RESUMO
The use of alkaline salt lands for crop production is hindered by a scarcity of knowledge and breeding efforts for plant alkaline tolerance. Through genome association analysis of sorghum, a naturally high-alkaline-tolerant crop, we detected a major locus, Alkaline Tolerance 1 (AT1), specifically related to alkaline-salinity sensitivity. An at1 allele with a carboxyl-terminal truncation increased sensitivity, whereas knockout of AT1 increased tolerance to alkalinity in sorghum, millet, rice, and maize. AT1 encodes an atypical G protein γ subunit that affects the phosphorylation of aquaporins to modulate the distribution of hydrogen peroxide (H2O2). These processes appear to protect plants against oxidative stress by alkali. Designing knockouts of AT1 homologs or selecting its natural nonfunctional alleles could improve crop productivity in sodic lands.
Assuntos
Álcalis , Produtos Agrícolas , Subunidades gama da Proteína de Ligação ao GTP , Proteínas de Plantas , Tolerância ao Sal , Sorghum , Produtos Agrícolas/genética , Produtos Agrícolas/fisiologia , Peróxido de Hidrogênio/metabolismo , Oryza/genética , Oryza/fisiologia , Estresse Oxidativo/genética , Melhoramento Vegetal , Salinidade , Álcalis/análise , Álcalis/toxicidade , Bicarbonato de Sódio/análise , Bicarbonato de Sódio/toxicidade , Carbonatos/análise , Carbonatos/toxicidade , Tolerância ao Sal/genética , Sorghum/genética , Sorghum/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Aquaporinas/metabolismo , Produção Agrícola , Loci Gênicos , Solo/químicaRESUMO
Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.
Assuntos
Adipócitos/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Tecido Adiposo/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Metabolismo Energético/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/deficiência , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mutations affecting the BSCL2 gene cause the most severe form of congenital generalised lipodystrophy (CGL). Affected individuals develop severe metabolic complications including diabetes and hepatic steatosis. Bscl2-deficient mice almost entirely reproduce the CGL phenotype. Adipose tissue-specific loss of Bscl2 is also sufficient to cause early-onset generalised lipodystrophy in mice. However, these mice do not show severe metabolic dysfunction, even when challenged with a high-fat diet. Germline Bscl2 loss in mice and BSCL2 disruption in humans causes severe hepatic steatosis, and the encoded protein, seipin, has acknowledged roles in lipid accumulation. Given the critical role of the liver in glucose regulation, we speculated that intact hepatic Bscl2 expression may protect adipose tissue-specific Bscl2-deficient mice from metabolic disease. To investigate this, we generated a novel mouse model in which Bscl2 has been deleted in both adipose tissue and hepatocytes simultaneously using an adeno-associated viral vector. Despite achieving efficient disruption of Bscl2 in the liver, hepatic lipid accumulation and metabolic homeostasis was unaffected in mice fed a high-fat diet for 4 weeks. We also investigated the consequences of BSCL2 ablation in the human hepatocyte HepG2 cell line using CRISPR/Cas9 genome editing. No significant increases in lipid accumulation were observed in BSCL2 knockout cell lines. Overall, we reveal that Bscl2/BSCL2 does not appear to play a cell-autonomous role in the regulation of lipid accumulation in the liver. Loss of hepatic BSCL2 is therefore unlikely to contribute significantly to the development of hepatic steatosis or metabolic dysfunction in this form of CGL.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Lipodistrofia Generalizada Congênita/metabolismo , Tecido Adiposo/metabolismo , Animais , Feminino , Células Hep G2 , Humanos , Masculino , CamundongosRESUMO
Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is the most severe form of human lipodystrophy and is caused by loss-of-function mutations in the BSCL2/seipin gene. Exactly how seipin may regulate adipogenesis remains unclear. A recent study in vitro suggested that seipin may function to inhibit the activity of glycerol-3-phosphate acyltransferases (GPATs), and increased GPAT activity may be responsible for the defective adipogenesis under seipin deficiency. Here we generated Seipin-/-Gpat3-/- mice, which had mild but significant recovery of white adipose tissue mass over Seipin-/- mice. The mass of brown adipose tissue (BAT) of the Seipin-/-Gpat3-/- mice was almost completely restored to normal level. Importantly, the Seipin-/-Gpat3-/- mice showed significant improvement in liver steatosis and insulin sensitivity over Seipin-/- mice, which is attributable to the increased BAT mass and to the enhanced browning of the subcutaneous fat of the Seipin-/-Gpat3-/- mice. Together, our results establish a functional link between seipin and GPAT3 in vivo and suggest that GPAT inhibitors may have beneficial effects on BSCL2 patients.