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1.
Science ; 367(6484): 1346-1352, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193322

RESUMO

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Glucagon/química , Receptores de Glucagon/química , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Glucagon/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Glucagon/metabolismo , Receptores de Glucagon/ultraestrutura , Transdução de Sinais
2.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
3.
Nat Commun ; 10(1): 4093, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501438

RESUMO

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on investigating the physiological properties and behavioral relevance of larval visual interneurons. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Gαo signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system.


Assuntos
Drosophila melanogaster/fisiologia , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Vias Visuais/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Cálcio/metabolismo , Drosophila melanogaster/efeitos da radiação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Interneurônios/efeitos da radiação , Larva/efeitos da radiação , Luz , Neurópilo/metabolismo , Neurópilo/efeitos da radiação , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/efeitos da radiação
4.
Mol Pharmacol ; 96(4): 463-474, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31399503

RESUMO

G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity, and in some cases is associated with G protein-independent signaling and endosomal receptor signaling. To date, internalization has been studied in great detail for class A GPCRs; whereas it is not well established to what extent the observations can be generalized to class C GPCRs, including the extracellular calcium-sensing receptor (CaSR). The CaSR is a prototypical class C GPCR that maintains stable blood calcium (Ca2+) levels by sensing minute changes in extracellular free Ca2+ It is thus necessary that the activity of the CaSR is tightly regulated, even while continuously being exposed to its endogenous agonist. Previous studies have used overexpression of intracellular proteins involved in GPCR trafficking, pathway inhibitors, and cell-surface expression or functional desensitization as indirect measures to investigate CaSR internalization. However, there is no general consensus on the processes involved, and the mechanism of CaSR internalization remains poorly understood. The current study provides new insights into the internalization mechanism of the CaSR. We have used a state-of-the-art time-resolved fluorescence resonance energy transfer-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that the CaSR displays both constitutive and concentration-dependent Ca2+-mediated internalization. For the first time, we conclusively show that CaSR internalization is sensitive to immediate positive and negative modulation by the CaSR-specific allosteric modulators N-(3-[2-chlorophenyl]propyl)-(R)-α-methyl-3-methoxybenzylamine (NPS R-568) and 2-chloro-6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino]propoxy]benzonitrile (NPS 2143), respectively. In addition, we provide compelling evidence that CaSR internalization is ß-arrestin-dependent while interestingly being largely independent of Gq/11 and Gi/o protein signaling. SIGNIFICANCE STATEMENT: A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is ß-arrestin-dependent and sensitive to modulation by allosteric ligands.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Receptores de Detecção de Cálcio/metabolismo , beta-Arrestinas/metabolismo , Regulação Alostérica , Cálcio/sangue , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Mutação , Naftalenos/farmacologia , Fenetilaminas/farmacologia , Propilaminas/farmacologia , Transporte Proteico , Receptores de Detecção de Cálcio/genética
5.
Mol Pharmacol ; 96(2): 233-246, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31189666

RESUMO

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-ß5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-ß5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-ßγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from ßγ Such action of GPR158 is expected to prolong the ßγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética
6.
PLoS One ; 14(6): e0218110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31173603

RESUMO

PURPOSE: We previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective. METHODS: Wild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET). RESULTS: Simultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility. CONCLUSIONS: Together, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.


Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Contração Miocárdica , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Toxina Pertussis/farmacologia
7.
Nature ; 571(7764): 279-283, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168089

RESUMO

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.


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 , Oxisteróis/química , Receptor Smoothened/química , Receptor Smoothened/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Ligantes , Modelos Moleculares , Oxisteróis/metabolismo , Receptor Patched-1/metabolismo , Conformação Proteica , Transdução de Sinais , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
8.
Nature ; 572(7767): 80-85, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243364

RESUMO

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/metabolismo
9.
Nat Commun ; 10(1): 2234, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110175

RESUMO

The ß2 adrenergic receptor (ß2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study ß2AR-Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the ß2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between ß2AR and competing G protein subtypes.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Lipídeos de Membrana/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Motivos de Aminoácidos , Animais , Cátions/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Lipídeos de Membrana/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática
10.
Nat Commun ; 10(1): 2008, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043612

RESUMO

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Ativação do Canal Iônico/fisiologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
11.
Can J Physiol Pharmacol ; 97(9): 872-879, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30925226

