RESUMO
Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Masculino , Células PC-3 , Receptores Acoplados a Proteínas G/genéticaRESUMO
Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.
Assuntos
Anafilaxia , Fibroblastos , Lisofosfolipídeos , Mastócitos , Camundongos Knockout , Comunicação Parácrina , Diester Fosfórico Hidrolases , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais , Animais , Mastócitos/imunologia , Mastócitos/metabolismo , Anafilaxia/imunologia , Anafilaxia/metabolismo , Camundongos , Fibroblastos/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Prostaglandina D2/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/genética , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/genética , Diferenciação Celular , Camundongos Endogâmicos C57BL , Proteína 1 Semelhante a Receptor de Interleucina-1 , LipocalinasRESUMO
Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of ß-glucosylceramide (ß-GlcCer). However, it remains unclear how ß-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that ß-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD.
Assuntos
Doença de Gaucher , Camundongos , Animais , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glucosilceramidase/uso terapêutico , Glucosilceramidas/metabolismo , Glucosilceramidas/uso terapêutico , Microglia/metabolismo , Neurônios/metabolismo , FagocitoseRESUMO
About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.
Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Processamento Pós-Transcricional do RNA , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais , Adenosina/genética , Adenosina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Coelhos , Receptor A3 de Adenosina/genéticaRESUMO
Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fosfolipase C beta/genética , Cálcio/metabolismo , Sinalização do Cálcio/genética , Citosol/metabolismo , Células HEK293 , Humanos , Ligação Proteica/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genéticaRESUMO
In addition to direct activation by pathogens and antigens, immune cell functions are further modulated by factors in their environment. Recent studies have revealed that lysophospholipids (LPL) derived from membrane glycerophospholipids are such environmental factors. They are produced by the action of various phospholipases and modulate immune responses positively or negatively via G-protein-coupled receptor-type receptors. These include lysophosphatidic acid, lysophosphatidylserine (LysoPS), and lysophosphatidylinositol. Here, we summarize what is known about the synthetic pathways, receptors, and immunomodulatory functions of these LPLs. Particular focus is given to LysoPS, which have recently been identified, and recent findings on their immunomodulatory actions are presented.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Lisofosfolipídeos/metabolismoRESUMO
BACKGROUND AND AIMS: The common genetic variant rs641738 C>T is a risk factor for metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis (MASH), including liver fibrosis, and is associated with decreased expression of the phospholipid-remodeling enzyme MBOAT7 (LPIAT1). However, whether restoring MBOAT7 expression in established metabolic dysfunction-associated steatotic liver disease dampens the progression to liver fibrosis and, importantly, the mechanism through which decreased MBOAT7 expression exacerbates MASH fibrosis remain unclear. APPROACH AND RESULTS: We first showed that hepatocyte MBOAT7 restoration in mice with diet-induced steatohepatitis slows the progression to liver fibrosis. Conversely, when hepatocyte-MBOAT7 was silenced in mice with established hepatosteatosis, liver fibrosis but not hepatosteatosis was exacerbated. Mechanistic studies revealed that hepatocyte-MBOAT7 restoration in MASH mice lowered hepatocyte-TAZ (WWTR1), which is known to promote MASH fibrosis. Conversely, hepatocyte-MBOAT7 silencing enhanced TAZ upregulation in MASH. Finally, we discovered that changes in hepatocyte phospholipids due to MBOAT7 loss-of-function promote a cholesterol trafficking pathway that upregulates TAZ and the TAZ-induced profibrotic factor Indian hedgehog (IHH). As evidence for relevance in humans, we found that the livers of individuals with MASH carrying the rs641738-T allele had higher hepatocyte nuclear TAZ, indicating higher TAZ activity and increased IHH mRNA. CONCLUSIONS: This study provides evidence for a novel mechanism linking MBOAT7-LoF to MASH fibrosis, adds new insight into an established genetic locus for MASH, and, given the druggability of hepatocyte TAZ for MASH fibrosis, suggests a personalized medicine approach for subjects at increased risk for MASH fibrosis due to inheritance of variants that lower MBOAT7.