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/fisiologia , Adipogenia , Modelos Animais de Doenças , Fígado Gorduroso/prevenção & controle , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Resistência à Insulina , Lipodistrofia Generalizada Congênita/complicações , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gßγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gßγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gßγ sink ßARK1-ct (C terminus of ß-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gßγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gßγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gßγ subunits and TRPM3. Since Gßγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gßγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gßγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gßγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Comportamento Animal , Feminino , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Hiperalgesia/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Nociceptores/efeitos dos fármacos , Toxina Pertussis/farmacologia , Receptor A2B de Adenosina/fisiologia , Receptor Muscarínico M1/fisiologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genéticaRESUMO
Heterotrimeric G proteins, which consist of Gα , Gß and Gγ subunits, function as molecular switches that regulate a wide range of developmental processes in plants. In this study, we characterised the function of rice RGG2, which encodes a type B Gγ subunit, in regulating grain size and yield production. The expression levels of RGG2 were significantly higher than those of other rice Gγ -encoding genes in all tissues tested, suggesting that RGG2 plays essential roles in rice growth and development. By regulating cell expansion, overexpression of RGG2 in Nipponbare (NIP) led to reduced plant height and decreased grain size. By contrast, two mutants generated by the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system in the Zhenshan 97 (ZS97) background, zrgg2-1 and zrgg2-2, exhibited enhanced growth, including elongated internodes, increased 1000-grain weight and plant biomass and enhanced grain yield per plant (+11.8% and 16.0%, respectively). These results demonstrate that RGG2 acts as a negative regulator of plant growth and organ size in rice. By measuring the length of the second leaf sheath after gibberellin (GA3 ) treatment and the GA-induced α-amylase activity of seeds, we found that RGG2 is also involved in GA signalling. In summary, we propose that RGG2 may regulate grain and organ size via the GA pathway and that manipulation of RGG2 may provide a novel strategy for rice grain yield enhancement.
Assuntos
Grão Comestível/crescimento & desenvolvimento , Subunidades gama da Proteína de Ligação ao GTP/genética , Oryza/genética , Proteínas de Plantas/genética , Sistemas CRISPR-Cas , Grão Comestível/genética , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Edição de Genes/métodos , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
Seipin is a widely expressed protein but with highest levels found in the brain and testes. Seipin function is not yet completely understood, therefore the aim of this study was to evaluate the expression of BSCL2 transcripts in the central nervous system (CNS) of humans and investigate the effect of their overexpression on a neuron model and their relationship with oxidative stress protection, as well as shed light on the pathogenic mechanisms of Celia's Encephalopathy. We analyzed the expression of BSCL2 transcripts using real-time RT-PCR in samples across the brain regions of subjects who underwent necropsy and from a case with Celia's Encephalopathy. The transcript encoding the long seipin isoform (BSCL2-203, 462 aa) is expressed primarily in the brain and its expression is inversely correlated with age in the temporal lobe, amygdala, and hypothalamus. Strong positive correlations were found between BSCL2 expression and some genes encoding protective enzymes against oxidative stress including SOD1 and SOD2, as well as peroxisome proliferator-activated receptor gamma (PPARG) in the amygdala. These results were experimentally corroborated by overexpressing BSCL2 transcripts in SH-SY5Y cells with lentiviral transduction and assessing their effects on neuron differentiated cells. Confocal microscopy studies showed that both seipin and PEX16 are closely expressed in the hypothalami of healthy human brains, and PEX16 was absent in the same region of the PELD case. We hypothesize that seipin has specific CNS functions and may play a role in peroxisome biogenesis.
Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Estresse Oxidativo , Peroxissomos/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Autopsia , Linhagem Celular Tumoral , Feminino , Subunidades gama da Proteína de Ligação ao GTP/biossíntese , Humanos , Masculino , Proteínas de Membrana/biossíntese , Pessoa de Meia-Idade , PPAR gama/biossíntese , Isoformas de Proteínas/biossíntese , Fatores Sexuais , Superóxido Dismutase/biossíntese , Superóxido Dismutase-1/biossíntese , Regulação para Cima , Adulto JovemRESUMO
The biogenesis of lipid droplets (LDs) and the development of adipocytes are two key aspects of mammalian fat storage. SEIPIN, an integral membrane protein of the endoplasmic reticulum (ER), plays a critical role in both LD formation and adipogenesis. The molecular function of SEIPIN, however, has yet to be elucidated. Here, we report the cryogenic electron microscopy structure of human SEIPIN at 3.8 Å resolution. SEIPIN exists as an undecamer, and this oligomerization state is critical for its physiological function. The evolutionarily conserved lumenal domain of SEIPIN forms an eight-stranded ß sandwich fold. Both full-length SEIPIN and its lumenal domain can bind anionic phospholipids including phosphatidic acid. Our results suggest that SEIPIN forms a scaffold that helps maintain phospholipid homeostasis and surface tension of the ER.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Gotículas Lipídicas/metabolismo , Adipócitos/metabolismo , Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Microscopia Crioeletrônica/métodos , Retículo Endoplasmático/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , FosfolipídeosRESUMO
Objective- In renal arteries, inhibitors of G protein ßγ subunits (Gßγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gßγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gßγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein ß subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gßγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gßγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Canais de Potássio KCNQ/fisiologia , Músculo Liso Vascular/fisiologia , Vasodilatação , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/fisiologia , Isoproterenol/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos Wistar , Artéria Renal/efeitos dos fármacos , Artéria Renal/fisiologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Xantenos/farmacologiaRESUMO
The oomycete Phytophthora infestans is a notorious plant pathogen with potato and tomato as its primary hosts. Previous research showed that the heterotrimeric G-protein subunits Gα and Gß have a role in zoospore motility and virulence, and sporangial development, respectively. Here, we present analyses of the gene encoding a Gγ subunit in P. infestans, Pigpg1. The overall similarity of PiGPG1 with non-oomycete Gγ subunits is low, with only the most conserved amino acids maintained, but similarity with its homologs in other oomycetes is high. Pigpg1 is expressed in all life stages and shows a similar expression profile as the gene encoding the Gß subunit, Pigpb1. To elucidate its function, transformants were generated in which Pigpg1 is silenced or overexpressed and their phenotypes were analyzed. Pigpg1-silenced lines produce less sporangia, which are malformed. Altogether, the results show that PiGPG1 is crucial for proper sporangia development and zoosporogenesis. PiGPG1 is a functional Gγ, and likely forms a dimer with PiGPB1 that mediates signaling.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Phytophthora infestans/crescimento & desenvolvimento , Esporângios/crescimento & desenvolvimento , Sequência Conservada , Subunidades gama da Proteína de Ligação ao GTP/genética , Micélio/metabolismo , Phytophthora infestans/genética , Interferência de RNA , Esporângios/genética , Esporos/metabolismoRESUMO
KEY POINTS: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. We found that the G-protein-gated inwardly rectifying K+ (GIRK) channel, especially GIRK2, is activated by IVM directly in a Gßγ -independent manner, but the activation is dependent on phosphatidylinositol-4,5-biphosphate (PIP2 ). We identified a critical amino acid residue of GIRK2 for activation by IVM, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD). The results demonstrate that the TM-CTD interface in GIRK channel, rather than the TMs, governs IVM-mediated activation and provide us with novel insights on the mode of action of IVM in ion channels. ABSTRACT: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. In this study, we found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. Electrophysiological recordings in Xenopus oocytes revealed that IVM activates GIRK channel in a phosphatidylinositol-4,5-biphosphate (PIP2 )-dependent manner, and that the IVM-mediated GIRK activation is independent of Gßγ subunits. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. Chimeric and mutagenesis analyses identified an amino acid residue unique to GIRK2 among the GIRK family, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM-CTD interface in GIRK channels, rather than the TMs, governs IVM-mediated activation. These findings provide us with novel insights on the mode of action of IVM in ion channels that could lead to identification of new pharmacophores which activate the GIRK channel.