RESUMO

Resveratrol (RV), a polyphenolic component of red wine, has been shown to attenuate high blood pressure (BP) in spontaneously hypertensive rats (SHRs). We previously found that the enhanced expression of Giα proteins plays a role in the pathogenesis of hypertension in SHRs. In the present study, we investigated whether this RV-induced decrease in BP in SHRs can be attributed to the ability of RV to inhibit the enhanced expression of Giα proteins and the upstream signaling molecules implicated in the overexpression of Giα proteins. Administration of RV (50 mg/kg per day) to prehypertensive 2-week-old SHRs for 6 weeks prevented the development of high BP and inhibited the enhanced expression of Giα proteins, the enhanced levels of superoxide anion (O2-) and NADPH oxidase activity, the enhanced activation (phosphorylation) of c-Src and growth factor receptors, as well as the enhanced levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) exhibited by vascular smooth muscle cells isolated from SHRs. In conclusion, these results indicate that RV attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Giα proteins, oxidative stress, and the upstream signaling molecules that contribute to the overexpression of Giα proteins. These findings suggest that RV could potentially be used as a therapeutic agent in the treatment of cardiovascular complications including hypertension.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/prevenção & controle , Resveratrol/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Hipertensão/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , NADPH Oxidases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos SHR , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo
12.
J Ethnopharmacol ; 235: 375-384, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30738114

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Either Aconite Lateralis Radix Praeparata (Fuzi) or Pinelliae Rhizoma (Banxia) exerts anti-inflammatory activity and their combination has long been used in China for treating cardiovascular diseases. However, combination of two drugs is controversially prohibited in clinical prescriptions because it serves a representative incompatible pairs in "eighteen antagonisms". Up to date, whether the combination of Fuzi and Banxia could be used for treating heart failure with preserved ejection fraction (HFpEF) especially charactered by systemic inflammation and the potential mechanisms have not been elucidated. AIM OF THE STUDY: The pros and cons of Fuzi in combination with Banxia were evaluated in pressure overload (PO) rat models of HF in vivo. MATERIALS AND METHODS: Male Sprague Dawley rats were subjected to abdominal aorta constriction or sham-operated procedure. From week 12, rats were administered with low dose Fuzi (5.4 g kg-1 d-1), Banxia (5.4 g kg-1 d-1), combination (5.4 g kg-1 d-1 + 5.4 g kg-1 d-1), high dose Fuzi (10.8 g kg-1 d-1) or with vehicle (n = 15 per group) orally for additional 6 weeks. RESULTS: Fuzi alone treatment led to exaggerated cardiac-renal response to PO, and occurred dramatically at high dose as manifested by markedly exacerbated cardiac-renal inflammation and myocardial fibrosis. Further studies revealed that cardiotoxicity of Fuzi may be associated with highly expression levels of ß2-AR and PKA. In contrast, coadministration of Fuzi and Banxia restored cardiac function, as indicated by relieving inflammation and fibrosis as well as normalizing electrocardiogram parameters, which were accompanied by PKA down-regulation. More importantly, both high dose Fuzi and combination treatment enhanced induction of apoptosis, which could be partially associated with inhibition of ß2-AR-Gi signaling. CONCLUSION: Thus, combination of Fuzi and Banxia elicited concurrent protective and toxic effects in PO induced HF. The protective effect appeared to predominate and was associated with suppression of PKA/ß2-AR-Gs signaling pathway. Unlike the eighteen antagonisms theory where Fuzi and Banxia combination was considered incompatible, in the present study, this herb pairs appeared to be benefit, and probably had potential therapeutic prospect in treating HFpEF and diseases associated with inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Pinellia/química , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/isolamento & purificação , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Extratos Vegetais/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Glia ; 67(6): 1076-1093, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30801845

RESUMO

G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of Gq and Gi/o GPCR activation on astroglial Ca2+ -based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while Gq GPCR activation led to cellular activation in both neurons and astrocytes, Gi/o GPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either Gq or Gi/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of Gq and Gi/o DREADDs in vivo increased astrocyte Ca2+ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.


Assuntos
Astrócitos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibição Neural/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antagonistas Muscarínicos/farmacologia , Inibição Neural/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo
14.
Methods Mol Biol ; 1929: 135-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710272

RESUMO

Protein aggregation and inclusion body formation have been a key causal phenomenon behind a majority of neurodegenerative disorders. Various approaches aimed at preventing the formation/elimination of protein aggregates are being developed to control these diseases. Molecular chaperones are a class of protein that not only direct the functionally relevant fold of the protein but also perform quality control against stress, misfolding/aggregation. Genes that encode molecular chaperones are induced and expressed in response to extreme stress conditions to "salvage" the cell by the "unfolded protein response" (UPR) signaling pathway. Here we describe in detail the various in vitro and in vivo assays involved in identifying the chaperone activity of proteins using human calnuc as a model protein. Calnuc is a Golgi resident, calcium-binding protein, identified as chaperone protein and is reported to protect the cells against the cytotoxicity caused by amyloidosis and ER stress. Calnuc is also reported to regulate Gαi activity and inflammation apart from the role of chaperoning against amyloid proteins.


