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1.
Nature ; 512(7513): 218-222, 2014 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-25043026

RESUMO

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.


Assuntos
Arrestinas/química , Arrestinas/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Estrutura Quaternária de Proteína , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Células Sf9 , beta-Arrestina 1 , beta-Arrestinas
2.
Nature ; 497(7447): 137-41, 2013 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-23604254

RESUMO

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.


Assuntos
Arrestinas/química , Arrestinas/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Receptores de Vasopressinas/química , Animais , Arrestinas/imunologia , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Ratos , Rotação , beta-Arrestina 1 , beta-Arrestinas
3.
Am J Physiol Cell Physiol ; 315(3): C367-C379, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29874111

RESUMO

Hypertension is a global health problem, and angiotensin I (ANG I)-converting enzyme (ACE) inhibitors are largely used to control this pathology. Recently, it has been shown that ACE can also act as a transducer signal molecule when its inhibitors or substrates bind to it. This new role of ACE could contribute to understanding some of the effects not explained by its catalytic activity only. In this study, we investigated signaling pathway activation in Chinese hamster ovary (CHO) cells stably expressing ACE (CHO-ACE) under different conditions. We also investigated gene modulation after 4 h and 24 h of captopril treatment. Our results demonstrated that CHO-ACE cells when stimulated with ANG I, ramipril, or captopril led to JNK and ERK1/2 phosphorylation. To verify any physiological role at the endogenous level, we made use of primary cultures of mesangial cells from spontaneously hypertensive rats (SHR) and Wistar rats. Our results showed that ERK1/2 activation occurred mainly in primary cultures of mesangial cells from SHR rats upon captopril stimulation, suggesting that this signaling pathway could be differentially regulated during hypertension. Our results also showed that captopril treatment leads to a decrease of cyclooxygenase 2, interleukin-1ß, and ß-arrestin2 and a significant increase of AP2 gene expression levels. Our findings strengthen the fact that, in addition to the blockage of enzymatic activity, ACE inhibitors also trigger signaling pathway activation, and this may contribute to their beneficial effects in the treatment of hypertension and other pathologies.


Assuntos
Angiotensina I/metabolismo , Captopril/farmacologia , Peptidil Dipeptidase A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetulus , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Wistar
4.
Biochim Biophys Acta ; 1832(10): 1591-604, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23643711

RESUMO

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500µM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with ß-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and ß-oxidation of fatty acids.


Assuntos
Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Resistência à Insulina , Mitocôndrias Musculares/fisiologia , Animais , Antioxidantes/metabolismo , Células Cultivadas , Masculino , Mitocôndrias Musculares/enzimologia , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Ácido Palmítico/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar
5.
Clin Sci (Lond) ; 127(3): 185-94, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24498923

RESUMO

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.


Assuntos
Bradicinina/análogos & derivados , Receptor B2 da Bradicinina/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Bradicinina/farmacologia , Cininas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/genética , RNA Mensageiro/metabolismo , Receptor B2 da Bradicinina/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Regulação para Cima
6.
Biochem Biophys Res Commun ; 434(3): 647-52, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583236

RESUMO

Mechanotransduction enables cells to sense and respond to stimuli, such as strain, pressure and shear stress (SS), critical for maintenance of cardiovascular homeostasis or pathological states. The angiotensin II type 1 receptor (AT1R) was the first G protein-coupled receptor described to display stretch-induced activation in cardiomyocytes independent of its ligand Ang II. Here, we assessed whether SS (15 dynes/cm(2), 10 min), an important mechanical force present in the cardiovascular system, activates AT1R independent of its ligand. SS induced extracellular signal-regulated kinase (ERK) activation, used as a surrogate of AT1R activation, in Chinese hamster ovary cells expressing the AT1R (CHO+AT1) but not in wild type cells (CHO). AT1R dependent SS-induced ERK activation involves Ca(2+) inflow and activation of Gαq since Ca(2+) chelator EGTA or Gαq-specific inhibitor YM-254890 decreased SS-induced ERK activation. On the other hand, the activation of JAK-2 and Src, two intracellular signaling molecules independent of G protein activation, were not differently modulated in the presence of AT1R. Also, ERK activation by SS was observed in CHO cells expressing the mutated AT1R DRY/AAY, which has impaired ability to activate Gαq dependent intracellular signaling. Altogether we provided evidence that SS activates AT1R in the absence of its ligand by both a G protein-dependent and -independent pathways. The biological relevance of these observations deserves to be further investigated since the novel mechanisms described extend the knowledge of the activation of GPCRs independent of its traditional ligand.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Estresse Fisiológico , Animais , Western Blotting , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Reação em Cadeia da Polimerase
7.
J Appl Crystallogr ; 56(Pt 5): 1361-1370, 2023 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-37791355

RESUMO

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.