Assuntos
Antiparasitários/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Ivermectina/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Ratos Wistar , Xenopus laevisRESUMO
KEY POINTS: The ascending brainstem transmitter acetylcholine depolarizes thalamocortical relay neurons while it induces hyperpolarization in local circuit inhibitory interneurons. Sustained K+ currents are modulated in thalamic neurons to control their activity modes; for the interneurons the molecular nature of the underlying ion channels is as yet unknown. Activation of TASK-1 K+ channels results in hyperpolarization of interneurons and suppression of their action potential firing. The modulation cascade involves a non-receptor tyrosine kinase, c-Src. The present study identifies a novel pathway for the activation of TASK-1 channels in CNS neurons that resembles cholinergic signalling and TASK-1 current modulation during hypoxia in smooth muscle cells. ABSTRACT: The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K2P ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein ßγ subunit (Gßγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cß as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons.
Assuntos
Acetilcolina/fisiologia , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Tálamo/fisiologia , Quinases da Família src/fisiologia , Animais , Proteína Tirosina Quinase CSK , Feminino , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Masculino , Camundongos Transgênicos , Agonistas Muscarínicos/farmacologia , Proteínas do Tecido Nervoso/genética , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/fisiologia , Canais de Potássio de Domínios Poros em Tandem/genética , Receptores Muscarínicos/fisiologia , Transdução de Sinais , Regulação para CimaRESUMO
Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein ßγ-subunit (Gßγ), GPCR-kinase 2, and ß-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gßγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gßγ signaling and ET system expression. Notably, systemic pharmacologic Gßγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gßγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gßγ in AKI and suggest GPCR-Gßγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.