Assuntos
Peptídeos beta-Amiloides/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Resposta a Proteínas não Dobradas , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Agregados Proteicos , Dobramento de Proteína
15.
PLoS One ; 14(1): e0211066, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682176

RESUMO

BACKGROUND: Infants and children with dominant de novo mutations in GNAO1 exhibit movement disorders, epilepsy, or both. Children with loss-of-function (LOF) mutations exhibit Epileptiform Encephalopathy 17 (EIEE17). Gain-of-function (GOF) mutations or those with normal function are found in patients with Neurodevelopmental Disorder with Involuntary Movements (NEDIM). There is no animal model with a human mutant GNAO1 allele. OBJECTIVES: Here we develop a mouse model carrying a human GNAO1 mutation (G203R) and determine whether the clinical features of patients with this GNAO1 mutation, which includes both epilepsy and movement disorder, would be evident in the mouse model. METHODS: A mouse Gnao1 knock-in GOF mutation (G203R) was created by CRISPR/Cas9 methods. The resulting offspring and littermate controls were subjected to a battery of behavioral tests. A previously reported GOF mutant mouse knock-in (Gnao1+/G184S), which has not been found in patients, was also studied for comparison. RESULTS: Gnao1+/G203R mutant mice are viable and gain weight comparably to controls. Homozygotes are non-viable. Grip strength was decreased in both males and females. Male Gnao1+/G203R mice were strongly affected in movement assays (RotaRod and DigiGait) while females were not. Male Gnao1+/G203R mice also showed enhanced seizure propensity in the pentylenetetrazole kindling test. Mice with a G184S GOF knock-in also showed movement-related behavioral phenotypes but females were more strongly affected than males. CONCLUSIONS: Gnao1+/G203R mice phenocopy children with heterozygous GNAO1 G203R mutations, showing both movement disorder and a relatively mild epilepsy pattern. This mouse model should be useful in mechanistic and preclinical studies of GNAO1-related movement disorders.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Transtornos dos Movimentos , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patologia , Epilepsia/fisiopatologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Força da Mão , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/fisiopatologia
16.
Int J Mol Med ; 43(1): 382-392, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30431055

RESUMO

Odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs) is a key process in tooth root formation and development. However, the molecular mechanisms underlying this process remain largely unknown. In the present study, it was identified that guanine and nucleotide binding protein 3 (GNAI3) was at least in part responsible for the odonto/osteogenic differentiation of SCAPs. GNAI3 was markedly induced in mouse tooth root development in vivo and in human SCAPs mineralization in vitro. Notably, knockdown of GNAI3 by lentiviral vectors expressing short­hairpin RNAs against GNAI3 significantly inhibited the proliferation, cell cycle progression and migration of SCAPs, as well as odonto/osteogenic differentiation of SCAPs in vitro, suggesting that GNAI3 may play an essential role in tooth root development. The promotive role of GNAI3 in odonto/osteogenic differentiation was further confirmed by downregulation of odonto/osteogenic makers in GNAI3­deficient SCAPs. In addition, knockdown of GNAI3 effectively suppressed activity of c­Jun N­terminal kinase (JNK) and extracellular­signal regulated kinase (ERK) signaling pathways that was induced during SCAPs differentiation, suggesting that GNAI3 promotes SCAPs mineralization at least partially via JNK/ERK signaling. Taken together, the present results implicate GNAI3 as a critical regulator of odonto/osteogenic differentiation of SCAPs in tooth root development, and suggest a possible role of GNAI3 in regeneration processes in dentin or other tissues.


Assuntos
Diferenciação Celular , Papila Dentária/citologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Odontogênese , Osteogênese , Células-Tronco/enzimologia , Animais , Antracenos/farmacologia , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Odontogênese/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Raiz Dentária/embriologia , Raiz Dentária/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
17.
Mol Neurobiol ; 56(7): 4778-4785, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30387076