8.
Commun Chem ; 6(1): 219, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37828292

RESUMO

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

9.
Am J Physiol Renal Physiol ; 302(7): F875-83, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22218590

RESUMO

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.


Assuntos
Angiotensina II/metabolismo , Túbulos Renais Proximais/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Neprilisina/metabolismo , Peptídeo Hidrolases/metabolismo , Peptidil Dipeptidase A/metabolismo , Multimerização Proteica , Transdução de Sinais , Suínos
10.
Front Mol Biosci ; 9: 890862, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35651816

RESUMO

Structure-function relationships of biological macromolecules, in particular proteins, provide crucial insights for fundamental biochemistry, medical research and early drug discovery. However, production of recombinant proteins, either for structure determination, functional studies, or to be used as biopharmaceutical products, is often hampered by their instability and propensity to aggregate in solution in vitro. Protein samples of poor quality are often associated with reduced reproducibility as well as high research and production expenses. Several biophysical methods are available for measuring protein aggregation and stability. Yet, discovering and developing means to improve protein behaviour and structure-function integrity remains a demanding task. Here, we discuss workflows that are made possible by adapting established biophysical methods to high-throughput screening approaches. Rapid identification and optimisation of conditions that promote protein stability and reduce aggregation will support researchers and industry to maximise sample quality, stability and reproducibility, thereby reducing research and development time and costs.

11.
Mol Vis ; 17: 2228-40, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21897745

RESUMO

PURPOSE: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. METHODS: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. RESULTS: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. CONCLUSIONS: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Transdução de Sinais/genética , eIF-2 Quinase/metabolismo , Animais , Apoptose/genética , Western Blotting , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/genética , Hibridização In Situ , Luciferases/análise , Masculino , MicroRNAs/genética , Ratos , Ratos Wistar , Células Ganglionares da Retina/citologia , Células Horizontais da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , eIF-2 Quinase/genética
12.
J Cell Physiol ; 225(2): 500-5, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20458750

RESUMO

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.


Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/citologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fator de Iniciação de Tradução Eucariótico 5A
13.
Biology (Basel) ; 9(11)2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33202740

RESUMO

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure-function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein-detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins' reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

14.
Sci Adv ; 6(33): eaav8207, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32851152

RESUMO

G protein-coupled receptors (GPCRs) are the largest and pharmaceutically most important class of membrane proteins encoded in the human genome, characterized by a seven-transmembrane helix architecture and a C-terminal amphipathic helix 8 (H8). In a minority of GPCR structures solved to date, H8 either is absent or adopts an unusual conformation. The controversial existence of H8 of the class A GPCR neurotensin receptor 1 (NTS1) has been examined here for the nonthermostabilized receptor in a functionally supporting membrane environment using electron paramagnetic resonance, molecular dynamics simulations, and circular dichroism. Lipid-protein interactions with phosphatidylserine and phosphatidylethanolamine lipids, in particular, stabilize the residues 374 to 390 of NTS1 into forming a helix. Furthermore, introduction of a helix-breaking proline residue in H8 elicited an increase in ß-arrestin-NTS1 interactions observed in pull-down assays, suggesting that the structure and/or dynamics of H8 might play an important role in GPCR signaling.


Assuntos
Arrestina , Receptores Acoplados a Proteínas G , Humanos , Lipídeos/química , Conformação Molecular , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G/metabolismo
15.
Int Immunopharmacol ; 8(2): 135-42, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18182216

RESUMO

The general description of kinins refers to these peptides as molecules involved in vascular tone regulation and inflammation. Nevertheless, in the last years a series of evidences has shown that local hormonal systems, such as the kallikrein-kinin system, may be differently regulated and are of pivotal importance to pathophysiological control. The combined interpretations of many recent studies allow us to conclude that the kallikrein-kinin system plays broader and richer roles than those classically described until recently. In this review, we report findings concerning the participation of the kallikrein-kinin system in inflammation, cancer, and in pathologies related to cardiovascular, renal and central nervous systems.


Assuntos
Doenças Cardiovasculares/etiologia , Doenças do Sistema Nervoso Central/etiologia , Inflamação/etiologia , Sistema Calicreína-Cinina/fisiologia , Nefropatias/etiologia , Neoplasias/etiologia , Animais , Humanos
16.
Regul Pept ; 140(1-2): 32-6, 2007 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-17239455

RESUMO

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.


Assuntos
Prolina/genética , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Ligação Competitiva , Células COS , Chlorocebus aethiops , Simulação por Computador , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Mutação , Prolina/química , Ligação Proteica , Ratos , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
17.
Regul Pept ; 141(1-3): 159-67, 2007 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-17320985

RESUMO

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.


