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1.
Plant Sci ; 306: 110858, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33775364

RESUMO

Drought represents a leading constraint over crop productivity worldwide. The plant response to this stress is centered on the behavior of the cell membrane, where the transduction of abscisic acid (ABA) signaling occurs. Here, the Ras-related small GTP-binding protein RabE1c has been shown able to bind to an ABA receptor in the Arabidopsis thaliana plasma membrane, thereby positively regulating ABA signaling. RabE1c is highly induced by drought stress and expressed abundantly in guard cells. In the loss-of-function rabe1c mutant, both stomatal closure and the whole plant drought stress response showed a reduced sensitivity to ABA treatment, demonstrating that RabE1c is involved in the control over transpirative water loss through the stomata. Impairment of RabE1c's function suppressed the accumulation of the ABA receptor PYL4. The over-expression of RabE1c in A. thaliana enhanced the plants' ability to tolerate drought, and a similar phenotypic effect was achieved by constitutively expressing the gene in Chinese cabbage (Brassica rapassp. pekinensis). The leading conclusion was that RabE1c promotes the degradation of PYL4, suggesting a possible genetic strategy to engineer crop plants to better withstand drought stress.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Proteínas de Arabidopsis/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estômatos de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
2.
Nucleic Acids Res ; 49(5): 2816-2834, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619562

RESUMO

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.


Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Animais , Domínio Catalítico , Citoplasma/enzimologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Modelos Moleculares , Mutação , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo , Células Sf9
3.
Nat Commun ; 12(1): 791, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542237

RESUMO

Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. Here, we report a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. We perform genetic, pharmacological and mechanical perturbation analyses to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. We also show that active Rho1 GTPase localizes at retracting free edge filopodia and that Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts, but likely plays an important role in the constriction of supracellular actin cables. Based on these findings, we propose that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster.


Assuntos
Movimento Celular/fisiologia , Morfogênese/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Ligação ao GTP/metabolismo , Microscopia Intravital , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
4.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498156

RESUMO

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aminoácido Oxirredutases/metabolismo , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Derme/citologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transglutaminases/metabolismo
5.
Nat Commun ; 12(1): 496, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479228

RESUMO

Chronic inflammation during many diseases is associated with bone loss. While interferons (IFNs) are often inhibitory to osteoclast formation, the complex role that IFN and interferon-stimulated genes (ISGs) play in osteoimmunology during inflammatory diseases is still poorly understood. We show that mice deficient in IFN signaling components including IFN alpha and beta receptor 1 (IFNAR1), interferon regulatory factor 1 (IRF1), IRF9, and STAT1 each have reduced bone density and increased osteoclastogenesis compared to wild type mice. The IFN-inducible guanylate-binding proteins (GBPs) on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, GBP7) are required to negatively regulate age-associated bone loss and osteoclastogenesis. Mechanistically, GBP2 and GBP5 both negatively regulate in vitro osteoclast differentiation, and loss of GBP5, but not GBP2, results in greater age-associated bone loss in mice. Moreover, mice deficient in GBP5 or chromosome 3 GBPs have greater LPS-mediated inflammatory bone loss compared to wild type mice. Overall, we find that GBP5 contributes to restricting age-associated and inflammation-induced bone loss by negatively regulating osteoclastogenesis.


Assuntos
Reabsorção Óssea/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Interferons/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Fatores Etários , Animais , Reabsorção Óssea/genética , Diferenciação Celular/genética , Fusão Celular , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/citologia , Osteogênese/genética , Transdução de Sinais/genética
6.
Life Sci ; 267: 118926, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33358901

RESUMO

Osteoarthritis (OA) is a degenerative disease, which has a high incidence in middle-aged and elderly people and tends to occur in weight-bearing or active joints. Current treatment can only relieve symptoms and delay the progression of OA in result of its indistinct pathogenesis. In recent years, more and more studies have focused on the pathogenesis of OA. Nucleolar GTP binding protein 3 (GNL3) is associated with chondrogenic differentiation and can participate in genomic regulation as RNA binding protein (RBP). We used RNA sequencing (RNA-seq) to analyze the overall transcription level of the human cervical cancer cell line HeLa after GNL3 deletion. The results showed that downstream genes IL24 and PTN were down-regulated. IL24 takes part in the progression of OA by inducing articular osteocyte apoptosis, while PTN conducts to the progression of OA by promoting angiogenesis. We validated the results in the human chondrosarcoma cell line SW1353 and OA patients. Compared with the control group, GNL3, IL24 and PTN genes were elevated in OA specimens. This study explored the relationship between GNL3 and these two downstream genes, hoping to find biomarkers in the pathogenesis of osteoarthritis that can be used as therapeutic targets in the future.


Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Interleucinas/metabolismo , Proteínas Nucleares/metabolismo , Osteoartrite/metabolismo , Adulto , Idoso , Proteínas de Transporte/genética , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/fisiologia , Citocinas/genética , Feminino , Cabeça do Fêmur/metabolismo , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Osteoartrite/genética , Osteoartrite/patologia , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
7.
J Chromatogr A ; 1637: 461835, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33383241

RESUMO

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (ß2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that ß2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to ß2-AR were calculated to be 2.02 × 104 M-1, 1.15 × 104 M-1, 1.75 × 104 M-1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.


Assuntos
Albuterol/metabolismo , Cromatografia em Papel/métodos , Receptores Adrenérgicos beta 2/metabolismo , Terbutalina/análogos & derivados , Terbutalina/metabolismo , Adsorção , Interações Medicamentosas , Proteínas de Ligação ao GTP/metabolismo , Ligantes
8.
Methods Mol Biol ; 2201: 109-116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975793

RESUMO

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR ) that are Gi coupled which make them suitable for studying the receptor functionality. The [35S]GTP γS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the a-subunit of the G-protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.


Assuntos
Autorradiografia/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/química , Receptores Opioides mu/metabolismo , Analgésicos Opioides/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Receptores Opioides/metabolismo , Transdução de Sinais , Radioisótopos de Enxofre/química , Radioisótopos de Enxofre/metabolismo
9.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140538, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916301

RESUMO

Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3•60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3•60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of kcat 1 min-1 and Km of 34 µM.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Cinética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Termodinâmica
10.
Nat Commun ; 11(1): 5510, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139737

RESUMO

In living cells, dynamics of the endoplasmic reticulum (ER) are driven by the cytoskeleton motor machinery as well as the action of ER-shaping proteins such as atlastin GTPases including RHD3 in Arabidopsis. It is not known if the two systems interplay, and, if so, how they do. Here we report the identification of ARK1 (Armadillo-Repeat Kinesin1) via a genetic screen for enhancers of the rhd3 mutant phenotype. In addition to defects in microtubule dynamics, ER organization is also defective in mutants lacking a functional ARK1. In growing root hair cells, ARK1 comets predominantly localize on the growing-end of microtubules and partially overlap with RHD3 in the cortex of the subapical region. ARK1 co-moves with RHD3 during tip growth of root hair cells. We show that there is a functional interdependence between ARK1 and RHD3. ARK1 physically interacts with RHD3 via its armadillo domain (ARM). In leaf epidermal cells where a polygonal ER network can be resolved, ARK1, but not ARK1ΔARM, moves together with RHD3 to pull an ER tubule toward another and stays with the newly formed 3-way junction of the ER for a while. We conclude that ARK1 acts together with RHD3 to move the ER on microtubules to generate a fine ER network.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas do Domínio Armadillo , Proteínas de Ligação ao GTP/genética , Mutação , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
11.
Nat Commun ; 11(1): 5111, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037216

RESUMO

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Estabilidade de RNA , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
12.
Proc Natl Acad Sci U S A ; 117(37): 23096-23105, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868434

RESUMO

The ß2-adrenergic receptor (ß2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the ß2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the ß2AR-Gs complex revealed the interaction interface of ß2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the ß2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the ß2AR in response to the full agonist BI-167107 and Gs and Gi1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the ß2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the ß2AR-Gs and the ß2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs These differences between the ß2AR-Gs and ß2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Benzoxazinas/farmacologia , Sítios de Ligação/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
13.
Proc Natl Acad Sci U S A ; 117(35): 21723-21730, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817560

RESUMO

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , AMP Cíclico/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Ligação Proteica/fisiologia , Receptores de Vasopressinas/metabolismo , Transdução de Sinais/fisiologia , Vasopressinas/metabolismo , beta-Arrestinas/metabolismo
14.
Nat Commun ; 11(1): 4121, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807782

RESUMO

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes.


