The genesis of neuropathic pain after peripheral nerve injury is associated with changes in gene expression and cell metabolism in sensory neurons and the release of inflammatory cytokines. Here, we connected glycolytic...The genesis of neuropathic pain after peripheral nerve injury is associated with changes in gene expression and cell metabolism in sensory neurons and the release of inflammatory cytokines. Here, we connected glycolytic metabolism induced by the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) to histone lactylation and changes in gene expression that promote chronic neuropathic pain. In both male and female mice subjected to peripheral nerve injury, the mRNA and protein abundance of AREG and its receptor EGFR was increased in dorsal root ganglia (DRGs). AREG-EGFR signaling induced glycolytic metabolism by activating the kinase PKM2. An increase in the glycolytic byproduct lactate facilitated lactylation of the histone lysines H3K18 and H4K12 by the lactyltransferase p300 in DRG neurons. These modifications promoted the expression of genes encoding various proinflammatory and pronociceptive proteins that contribute to the development and maintenance of pain. Deletion or knockdown of AREG or pharmacologically inhibiting EGFR, PKM2, or p300 alleviated neuropathic pain in mice and attenuated the injury-induced hyperexcitability of nociceptive neurons. Targeting this metabolically driven epigenetic mechanism may be a way to treat neuropathic pain in patients.
The chemokine CXCL12 signals through its receptor CXCR4 to induce the migration of all leukocyte types and multiple other cell types. Here, we report that CXCR4 is expressed in mouse erythroblasts, the bone marrow erythr...The chemokine CXCL12 signals through its receptor CXCR4 to induce the migration of all leukocyte types and multiple other cell types. Here, we report that CXCR4 is expressed in mouse erythroblasts, the bone marrow erythroid precursors, in which it stimulates erythrocyte generation instead of chemotaxis. CXCR4 signaling promoted homeostatic erythroblast maturation and increased the expression of genes mainly involved in metabolism and chromatin organization. Consequently, genetic depletion of CXCR4 in erythroblasts inhibited late erythropoiesis and diminished bone marrow erythroid outputs. Binding of CXCL12 to CXCR4 stimulated its rapid endocytosis and translocation together with Gα or phosphorylated β-arrestin1 into distinct intracellular compartments, including the nuclear envelope and nucleus. CXCL12 signaling promoted erythroblast elongation and the condensation and excentric positioning of nuclei and stimulated rapid perinuclear Ca transients that immediately preceded erythroblast enucleation. These findings highlight previously uncharacterized physiological roles for CXCR4 and bone marrow-derived CXCL12 in erythropoiesis.
The cryo-EM structures of human sweet taste receptors reveal the molecular basis of sweet taste detection.The cryo-EM structures of human sweet taste receptors reveal the molecular basis of sweet taste detection.
Lysosomes are versatile organelles that play pivotal roles in cellular recycling and signal transduction. They are crucial for the autophagic degradation and recycling of macromolecules, which facilitates the efficient t...Lysosomes are versatile organelles that play pivotal roles in cellular recycling and signal transduction. They are crucial for the autophagic degradation and recycling of macromolecules, which facilitates the efficient turnover of cellular components. Beyond their intracellular roles, lysosomes also regulate the degradation and assembly of extracellular matrix (ECM) constituents, affecting ECM remodeling and the processing of signaling molecules essential for cellular communication and adaptation to the microenvironment. Conversely, the ECM regulates key lysosomal functions, including biogenesis, acidification, and subcellular positioning. In this Review, we discuss the bidirectional interaction between lysosomes and the ECM and explore its implications in the development and treatment of neurodegenerative disease.
Notch proteins are single-pass transmembrane receptors activated by sequential extracellular and intramembrane cleavages to release the cytosolic domains that function as transcription factors. Transmembrane ligands of t...Notch proteins are single-pass transmembrane receptors activated by sequential extracellular and intramembrane cleavages to release the cytosolic domains that function as transcription factors. Transmembrane ligands of the Delta/Serrate/LAG-2 (DSL) family activate Notch on neighboring cells by exerting a pulling force across the intercellular ligand-receptor bridge. This force is generated by Epsin-mediated endocytosis of the ligand into the signal-sending cell and results in the extracellular cleavage of the force-sensing negative regulatory region (NRR) of the receptor by an ADAM10 protease on the signal-receiving cell. Here, we used chimeric Notch and DSL proteins to screen for other domains that could function as ligand-dependent proteolytic switches in place of the NRR in the developing wing. The domains that could functionally substitute for the NRR in vivo derived from diverse source proteins, varied in sequence, and had different predicted structures, yet all depended on cleavage that was catalyzed by the ADAM10 homolog Kuzbanian (Kuz) and stimulated by Epsin-mediated ligand endocytosis. The large sequence space of protein domains that can serve as force-sensing proteolytic switches suggests a widespread potential role for force-dependent, ADAM10-mediated proteolysis in other cell contact-dependent signaling mechanisms.
