisms. In addition, DUOX2 was activated in vitro by phosphorylation of threonine-789 mediated by PKA Cβ or PKCθ, and genetic deficiency of PKA Cβ2 or PKCθ decreased NAADP-dependent Ca microdomain formation in T cells. PKA Cβ2 was activated downstream of adenosine A receptors, independently of the TCR. In contrast, PKCθ was activated by the tyrosine kinase LCK downstream of TCR stimulation. Inhibition of A receptors or PKCθ to prevent full DUOX2 activation decreased the production of the proinflammatory cytokine IL-17 by effector T cells. Thus, full stimulation of NAADP signaling that is critical for T cell activation requires integration of multiple TCR-independent and -dependent signals with different spatiotemporal characteristics by DUOX2, a fine-tuning mechanism that could be relevant for inflammation.
Hepatic lipid metabolism is regulated by circadian rhythms and dynamically responds to nutrient availability, such that lipid synthesis, oxidation, and storage are temporally coordinated. We demonstrated that the endopla...Hepatic lipid metabolism is regulated by circadian rhythms and dynamically responds to nutrient availability, such that lipid synthesis, oxidation, and storage are temporally coordinated. We demonstrated that the endoplasmic reticulum (ER)-localized E3 ubiquitin ligase HRD1 stimulated lipid accumulation in the liver by decreasing the -methyladenosine (m6A) methylation and expression of mRNAs encoding factors involved in lipid metabolism. In mouse livers, m6A RNA modification and the expression of mRNAs encoding the m6A writer METTL14 and the m6A reader YTHDF3 were under circadian control and inversely correlated with the abundance of HRD1. m6A RNA sequencing analyses revealed that HRD1 and the m6A writer METTL14 had opposing roles in the m6A modification and expression of mRNAs encoding factors involved in fatty acid metabolism. In vivo, hepatic lipid accumulation and triglyceride amounts were decreased in mice with hepatic HRD1 deficiency fed a high-fat diet but increased in mice with hepatic METTL14 or YTHDF deficiency fed normal chow. Mechanistically, HRD1 mediated the polyubiquitination and degradation of PPARα, which transcriptionally activated and expression in the liver. Our work identifies a pathway regulated by circadian rhythms or nutrients in which HRD1 promotes the degradation of PPARα to decrease the m6A modification and expression of hepatic mRNAs encoding factors involved in lipid metabolism.
Hydroxycarboxylic acid receptor 1 (HCAR1) is a class A G protein-coupled receptor (GPCR) that is activated by the endogenous metabolite l-lactate and that plays an important role in various metabolic and inflammatory dis...Hydroxycarboxylic acid receptor 1 (HCAR1) is a class A G protein-coupled receptor (GPCR) that is activated by the endogenous metabolite l-lactate and that plays an important role in various metabolic and inflammatory disorders. HCAR1 uses distinct ligand recognition and self-activation mechanisms to mediate specific pathophysiological functions through Gα and β-arrestin signaling pathways. To support effective drug development targeting HCAR1, we investigated ligand recognition and activation mechanisms through cryo-electron microscopy (cryo-EM) structures of the HCAR1-Gα complex in the apo state or with l-lactate or with the synthetic agonist CHBA. Compared with other HCARs, HCAR1 has a more compact binding pocket, which is stabilized by three unique disulfide bonds. l-lactate exhibited a flexible binding mode and relatively weak intermolecular interactions, thus requiring millimolar concentrations for receptor activation. In contrast, the binding of CHBA was more stable because of its chlorinated benzene ring, thus resulting in improved agonist potency. Structural comparisons with HCAR2 identified critical residues that restrict the size of the binding pocket of HCAR1 and influence ligand selectivity. Self-activation of HCAR1 is driven by conformational rearrangements within extracellular loop 2, with Phe168 playing a pivotal role as the key agonist. Together, these results clarify the mechanisms underlying HCAR1 activation, self-activation, and ligand selectivity, providing a structural framework for the design of high-affinity, selective agonists and inverse agonists with minimized off-target effects.
Boulton AM, Grund ME, Wang Y
… +11 more, Orsini EM, Liu Y, Abraham S, Grove LM, Musich R, Snyder CM, Ricci H, Cardani-Boulton A, Vachharajani V, Olman MA, Scheraga RG
The nuclear factor κB (NF-κB) signaling pathway plays a critical role in activating macrophages in the pathogenesis of many inflammatory diseases. Tissue mechanical properties are important in modulating key cellular pro...The nuclear factor κB (NF-κB) signaling pathway plays a critical role in activating macrophages in the pathogenesis of many inflammatory diseases. Tissue mechanical properties are important in modulating key cellular proinflammatory responses. Here, we investigated how the mechanosensitive membrane cation channel TRPV4 (transient receptor potential vanilloid 4) limits macrophage proinflammatory responses in bacterial pneumonia. We found that TRPV4 suppressed proinflammatory gene expression in alveolar macrophages in response to pneumonia in mice and in response to agonists of various Toll-like receptors (TLRs) in vitro. TRPV4 suppressed proinflammatory gene expression in macrophages by decreasing the activity of the NF-κB subunit p65. Upon stimulation of macrophages with bacterial lipopolysaccharide, a fraction of TRPV4 translocated from the endoplasmic reticulum to the plasma membrane, releasing p65 for nuclear translocation. TRPV4 interacted with p65 through an N-terminal cytoplasmic ankyrin repeat domain (ANKRD) that shares sequence homology with the p65-binding ANKRD of the NF-κB inhibitor IκBα. Given the diverse roles of TRPV4 and NF-κB in various cell types, our identification of cross-talk between a mechanosensitive channel and p65 in macrophages suggests application to many NF-κB-dependent diseases, such as cancer and atherosclerosis.