RESUMO

The two most common polymorphisms of the human DRD4 gene encode a dopamine D4 receptor (D4R) with four or seven repeats of a proline-rich sequence of 16 amino acids (D4.4R or D4.7R). Although the seven-repeat polymorphism has been repeatedly associated with attention-deficit hyperactivity disorder and substance use disorders, the differential functional properties between D4.4R and D4.7R remained enigmatic until recent electrophysiological and optogenetic-microdialysis experiments indicated a gain of function of D4.7R. Since no clear differences in the biochemical properties of individual D4.4R and D4.7R have been reported, it was previously suggested that those differences emerge upon heteromerization with dopamine D2 receptor (D2R), which co-localizes with D4R in the brain. However, contrary to a gain of function, experiments in mammalian transfected cells suggested that heteromerization with D2R results in lower MAPK signaling by D4.7R as compared to D4.4R. In the present study, we readdressed the question of functional differences of D4.4R and D4.7R forming homomers or heteromers with the short isoform of D2R (D2SR), using a functional bioluminescence resonance energy transfer (BRET) assay that allows the measurement of ligand-induced changes in the interaction between G protein-coupled receptors (GPCRs) forming homomers or heteromers with their cognate G protein. Significant functional and pharmacological differences between D4.4R and D4.7R were only evident upon heteromerization with the short isoform of D2R (D2SR). The most dramatic finding was a significant increase and decrease in the constitutive activity of D2S upon heteromerization with D4.7R and D4.4R, respectively, providing the first clear mechanism for a functional difference between both products of polymorphic variants and for a gain of function of the D4.7R.


Assuntos
Mutação com Ganho de Função/genética , Polimorfismo Genético , Multimerização Proteica , Receptores de Dopamina D4/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Racloprida/farmacologia
18.
Biochemistry ; 57(47): 6562-6569, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30406994

RESUMO

GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gαi1·GDP/RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/ dPins and GPSM1/AGS3. We explored the mechanism of differential interactions of GoLoco protein LGN with hGαi3 and hGαo. By combining mutagenesis experiments and molecular dynamics simulations, we identified a residue (Asp229 in hGαi3) away from the binding interface that remarkably affects the interaction between LGN and hGαi/o. A negatively charged residue at this position is required for high binding affinity. This affinity regulation mechanism was further verified by the cases of hGαi2 and dGαo, suggesting that this pathway is conserved among members of the Gα family.


Assuntos
Proteínas de Transporte/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Ciclo Celular , Cristalografia por Raios X , Drosophila , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Inibidores de Dissociação do Nucleotídeo Guanina/química , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Homologia de Sequência
19.
Theranostics ; 8(17): 4695-4709, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30279732

RESUMO

VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis. Methods: Using genetic strategies, we tested the requirement of α subunits of heterotrimeric G proteins (Gαi1/3) in the process. Results: Gαi1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. Gαi1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that Gαi1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. In vivo, Gαi1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of Gαi1/3 shRNA lentivirus. Moreover, in vivo angiogenesis by alkali burn and OIR was significantly attenuated in Gαi1/3 double knockout mice. Significantly, Gαi1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients. Conclusion: These results provide mechanistic insights into the critical role played by Gαi1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.


Assuntos
Endocitose , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Modelos Animais , Modelos Teóricos , Ligação Proteica
20.
Sci Signal ; 11(552)2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327411

RESUMO

Chemokines and some chemical analogs of chemokines prevent cellular HIV-1 entry when bound to the HIV-1 coreceptors C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4), which are G protein-coupled receptors (GPCRs). The ideal HIV-1 entry blocker targeting the coreceptors would display ligand bias and avoid activating G protein-mediated pathways that lead to inflammation. We compared CCR5-dependent activation of second messenger pathways in a single cell line. We studied two endogenous chemokines [RANTES (also known as CCL5) and MIP-1α (also known as CCL3)] and four chemokine analogs of RANTES (5P12-, 5P14-, 6P4-, and PSC-RANTES). We found that CCR5 signaled through both Gi/o and Gq/11 IP1 accumulation and Ca2+ flux arose from Gq/11 activation, rather than from Gßγ subunit release after Gi/o activation as had been previously proposed. The 6P4- and PSC-RANTES analogs were superagonists for Gq/11 activation, whereas the 5P12- and 5P14-RANTES analogs displayed a signaling bias for Gi/o These results demonstrate that RANTES analogs elicit G protein subtype-specific signaling bias and can cause CCR5 to couple preferentially to Gq/11 rather than to Gi/o signaling pathways. We propose that G protein subtype-specific signaling bias may be a general feature of GPCRs that can couple to more than one G protein family.


Assuntos
Quimiocinas/metabolismo , Receptores CCR5/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Quimiocina CCL3/farmacologia , Quimiocina CCL5/farmacologia , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Inflamação , Fosfatos de Inositol/metabolismo , Ligantes , Peptídeos Cíclicos/farmacologia , Transfecção
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