Natriuretic peptide receptor 2 (Npr2; also termed guanylyl cyclase B) is a transmembrane guanylyl cyclase that is highly abundant in nociceptors. Here, we investigated the role of production of cyclic GMP (cGMP) by Npr2...Natriuretic peptide receptor 2 (Npr2; also termed guanylyl cyclase B) is a transmembrane guanylyl cyclase that is highly abundant in nociceptors. Here, we investigated the role of production of cyclic GMP (cGMP) by Npr2 in pain processing. Adult mice with a deletion of Npr2 specifically in nociceptive sensory neurons exhibited deficits in noxious heat sensing, which can activate the nonselective cation channels TRPV1 and TRPA1. In parallel, Npr2-deficient mice showed a reduction in TRPV1-mediated nocifensive behavior and Ca influx into sensory neurons. Furthermore, Npr2-deficient mice had considerably reduced hypersensitivity after hindpaw injection of TRPA1 and TRPV1 activators or after hindpaw injection of complete Freund adjuvant, a model of persistent inflammatory pain. These results indicate that Npr2 contributes to the pain sensitization that can lead to chronic pain. Patch-clamp recordings revealed that the endogenous Npr2 ligand, C-type natriuretic peptide (CNP), enhanced the excitability of nociceptive sensory neurons through Npr2. CNP/Npr2 signaling led to the phosphorylation of cysteine-rich LIM-only protein 4 (CRP4), a substrate of cGMP-dependent protein kinase I. Behavioral and electrophysiological analyses using CRP4-deficient mice revealed that CRP4 limited CNP/Npr2-mediated pain sensitization. Our findings reveal a role for CNP/Npr2 signaling in sensory neurons in acute nociceptive and chronic pain and suggest that CRP4 is a downstream target that attenuates pain sensitization.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor and frequently has mutations in the transcription factor p53. TAp63α is a member of the p53 protein family that is generally tumor suppressive in various oth...Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor and frequently has mutations in the transcription factor p53. TAp63α is a member of the p53 protein family that is generally tumor suppressive in various other p53-mutant or p53-deficient cancers. Here, we found that TAp63α inhibited cell proliferation, epithelial-mesenchymal transition (EMT), and migration in several p53-mutant PDAC cell lines. TAp63α transcriptionally repressed (which encodes a subunit of the interleukin-20 receptor), potentially by inducing the methylation of its promoter. However, mutations in p53 or KRAS that are common in PDAC increased the abundance of the E3 ligase TRIM21, which promoted the ubiquitin-dependent degradation of TAp63α. Thus, the degradation of TAp63α enabled increases in expression and formation of IL-20 receptors, resulting in the activation of downstream JAK1-STAT3 signaling that stimulated the proliferation, EMT, migration, and in vivo metastatic seeding of PDAC cells. Our findings identify a signaling axis involving TRIM21, TAp63α, and IL-20RB in PDAC progression.
The transcriptional regulators SMAD2 and SMAD3 share the same primary signaling pathway in response to the cytokine TGFβ. However, whereas SMAD2 stimulates the differentiation of naive CD4 T cells into proinflammatory T...The transcriptional regulators SMAD2 and SMAD3 share the same primary signaling pathway in response to the cytokine TGFβ. However, whereas SMAD2 stimulates the differentiation of naive CD4 T cells into proinflammatory T helper 17 cells (T17 cells), SMAD3 stimulates the differentiation of anti-inflammatory regulatory T cells (T cells). Here, we report a dynamic SMAD2-specific posttranslational modification important for T17 cell differentiation. SMAD2, but not SMAD3, was reversibly S-palmitoylated at cysteine-41 and cysteine-81 by the palmitoyltransferase DHHC7 and depalmitoylated by the acyl protein thioesterase APT2. As a result, SMAD2 was recruited to intracellular membranes where its linker region was phosphorylated, leading to its interaction with the transcriptional regulator STAT3. Nuclear translocation of the SMAD2-STAT3 complex induced the expression of their target genes that promoted T17 cell differentiation. Perturbation of SMAD2-STAT3 binding by inhibiting the palmitoylation-depalmitoylation cycle suppressed T17 cell differentiation and reduced disease severity in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis (MS). Thus, the S-palmitoylation-depalmitoylation cycle mediated by DHHC7 and APT2 specifically regulates SMAD2, providing insights into the functional differences between SMAD2 and SMAD3 and the distinct role of SMAD2 in T17 cell differentiation. The findings further highlight DHHC7 and APT2 as potential therapeutic targets for the treatment of T17 cell-mediated inflammatory diseases, including MS.