Assuntos
Síndrome Cardiorrenal/etiologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Insuficiência Cardíaca/complicações , Rim/patologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Fibrose/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisAssuntos
Subunidades gama da Proteína de Ligação ao GTP/genética , Doença dos Neurônios Motores/genética , Idade de Início , Criança , Progressão da Doença , Eletromiografia , Feminino , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Transtornos Neurológicos da Marcha/etiologia , Heterozigoto , Humanos , Deficiência Intelectual/etiologia , Deficiências da Aprendizagem/etiologia , Doença dos Neurônios Motores/complicações , Transtornos das Habilidades Motoras/etiologia , Atrofia Muscular/etiologia , MutaçãoRESUMO
Heterotrimeric G proteins are intracellular membrane-attached signal transducers involved in various cellular processes in both plants and animals. They consist of three subunits denoted as α, ß and γ. The γ-subunits of the so-called AGG3 type, which comprise a transmembrane domain, are exclusively found in plants. In model species, these proteins have been shown to participate in the control of plant height, branching and seed size and could therefore impact the harvestable yield of various crop plants. Whether AGG3-type γ-subunits influence yield in temperate cereals like barley and wheat remains unknown. Using a transgenic complementation approach, we show here that the Scottish malting barley cultivar (cv.) Golden Promise carries a loss-of-function mutation in HvDep1, an AGG3-type subunit encoding gene that positively regulates culm elongation and seed size in barley. Somewhat intriguingly, agronomic field data collected over a 12-year period reveals that the HvDep1 loss-of-function mutation in cv. Golden Promise has the potential to confer either a significant increase or decrease in harvestable yield depending on the environment. Our results confirm the role of AGG3-type subunit-encoding genes in shaping plant architecture, but interestingly also indicate that the impact HvDep1 has on yield in barley is both genotypically and environmentally sensitive. This may explain why widespread exploitation of variation in AGG3-type subunit-encoding genes has not occurred in temperate cereals while in rice the DEP1 locus is widely exploited to improve harvestable yield.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Hordeum/genética , Proteínas de Plantas/fisiologia , Mapeamento Cromossômico , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Estudos de Associação Genética , Genótipo , Hordeum/crescimento & desenvolvimento , Mutação , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Locos de Características Quantitativas , Sementes/anatomia & histologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Transdução de SinaisRESUMO
OBJECTIVE: Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein ßγ subunit (Gßγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gßγ signaling ameliorates disease in a mouse model of SLE. METHODS: Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gßγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay. RESULTS: Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation. CONCLUSION: Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gßγ signaling in SLE treatment.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/prevenção & controle , Nefrite/prevenção & controle , Xantenos/uso terapêutico , Animais , Feminino , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos NZB , Nefrite/imunologia , Transdução de SinaisRESUMO
Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by µ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human µ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of µ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via µ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein ßγ (Gßγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[ß-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gßγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gßγ subunits in a voltage-independent manner.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo R/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Receptores Opioides delta/fisiologia , Receptores Opioides kappa/fisiologia , Receptores Opioides mu/fisiologia , Analgésicos Opioides/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Células HEK293 , Humanos , Subunidades Proteicas/fisiologia , Receptores Opioides delta/agonistas , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistasRESUMO
Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER-LD contact sites.
Assuntos
Retículo Endoplasmático/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fosfolipídeos/biossíntese , Transporte Proteico , Saccharomyces cerevisiae/ultraestruturaRESUMO
We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein ßγ subunits. Gßγ inhibition blocked ET-1-stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gßγ at the Golgi inhibited ET-1-dependent PI4P depletion and nuclear PKD activation. Translocation of Gßγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. Finally, blocking Gßγ at the Golgi or PM blocked ET-1-dependent cardiomyocyte hypertrophy. These data indicate that Gßγ regulation of the perinuclear Golgi PI4P pathway and a separate pathway at the PM is required for ET-1-stimulated hypertrophy, and the efficacy of Gßγ inhibition in preventing heart failure maybe due in part to its blocking both these pathways.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Complexo de Golgi/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Cardiomegalia/metabolismo , Células Cultivadas , Hidrólise , Fosfatos de Fosfatidilinositol , Transporte Proteico , Ratos Sprague-Dawley , Sistemas do Segundo MensageiroRESUMO
Seipin is necessary for both adipogenesis and lipid droplet (LD) organization in nonadipose tissues; however, its molecular function is incompletely understood. Phenotypes in the seipin-null mutant of Saccharomyces cerevisiae include aberrant droplet morphology (endoplasmic reticulum-droplet clusters and size heterogeneity) and sensitivity of droplet size to changes in phospholipid synthesis. It has not been clear, however, whether seipin acts in initiation of droplet synthesis or at a later step. Here we utilize a system of de novo droplet formation to show that the absence of seipin results in a delay in droplet appearance with concomitant accumulation of neutral lipid in membranes. We also demonstrate that seipin is required for vectorial budding of droplets toward the cytoplasm. Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology. Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant. We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.