Assuntos
Mutação , Proto-Oncogenes/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Células CHO , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Fluoresceínas , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Indóis , Concentração Inibidora 50 , Fosfatos de Inositol/análise , Fosfatos de Inositol/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptor Tipo 1 de Angiotensina/química , Receptores Acoplados a Proteínas G/genética , Transfecção
18.
Sci Rep ; 6: 22078, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26898917

RESUMO

Melanoma is a very aggressive tumor that arises from melanocytes. Late stage and widely spread diseases do not respond to standard therapeutic approaches. The kallikrein-kinin system (KKS) participates in biological processes such as vasodilatation, pain and inflammatory response. However, the role of KKS in tumor formation and progression is not completely understood. The role of the host kinin B1 receptor in melanoma development was evaluated using a syngeneic melanoma model. Primary tumors and metastasis were respectively induced by injecting B16F10 melanoma cells, which are derived from C57BL/6 mice, subcutaneously or in the tail vein in wild type C57BL/6 and B1 receptor knockout mice (B1(-/-)). Tumors developed in B1(-/-) mice presented unfavorable prognostic factors such as increased incidence of ulceration, higher levels of IL-10, higher activation of proliferative pathways such as ERK1/2 and Akt, and increased mitotic index. Furthermore, in the metastasis model, B1(-/-) mice developed larger metastatic colonies in the lung and lower CD8(+)immune effector cells when compared with WT animals. Altogether, our results provide evidences that B1(-/-) animals developed primary tumors with multiple features associated with poor prognosis and unfavorable metastatic onset, indicating that the B1 receptor may contribute to improve the host response against melanoma progression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Receptor B1 da Bradicinina/genética , Neoplasias Cutâneas/genética , Animais , Progressão da Doença , Feminino , Interleucina-10/genética , Interleucina-10/metabolismo , Sistema Calicreína-Cinina/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Índice Mitótico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B1 da Bradicinina/deficiência , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
19.
Int J Cardiol ; 167(4): 1199-205, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22483258

RESUMO

BACKGROUND: Hyperactivation of the renin-angiotensin system contributes to hypertension-induced upregulation of vascular matrix metalloproteinases (MMPs) and remodeling, especially in the two kidney, one clip (2K1C) hypertension model. We hypothesized that the AT1R antagonist losartan or the renin inhibitor aliskiren, given at doses allowing similar antihypertensive effects, could prevent in vivo vascular MMPs upregulation and remodeling, and collagen/elastin deposition found in 2K1C hypertension by preventing the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and transforming growth factor-ß1 (TGF-ß1). We also hypothesized that aliskiren could enhance the effects of losartan. METHODS: 2K1C rats were treated with aliskiren (50mg.kg(-1).day(-1)), or losartan (10mg.kg(-1).day(-1)), or both by gavage during 4 weeks. RESULTS: Aliskiren, losartan, or both drugs exerted similar antihypertensive effects when compared with 2K-1C rats treated with water. Aliskiren reduced plasma renin activity in both sham and 2K-1C rats. Losartan alone or combined with aliskiren, but not aliskiren alone, abolished 2K1C-induced aortic hypertrophy and hyperplasia, and prevented the increases in aortic collagen/elastin content, MMP-2 levels, gelatinolytic activity, and expression of phospho-ERK 1/2 and TGF-ß1. No significant differences were found in the aortic expression of the (pro)renin receptor. CONCLUSIONS: These findings show that although losartan and aliskiren exerted similar antihypertensive effects, only losartan prevented the activation of vascular profibrotic mechanisms and MMP upregulation associated with vascular remodeling in 2K1C hypertension. Our findings also suggest that aliskiren does not enhance the protective effects exerted by losartan.


Assuntos
Amidas/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Aorta/efeitos dos fármacos , Fumaratos/uso terapêutico , Hipertensão/tratamento farmacológico , Losartan/uso terapêutico , Remodelação Ventricular/efeitos dos fármacos , Amidas/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Aorta/metabolismo , Aorta/patologia , Fumaratos/farmacologia , Hipertensão/metabolismo , Hipertensão/patologia , Losartan/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Resultado do Tratamento , Remodelação Ventricular/fisiologia
20.
PLoS One ; 8(5): e64453, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691222

RESUMO

Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.


Assuntos
Melanoma/prevenção & controle , Metástase Neoplásica/prevenção & controle , Receptor B1 da Bradicinina/metabolismo , Análise de Variância , Western Blotting , Linhagem Celular Tumoral , Corantes Fluorescentes , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Kisspeptinas/metabolismo , Melanoma/fisiopatologia , Receptor B1 da Bradicinina/agonistas , Sais de Tetrazólio , Tiazóis
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