ROS generated by sleep-promoting neurons tracks the need for sleep and initiates sleep.ROS generated by sleep-promoting neurons tracks the need for sleep and initiates sleep.
Shah SR, Ren C, Tippens ND
… +9 more, Park J, Mohyeldin A, Wang S, Vela G, Martinez-Gutierrez JC, Margolis SS, Schmidt S, Quiñones-Hinojosa A, Levchenko A
Delineating the mechanisms that control the movement of cells is central to understanding diverse physiological and pathophysiological processes. The transcriptional coactivator YAP is important during development and as...Delineating the mechanisms that control the movement of cells is central to understanding diverse physiological and pathophysiological processes. The transcriptional coactivator YAP is important during development and associated with cancer metastasis. Here, we found that YAP promoted cell migration by modulating a Rho family guanosine triphosphatase (GTPase) switch involving Rac1 and RhoA, which are key regulators of cytoskeletal dynamics. YAP transcriptionally transactivated the gene encoding the Rac1 guanine nucleotide exchange factor TRIO by directly binding to its intronic enhancer. This led to the activation of Rac1 and inhibition of RhoA, which increased cell migration and invasion in vitro and in vivo. This YAP-dependent program was observed across many cell types, including human breast epithelial cells and astrocytes, but it was particularly enhanced in a patient-specific manner in glioblastoma (GBM), the most common malignant brain tumor. Additionally, YAP-TRIO signaling activated STAT3, a transcription factor implicated in invasive growth in cancer, suggesting potential for cross-talk with this pathway to exacerbate invasive behavior. Clinically, hyperactivation of YAP, TRIO, and STAT3 gene signatures in GBM were associated with poor survival outcomes in patients. Our findings suggest that the YAP-TRIO-Rho-GTPase signaling network regulates invasive cell spread in both physiological and pathological contexts.
G protein-coupled receptors (GPCRs) are transmembrane detectors of extracellular signals that activate heterotrimeric G proteins to regulate intracellular responses. Because there are only 16 Gα proteins that can couple...G protein-coupled receptors (GPCRs) are transmembrane detectors of extracellular signals that activate heterotrimeric G proteins to regulate intracellular responses. Because there are only 16 Gα proteins that can couple to GPCRs, variation in a single Gα can affect the function of numerous receptors. Here, we investigated two mutant forms of Gα (L388R and E392K) that are associated with pseudohypoparathyroidism type Ic (PHPIc), a maternally inherited rare disease. Gα is encoded by an imprinted gene, resulting in the mutant form of Gα being the only version of the protein present in certain tissues, which leads to tissue-specific disease manifestations. By integrating data from three-dimensional structures, GPCR-G protein coupling specificity, transcriptomics, biophysics, and molecular dynamics with systems pharmacology modeling, we identified GPCRs whose signaling could be altered by Gα mutations in the kidney, a tissue involved in the pathophysiology of PHPIc. Analysis of G protein activation by the parathyroid hormone receptor 1 (PTH1R) revealed that L388R impaired Gα interaction with the receptor, whereas E392K reduced the receptor-induced activation of heterotrimeric G. This indicates that different signal transduction steps can be altered by specific Gα mutants associated with the same disease. These findings highlight the importance of investigating mutation-specific perturbations in GPCR signaling to suggest patient-specific treatment strategies. Furthermore, our methods provide a blueprint for interrogating GPCR signaling diversity in different physiological and pathophysiological contexts.