Assuntos
Microscopia Crioeletrônica/métodos , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Difusão Dinâmica da Luz , Humanos , Microscopia Eletrônica , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(28): 16346-16355, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601232

RESUMO

Agonists to the µ-opioid G protein-coupled receptor (µOR) can alleviate pain through activation of G protein signaling, but they can also induce ß-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to µOR that induce G protein signaling without inducing ß-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating ß-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated ß-arrestin2 stabilized by phosphorylated µOR bound to the morphine and D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the ß-arrestin2 couples to the phosphorylated µOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and ß-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of µOR, hindering ß-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between µOR and ß-arrestin2.


Assuntos
Receptores Opioides mu/metabolismo , beta-Arrestina 2/metabolismo , Analgésicos Opioides/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Citoplasma/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Simulação de Dinâmica Molecular , Morfina/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Transdução de Sinais , Compostos de Espiro/metabolismo , Tiofenos/metabolismo , beta-Arrestina 2/química
16.
Mol Cell ; 79(4): 603-614.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32579943

RESUMO

Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Livre de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , Coelhos , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , Ubiquitinação
17.
Nat Commun ; 11(1): 3276, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581219

RESUMO

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.


Assuntos
Caspases Iniciadoras/metabolismo , Citosol/microbiologia , Proteínas de Ligação ao GTP/metabolismo , Lipopolissacarídeos/metabolismo , Salmonella/metabolismo , Linhagem Celular , Ativação Enzimática , Células Epiteliais/metabolismo , Células HeLa , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Ligação Proteica , Piroptose , Eletricidade Estática
18.
Proc Natl Acad Sci U S A ; 117(27): 15414-15422, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571939

RESUMO

We report on a combined activation mechanism for a class B G-protein-coupled receptor (GPCR), the glucagon receptor. By computing the conformational free-energy landscape associated with the activation of the receptor-agonist complex and comparing it with that obtained with the ternary complex (receptor-agonist-G protein) we show that the agonist stabilizes the receptor in a preactivated complex, which is then fully activated upon binding of the G protein. The proposed mechanism contrasts with the generally assumed GPCR activation mechanism, which proceeds through an opening of the intracellular region allosterically elicited by the binding of the agonist. The mechanism found here is consistent with electron cryo-microscopy structural data and might be general for class B GPCRs. It also helps us to understand the mode of action of the numerous allosteric antagonists of this important drug target.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Domínios Proteicos , Receptores de Glucagon/metabolismo , Regulação Alostérica , Membrana Celular/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Glucagon/análogos & derivados , Glucagon/metabolismo , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade
19.
Nat Commun ; 11(1): 3160, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572026

RESUMO

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the ß2-adrenergic receptor (ß2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling.


Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas-G , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Estrutura Molecular , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais/fisiologia
20.
Oncogene ; 39(27): 5042-5055, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32518375

RESUMO

Guanylate-binding protein 2 (GBP2) is an interferon-inducible large GTPase which is crucial to the protective immunity against microorganisms. However, its biological function in cancer remains largely unknown. Glioblastoma multiforme (GBM) is the most common and deadly brain tumor in adults. Here we show that GBP2 expression is highly elevated in GBM tumor and cell lines, particularly in those of the mesenchymal subtype. High GBP2 expression is associated with poor prognosis. GBP2 overexpression significantly promotes GBM cell migration and invasion in vitro, and GBP2 silencing by RNA interference exhibits opposite effects. We further show that fibronectin (FN1) is dramatically induced by GBP2 expression at both mRNA and protein levels, and FN1 is essential for GBP2-promoted GBM invasiveness. Inhibition of Stat3 pathway prevents GBP2-promoted FN1 induction and cell invasion. Consistently, GBP2 dramatically promotes GBM tumor growth and invasion in mice and significantly reduces the survival time of the mice with tumor. Taken together, these findings establish the role of GBP2/Stat3/FN1 signaling cascade in GBM invasion and suggest GBP2 may serve as a potential therapeutic target for inhibiting GBM invasion.


Assuntos
Neoplasias Encefálicas/patologia , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glioblastoma/patologia , Fator de Transcrição STAT3/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação ao GTP/genética , Glioblastoma/genética , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Transplante Heterólogo
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