A bacterial virulence factor subverts host cell apoptosis by acting as a protein recombinase.A bacterial virulence factor subverts host cell apoptosis by acting as a protein recombinase.
Hu Q, Remsing Rix LL, Desai B
… +12 more, Miroshnychenko D, Li X, Welsh EA, Fang B, Wright GM, Chaudhary N, Kroeger JL, Doebele RC, Koomen JM, Haura EB, Marusyk A, Rix U
Cancer-associated fibroblasts (CAFs) are associated with tumor progression and drug resistance. Here, we investigated the mechanisms underlying the cross-talk between CAFs and tumor cells in non-small cell lung cancer (N...Cancer-associated fibroblasts (CAFs) are associated with tumor progression and drug resistance. Here, we investigated the mechanisms underlying the cross-talk between CAFs and tumor cells in non-small cell lung cancer (NSCLC). In NSCLC cell lines with fusions, we observed substantial CAF-mediated drug resistance to clinically used inhibitors of the tyrosine kinase ALK. Array-based cytokine profiling of CAF-derived conditioned medium indicated that a major contributor to the phenomenon was the secreted growth factor HGF, and blocking its receptor MET overcame paracrine resistance to ALK inhibition. However, cell-selective labeling of the proteome in cocultures also revealed an equally important contribution by the fibronectin-integrin pathway, specifically integrin β, which was confirmed through pharmacological inhibition and cell-specific silencing or knockout. Concurrent targeting of MET and integrin signaling effectively abrogated ALK inhibitor resistance in NSCLC cells cocultured with CAFs. Moreover, the combination of the ALK inhibitor alectinib with the MET inhibitor capmatinib and/or the integrin inhibitor cilengitide was more effective than single-agent treatment in suppressing tumor growth in allografted mice. The findings illustrate a previously unappreciated complex nature of concurrent paracrine and juxtacrine mechanisms of CAF-driven resistance that may inform the development of more effective therapeutic approaches.
Lipid homeostasis is subject to control by posttranslational modification machinery, such as sirtuin deacetylases that reverse coenzyme A (CoA)-dependent acetylation. Here, we showed that a mammalian denitrosylase (SCoR2...Lipid homeostasis is subject to control by posttranslational modification machinery, such as sirtuin deacetylases that reverse coenzyme A (CoA)-dependent acetylation. Here, we showed that a mammalian denitrosylase (SCoR2), which counteracts CoA-dependent S-nitrosylation, promoted both fat storage and lipogenesis to impair metabolic health. In mice, SCoR2 protein abundance correlated with body mass, and deleting or pharmacologically inhibiting SCoR2 prevented both diet-induced obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Loss of in adipocytes promoted the S-nitrosylation of the actin cytoskeletal regulator myosin 9, which inhibited the activity of the lipogenesis-promoting transcription factors PPARγ, SREBP1, and CEBPα to prevent fat storage. In hepatocytes, inhibition of SCoR2-mediated denitrosylation of lipogenic enzymes reduced fat synthesis and induced fat oxidation. In humans, an obesity-linked polymorphism was associated with increased mRNA expression, and in patient adipose and liver tissues, SCoR2 protein or mRNA abundance directly correlated with adipocyte size or MASLD. These results indicate that SCoR2 regulates nutrient metabolism, similar to sirtuins, and is a potential drug target for obesity and MASLD.