The extension of lamellipodia, which are thin, fanlike projections at the cell periphery, requires the assembly of branched actin networks under the control of the small GTPase Rac1. In melanoma, a hyperactive P29S Rac1...The extension of lamellipodia, which are thin, fanlike projections at the cell periphery, requires the assembly of branched actin networks under the control of the small GTPase Rac1. In melanoma, a hyperactive P29S Rac1 mutant is associated with resistance to inhibitors that target the kinases BRAF and MAPK and with more aggressive disease because it sequesters and inactivates the tumor suppressor merlin (encoded by ) inside abnormally large lamellipodia. Here, we investigated how these merlin-inactivating lamellipodia are maintained using quantitative, live cell imaging of cell morphology and signaling dynamics. We showed that Rac1 and merlin activity were regulated in spatially confined regions or microdomains within the lamellipodium. The role of merlin as a proliferation-limiting tumor suppressor required its ability to inhibit lamellipodial extension and to locally inhibit Rac1 signaling. Conversely, local inactivation of merlin in lamellipodia released these restraints on morphology and signaling, leading to enhanced proliferation. Merlin and Rac1 are thus in a morphologically and dynamically regulated double-negative feedback loop, a signaling motif that can amplify and stabilize modest stimuli of lamellipodia extensions that enable melanoma to sustain mitogenic signaling under growth challenge. This represents an example of how acute oncogenicity is promoted by collaborations between cell morphological programs and biochemical signaling.
Schebb NH, Kampschulte N, Hagn G
… +92 more, Plitzko K, Meckelmann SW, Ghosh S, Joshi R, Kuligowski J, Vuckovic D, Botana MT, Sánchez-Illana Á, Zandkarimi F, Das A, Yang J, Schmidt L, Checa A, Roche HM, Armando AM, Edin ML, Lih FB, Aristizabal-Henao JJ, Miyamoto S, Giuffrida F, Moussaieff A, Domingues R, Rothe M, Hinz C, Das US, Rund KM, Taha AY, Hofstetter RK, Werner M, Werz O, Kahnt AS, Bertrand-Michel J, Le Faouder P, Gurke R, Thomas D, Torta F, Milic I, Dias IHK, Spickett CM, Biagini D, Lomonaco T, Idborg H, Liu JY, Fedorova M, Ford DA, Barden A, Mori TA, Kennedy PD, Maxey K, Ivanisevic J, Gallart-Ayala H, Gladine C, Wenk M, Galano JM, Durand T, Stark KD, Barbas C, Garscha U, Gelhaus SL, Ceglarek U, Flamand N, Griffin JL, Ahrends R, Arita M, Zeldin DC, Schopfer FJ, Quehenberger O, Julian R, Nicolaou A, Blair IA, Murphy MP, Hammock BD, Freeman B, Liebisch G, Serhan CN, Köfeler HC, Jakobsson PJ, Steinhilber D, Gelb MH, Holčapek M, Andrew R, Giera M, FitzGerald GA, Murphy RC, Newman JW, Dennis EA, Ekroos K, Milne GL, Gijón MA, Vesper HW, Wheelock CE, O'Donnell VB
Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenge...Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenges. As members of the lipidomics community, we developed technical recommendations to ensure best practices when quantifying oxylipins by LC-MS.
In this issue of , Houtekamer . report a mechanism by which adherens junctions enable epithelia to respond to mechanical stress. They demonstrate that E-cadherin-containing adhesions stimulate ERK signaling in response t...In this issue of , Houtekamer . report a mechanism by which adherens junctions enable epithelia to respond to mechanical stress. They demonstrate that E-cadherin-containing adhesions stimulate ERK signaling in response to tissue tension through a metalloproteinase pathway that releases EGF receptor ligands.
Chronic stress impairs intestinal stem cell function through the parasympathetic nervous system.Chronic stress impairs intestinal stem cell function through the parasympathetic nervous system.
Houtekamer RM, van der Net MC, Vliem MJ
… +12 more, Noordzij TEJC, van Uden L, van Es RM, Sim JY, Deguchi E, Terai K, Hopcroft MA, Vos HR, Pruitt BL, Matsuda M, Pannekoek WJ, Gloerich M
The behavior of cells is governed by signals originating from their local environment, including mechanical forces exerted on the cells. Forces are transduced by mechanosensitive proteins, which can impinge on signaling...The behavior of cells is governed by signals originating from their local environment, including mechanical forces exerted on the cells. Forces are transduced by mechanosensitive proteins, which can impinge on signaling cascades that are also activated by growth factors. We investigated the cross-talk between mechanical and biochemical signals in the regulation of intracellular signaling networks in epithelial monolayers. Phosphoproteomic and transcriptomic analyses on epithelial monolayers subjected to mechanical strain revealed the activation of extracellular signal-regulated kinase (ERK) downstream of the epidermal growth factor receptor (EGFR) as a predominant strain-induced signaling event. Strain-induced EGFR-ERK signaling depended on mechanosensitive E-cadherin adhesions. Proximity labeling showed that the metalloproteinase ADAM17, an enzyme that mediates shedding of soluble EGFR ligands, was closely associated with E-cadherin. A probe that we developed to monitor ADAM-mediated shedding demonstrated that mechanical strain induced ADAM activation. Mechanically induced ADAM activation was essential for mechanosensitive, E-cadherin-dependent EGFR-ERK signaling. Together, our data demonstrate that mechanical strain transduced by E-cadherin adhesion triggers the shedding of EGFR ligands that stimulate downstream ERK activity. Our findings illustrate how mechanical signals and biochemical ligands can operate within a linear signaling cascade.