RESUMO
Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of airway obstruction, hyperresponsiveness, remodeling, and eosinophilia. Phospholipase A2 s (PLA2 s), which release fatty acids and lysophospholipids from membrane phospholipids, have been implicated in exacerbating asthma by generating pro-asthmatic lipid mediators, but an understanding of the association between individual PLA2 subtypes and asthma is still incomplete. Here, we show that group III-secreted PLA2 (sPLA2 -III) plays an ameliorating, rather than aggravating, role in asthma pathology. In both mouse and human lungs, sPLA2 -III was expressed in bronchial epithelial cells and decreased during the asthmatic response. In an ovalbumin (OVA)-induced asthma model, Pla2g3-/- mice exhibited enhanced airway hyperresponsiveness, eosinophilia, OVA-specific IgE production, and type 2 cytokine expression as compared to Pla2g3+/+ mice. Lipidomics analysis showed that the pulmonary levels of several lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (LPA), were decreased in OVA-challenged Pla2g3-/- mice relative to Pla2g3+/+ mice. LPA receptor 2 (LPA2 ) agonists suppressed thymic stromal lymphopoietin (TSLP) expression in bronchial epithelial cells and reversed airway hyperresponsiveness and eosinophilia in Pla2g3-/- mice, suggesting that sPLA2 -III negatively regulates allergen-induced asthma at least by producing LPA. Thus, the activation of the sPLA2 -III-LPA pathway may be a new therapeutic target for allergic asthma.
Assuntos
Asma , Eosinofilia , Fosfolipases A2 Secretórias , Hipersensibilidade Respiratória , Humanos , Animais , Camundongos , Lisofosfolipídeos , Fosfolipases A2 Secretórias/genética , CitocinasRESUMO
Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.
Assuntos
Bactérias/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Extensões da Superfície Celular/metabolismo , Intestino Delgado/citologia , Intestino Delgado/microbiologia , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bactérias/imunologia , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/imunologia , Feminino , Células HEK293 , Humanos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Ácido Láctico/farmacologia , Lactobacillus helveticus/metabolismo , Masculino , Metanol , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ácido Pirúvico/farmacologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Salmonella/imunologia , Salmonella/metabolismoRESUMO
Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Receptores de Neurotensina/química , Receptores de Neurotensina/ultraestrutura , Sítios de Ligação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/metabolismoRESUMO
Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.
Assuntos
Fibras Musculares de Contração Rápida , Músculo Esquelético , Fosfolipídeos , Animais , Camundongos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/química , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Estearatos/metabolismo , Plasmalogênios , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMO
Functional mass spectrometry imaging (fMSI) is a potent tool for elucidating the spatial distribution of enzyme activities in tissues at high resolution. In this study, we applied fMSI to probe the intricate biosynthesis of phospholipids, which exist as thousands of molecular species in tissues and exhibit a unique distribution specific to cell type. By using deuterium- and 13C-labeled substrates, we visualized the activities of key enzymes involved in phospholipid synthesis, including glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferases (LPAAT), lysophospholipid acyltransferases (LPLAT), and long-chain acyl-CoA synthetase (ACSL). Additionally, we were able to visualize a two-step sequential enzyme reaction involving ACSL and LPLAT. This novel approach unveiled significant variations in enzyme activity distribution depending on the type of fatty acids used as substrates. It will also help to reveal the mechanisms underlying the formation of numerous phospholipid species.
Assuntos
Espectrometria de Massas , Fosfolipídeos , Fosfolipídeos/metabolismo , Fosfolipídeos/análise , Animais , CamundongosRESUMO
Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.
Assuntos
Cromolina Sódica , Estabilizadores de Mastócitos , Ratos , Camundongos , Animais , Cromolina Sódica/farmacologia , Estabilizadores de Mastócitos/farmacologia , Mastócitos , Receptores Acoplados a Proteínas G/metabolismo , Imunoglobulina E/metabolismo , Imunoglobulina E/farmacologia , Degranulação CelularRESUMO
G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310 . Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308 , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of ß-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2-GRK pathway, resulting in a ß-arrestin-bound low-affinity state. This configuration, referred to as the "low-LTB4 -induced complex," necessitates the finger loop region and the phosphoinositide-binding motif of ß-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB4 -induced complex into a different form termed "high-LTB4 -induced complex." This change is propelled by The308 -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, ß-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein ß-arrestins perform distinct functions.
Assuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Fosforilação , beta-Arrestinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligantes , beta-Arrestina 1/metabolismo , Receptores do Leucotrieno B4/metabolismo , Leucotrieno B4/metabolismoRESUMO
The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.
Assuntos
Ácidos Graxos Voláteis , Leucócitos , Receptores de Superfície Celular , Humanos , Ácidos Graxos Voláteis/metabolismo , Leucócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Diferenciação Celular , Células HL-60RESUMO
Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.
Assuntos
Proteoglicanas/metabolismo , Sinapses/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Eletroencefalografia , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Proteoglicanas/análise , Proteoglicanas/genética , Receptores de AMPA/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genéticaRESUMO
Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.
Assuntos
Gangliosídeo G(M3) , Melanoma , Humanos , Gangliosídeo G(M3)/metabolismo , Membrana Celular/metabolismo , Anticorpos Monoclonais , Melanoma/metabolismo , Contagem de CélulasRESUMO
Activated microglia contribute to many neuroinflammatory diseases in the central nervous system. In this study, we attempted to identify an anti-inflammatory compound that could suppress microglial activation. We performed high-throughput screening with a chemical library developed at our institute. We performed a luciferase assay of nuclear factor-kappa B (NF-κB) reporter stable HT22 cells and identified a compound that was confirmed to inhibit the anti-inflammatory response in BV2 microglial cells. The selected dihydropyridine derivative can suppress the expression response of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), as well as NF-κB phosphorylation and nuclear translocation, and reduce the intracellular calcium level. Thus, our identified compound has a potential role in suppressing microglial activation and may contribute to the development of a new therapeutic molecule against neuroinflammatory diseases.
Assuntos
Cálcio , Di-Hidropiridinas , Microglia , NF-kappa B , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Camundongos , NF-kappa B/metabolismo , Cálcio/metabolismo , Linhagem Celular , Di-Hidropiridinas/farmacologia , Fosforilação/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacosRESUMO
Phosphatidylserine (PS) is an acidic phospholipid that is involved in various cellular events. Heterologous dominant mutations have been identified in the gene encoding PS synthase 1 (PSS1) in patients with a congenital disease called Lenz-Majewski syndrome (LMS). Patients with LMS show various symptoms, including craniofacial/distal-limb bone dysplasia and progressive hyperostosis. The LMS-causing gain-of-function mutants of PSS1 (PSS1LMS) have been shown to synthesize PS without control, but why the uncontrolled synthesis would lead to LMS is unknown. Here we investigated the effect of PSS1LMS on osteoclasts (OCs) to elucidate the causative mechanism of LMS. PSS1LMS did not affect the expression of OC-related genes but inhibited the formation, multinucleation, and activity of OCs. Especially, OCs expressing PSS1LMS showed abnormal patterns and dynamics of actin podosome clusters, which have roles in OC migration and fusion. PSS1LMS did not affect the level of PS but changed the acyl chain compositions of PS and phosphatidylethanolamine, and decreased the level of phosphatidylinositol. The introduction of a catalytically inactive mutation into PSSLMS canceled the changes in phospholipids and the phenotypes observed in OCs expressing PSS1LMS. A gain-of-function mutant of PSS2 (PSS2 R97K) also impaired OC formation and caused changes in phospholipid composition similar to the changes caused by PSS1LMS. Our results suggest that uncontrolled PS synthesis by PSS1LMS causes changes in the quantity or fatty acid composition of certain phospholipid classes, impairing OC formation and function, which might be a cause of osteosclerosis in patients with LMS.
Assuntos
Anormalidades Múltiplas , Deficiência Intelectual , Humanos , Osteoclastos/metabolismo , Fosfolipídeos/metabolismoRESUMO
The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.