The central melanocortin system links nutrition to energy expenditure. Melanocortin-4 receptor (MC4R) controls appetite and food intake, and its signaling is potentiated by melanocortin-2 receptor accessory protein 2 (MR...The central melanocortin system links nutrition to energy expenditure. Melanocortin-4 receptor (MC4R) controls appetite and food intake, and its signaling is potentiated by melanocortin-2 receptor accessory protein 2 (MRAP2). Human mutations in and are associated with obesity. Here, we sought to determine whether MRAP2 affected the activity of MC3R, which is structurally similar to MC4R and which regulates sexual maturation, linear growth rate, and lean mass accumulation. Single-molecule pull-down assays showed that MC3R and MRAP2 interacted in HEK293 cells. Analysis of fluorescence photobleaching steps showed that MC3R and MRAP2 readily formed heterodimers, most commonly with a 1:1 stoichiometry. Mining of previously published human single-nucleus and spatial transcriptomic data showed coexpression of and in hypothalamic neurons that function in energy homeostasis and appetite control. In HEK293 cells, MRAP2 enhanced cAMP signaling downstream of MC3R, impaired β-arrestin recruitment to MC3R, and reduced MC3R internalization. The ability of MRAP2 to promote MC3R signaling was suppressed by alanine mutagenesis of five MRAP2 and two MC3R transmembrane residues identified by structural homology models as important for the interaction. We showed that variants of MRAP2 found in individuals who are overweight or obese did not enhance MC3R-driven signaling. Thus, these studies implicate MRAP2 as an important regulator of MC3R function and provide further evidence for the crucial role of MRAP2 in energy homeostasis.
The highly addictive psychostimulant methamphetamine increases the release of dopamine in the brain's reward circuitry, where it also promotes the release of cytokines, including TNF-α, that contribute to neuroinflammati...The highly addictive psychostimulant methamphetamine increases the release of dopamine in the brain's reward circuitry, where it also promotes the release of cytokines, including TNF-α, that contribute to neuroinflammation associated with methamphetamine abuse. Here, we found a dynamic interplay between methamphetamine and TNF-α in facilitating dopamine transmission within the ventral tegmental area (VTA) in mice. In ex vivo mouse brain slices and dopaminergic neurons, methamphetamine or TNF-α treatment increased dopamine release, intracellular Ca concentrations, and the firing activity of VTA dopaminergic neurons. These effects depended on the activity of dopamine transporter (DAT) and L-type voltage-gated Ca channels. Pharmacological inhibition of either DAT or TNF-α signaling mitigated these effects, suggesting that methamphetamine-induced alterations in VTA dopaminergic neurons are partially TNF-α dependent. These results underscore the role of neuroimmune signaling in modulating the dopaminergic circuitry and may inform therapeutic strategies for addressing methamphetamine addiction and its associated neuroinflammatory disorders.
Dopamine drives the neuronal activity and synaptic plasticity required for various forms of learning. It supports short-term working memory through activation of the D-like dopamine receptors D and D. Here, we found that...Dopamine drives the neuronal activity and synaptic plasticity required for various forms of learning. It supports short-term working memory through activation of the D-like dopamine receptors D and D. Here, we found that the L-type Ca channel Ca1.2 was a critical mediator of D signaling in hippocampal pyramidal neurons. In cultured mouse hippocampal neurons, a D agonist augmented currents through Ca1.2 in the soma and Ca influx in the dendrites. This effect was mediated through the second messenger cAMP and cAMP-dependent protein kinase (PKA), which phosphorylated the Ca1.2 α subunit at Ser. Ca1.2 and D colocalized, suggesting that this signaling was spatially restricted. In mice, D agonism facilitated spatial working memory in wild-type but not litter-matched Ca1.2 α S1928A knockin animals. These findings identify Ca1.2 as a key D signaling effector that supports dopamine-driven executive functions in cognition.
Singh A, Vijver SV, Aglmous-Talibi H
… +17 more, Jukic N, Chen P, Crawley S, Mondal K, Zhou J, Niederauer C, Matharu Z, Li B, Fan B, van der Vlist M, Kaplan DD, Rivera LB, Sissons J, Sitrin J, Ganzinger KA, Inês Pascoal Ramos M, Meyaard L
LAIR-1 is an inhibitory receptor on immune cells that recognizes collagens and collagen domain-containing proteins. The high abundance of both LAIR-1 and its ligands suggests tight regulation of this interaction. MARCO i...LAIR-1 is an inhibitory receptor on immune cells that recognizes collagens and collagen domain-containing proteins. The high abundance of both LAIR-1 and its ligands suggests tight regulation of this interaction. MARCO is a scavenger receptor with a collagen-like domain that is highly expressed on immunosuppressive macrophages. Here, we identified MARCO as a ligand for LAIR-1. MARCO interacted with LAIR-1 in trans and induced inhibitory signaling by LAIR-1 in human natural killer (NK) cells. MARCO and LAIR-1 were coexpressed by human macrophages in tumors and after stimulation of monocyte-derived macrophages with the cytokine interleukin-10 (IL-10) in vitro. Single-molecule fluorescence microscopy demonstrated that MARCO and LAIR-1 interacted in cis on THP-1 macrophages. Whereas the interaction did not affect the scavenger function of MARCO on human macrophages, it reduced both LAIR-1 binding and the LAIR-1 signaling response to collagen. LAIR-1-mediated inhibitory function was increased after CRISPR-Cas9-mediated knockout of MARCO in IL-10-polarized primary human monocyte-derived macrophages. Our results identify MARCO as a regulator of LAIR-1 signaling and suggest that the induction of MARCO on immunosuppressive macrophages could enhance their function by releasing LAIR-1-mediated inhibition.