Homeostatic synaptic plasticity is a negative feedback mechanism through which neurons modify their synaptic strength to counteract chronic increases or decreases in activity. In response to activity deprivation, synapti...Homeostatic synaptic plasticity is a negative feedback mechanism through which neurons modify their synaptic strength to counteract chronic increases or decreases in activity. In response to activity deprivation, synaptic strength is enhanced by increasing the number of AMPA receptors (AMPARs), particularly Ca-permeable AMPARs, at the synapse. Here, we found that this increase in Ca-permeable AMPARs during homeostatic upscaling was mediated by decreased posttranscriptional editing of mRNA encoding the AMPAR subunit GluA2. In cultured neurons, activity deprivation resulted in increases in the amount of unedited GluA2, such that its ion channel pore contains a glutamine (Q) codon instead of arginine (R), and in the number of Ca-permeable AMPARs at the synapse. These effects were mediated by a splicing factor-dependent decrease in ADAR2 abundance and activity in the nucleus. Overexpression of ADAR2 or CRISPR-Cas13-directed editing of GluA2 transcripts blocked homeostatic upscaling in activity-deprived primary neurons. In mice, dark rearing resulted in decreased Q-to-R editing of GluA2-encoding transcripts in the primary visual cortex (V1), and viral overexpression of ADAR2 in the V1 blocked the induction of homeostatic synaptic plasticity. The findings indicate that activity-dependent regulation of GluA2 editing contributes to homeostatic synaptic plasticity.
Specialized intramembrane proteases, known as iCLiPs, regulate the processing of transmembrane proteins by releasing intracellular domains, which can function as transcriptional regulators. The signal peptide peptidase-l...Specialized intramembrane proteases, known as iCLiPs, regulate the processing of transmembrane proteins by releasing intracellular domains, which can function as transcriptional regulators. The signal peptide peptidase-like (SPPL) family of iCLiPs, particularly SPPL2b, has roles in immune regulation, neuronal function, and disease pathogenesis. In the brain, SPPL2b localizes mainly in the plasma membrane of neurons and microglia and is abundant in the cortex and hippocampus. Its known substrates regulate neuronal growth, inflammation, and synaptic function, and increased amounts of SPPL2b have been found in postmortem brain tissue from patients with Alzheimer's disease. In this review, we discuss the currently known roles of SPPL2b, its substrates, and its disease implications. Understanding the downstream effects of SPPL2b-cleaved substrates will provide clearer insights into the impact of SPPL2b on cellular homeostasis and disease, potentially leading to new therapeutic strategies.
Bacterial serine-threonine kinases (STKs) regulate diverse cellular processes associated with cell growth, virulence, and pathogenicity and are evolutionarily related to the druggable eukaryotic STKs. A deeper understand...Bacterial serine-threonine kinases (STKs) regulate diverse cellular processes associated with cell growth, virulence, and pathogenicity and are evolutionarily related to the druggable eukaryotic STKs. A deeper understanding of how bacterial STKs differ from their eukaryotic counterparts and how they have evolved to regulate diverse bacterial signaling functions is crucial for advancing the discovery and development of new antibiotic therapies. Here, we classified more than 300,000 bacterial STK sequences from the NCBI RefSeq nonredundant and UniProt protein databases into 35 canonical and seven pseudokinase families on the basis of the patterns of evolutionary constraints in the conserved catalytic domain and flanking regulatory domains. Through statistical comparisons, we identified features distinguishing bacterial STKs from eukaryotic STKs, including an arginine residue in a regulatory helix (C helix) that dynamically couples the ATP- and substrate-binding lobes of the kinase domain. Biochemical and peptide library screens demonstrated that evolutionarily constrained residues contributed to substrate specificity and kinase activation in the kinase PknB. Together, these findings open previously unidentified avenues for investigating bacterial STK functions in cellular signaling and for developing selective bacterial STK inhibitors.