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1.
PLoS One ; 14(12): e0225729, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815963

RESUMO

Restricted and controlled drug delivery to the heart remains a challenge giving frequent off-target effects as well as limited retention of drugs in the heart. There is a need to develop and optimize tools to allow for improved design of drug candidates for treatment of heart diseases. Over the last decade, novel drug platforms and nanomaterials were designed to confine bioactive materials to the heart. Yet, the research remains in its infancy, not only in the development of tools but also in the understanding of effects of these materials on cardiac function and tissue integrity. Upconverting nanoparticles are nanomaterials that recently accelerated interest in theranostic nanomedicine technologies. Their unique photophysical properties allow for sensitive in vivo imaging that can be combined with spatio-temporal control for targeted release of encapsulated drugs. Here we synthesized upconverting NaYF4:Yb,Tm nanoparticles and show for the first time their innocuity in the heart, when injected in the myocardium or in the pericardial space in mice. Nanoparticle retention and upconversion in the cardiac region did not alter heart rate variability, nor cardiac function as determined over a 15-day time course ensuing the sole injection. Altogether, our nanoparticles show innocuity primarily in the pericardial region and can be safely used for controlled spatiotemporal drug delivery. Our results support the use of upconverting nanoparticles as potential theranostics tools overcoming some of the key limitations associated with conventional experimental cardiology.

2.
Ann Phys Rehabil Med ; 62(5): 321-328, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31352063

RESUMO

BACKGROUND: Exaggerated sympathetic nervous system activity associated with low heart rate variability (HRV) is considered to trigger cardiac arrhythmias and sudden death. Regular exercise training is efficient to improve autonomic balance. OBJECTIVE: We aimed to verify the superiority of high-intensity interval training (HIIT) to enhance HRV, cardiorespiratory fitness and cardiac function as compared with moderate intensity continuous training (MICT) in a short, intense cardiac rehabilitation program. METHODS: This was a prospective, monocentric, evaluator-blinded, randomised (1:1) study with a parallel two-group design. Overall, 31 individuals with voluntary chronic heart failure (CHF) (left ventricular ejection fraction [LVEF]<45%) were allocated to MICT (n=15) or HIIT (n=16) for a short rehabilitation program (mean [SD] 27 [4] days). Participants underwent 24-hr electrocardiography, echocardiography and a cardiopulmonary exercise test at entry and at the end of the study. RESULTS: High-frequency power in normalized units (HFnu%) measured as HRV increased with HIIT (from 21.2% to 26.4%, P<0.001) but remained unchanged with MICT (from 23.1% to 21.9%, P=0.444, with a significant intergroup difference, P=0.003). Resting heart rate (24-hr Holter electrocardiography) decreased significantly for both groups (from 68.2 to 64.6 bpm and 66.0 to 63.5 bpm for MICT and HIIT, respectively, with no intergroup difference, P=0.578). The 2 groups did not differ in premature ventricular contractions. Improvement in peak oxygen uptake was greater with HIIT than MICT (+21% vs. +5%, P=0.009). LVEF improved with only HIIT (from 36.2% to 39.5%, P=0.034). CONCLUSIONS: In this short rehabilitation program, HIIT was significantly superior to the classical MICT program for enhancing parasympathetic tone and peak oxygen uptake. CLINICALTRIALS. GOV IDENTIFIER: NCT03603743.

3.
Cardiovasc Res ; 115(6): 1078-1091, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30329023

RESUMO

AIMS: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.

4.
Cell Mol Life Sci ; 76(3): 561-576, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30406277

RESUMO

P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit-risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Ticagrelor/farmacologia , Monofosfato de Adenosina/farmacologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Modelos Biológicos , Mutação , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/ultraestrutura , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética , Transdução de Sinais/efeitos dos fármacos , Trombose/tratamento farmacológico , Trombose/fisiopatologia
5.
Front Immunol ; 10: 2970, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921208

RESUMO

The CXCL12-CXCR4 axis plays a key role in the retention of stem cells and progenitors in dedicated bone marrow niches. It is well-known that CXCR4 responsiveness in B lymphocytes decreases dramatically during the final stages of their development in the bone marrow. However, the molecular mechanism underlying this regulation and whether it plays a role in B-cell homeostasis remain unknown. In the present study, we show that the differentiation of pre-B cells into immature and mature B cells is accompanied by modifications to the relative expression of chemokine receptors, with a two-fold downregulation of CXCR4 and upregulation of CCR7. We demonstrate that expression of CCR7 in B cells is involved in the selective inactivation of CXCR4, and that mature B cells from CCR7-/- mice display higher responsiveness to CXCL12 and improved retention in the bone marrow. We also provide molecular evidence supporting a model in which upregulation of CCR7 favors the formation of CXCR4-CCR7 heteromers, wherein CXCR4 is selectively impaired in its ability to activate certain G-protein complexes. Collectively, our results demonstrate that CCR7 behaves as a novel selective endogenous allosteric modulator of CXCR4.

6.
Proc Natl Acad Sci U S A ; 115(17): 4501-4506, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632174

RESUMO

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.


Assuntos
Dopamina/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Multimerização Proteica , Receptores de Dopamina D2/química , Receptores de Grelina/química , Transdução de Sinais , Dopamina/genética , Dopamina/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo
7.
Sci Rep ; 7(1): 7885, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801617

RESUMO

Biased agonism at G protein coupled receptors emerges as an opportunity for development of drugs with enhanced benefit/risk balance making biased ligand identification a priority. However, ligand biased signature, classically inferred from ligand activity across multiple pathways, displays high variability in recombinant systems. Functional assays usually necessity receptor/effector overexpression that should be controlled among assays to allow comparison but this calibration currently fails. Herein, we demonstrate that Gα expression level dictates the biased profiling of agonists and, to a lesser extent of ß-blockers, in a Gα isoform- and receptor-specific way, depending on specific G protein activity in different membrane territories. These results have major therapeutic implications since they suggest that the ligand bias phenotype is not necessarily maintained in pathological cell background characterized by fluctuations in G protein expression. Thus, we recommend implementation of G protein stoichiometry as a new parameter in biased ligand screening programs.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Células HEK293 , Humanos , Ligantes , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Acoplados a Proteínas-G/genética
8.
J Struct Biol ; 198(1): 28-37, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28263874

RESUMO

PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolemmal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases.


Assuntos
Microscopia de Força Atômica/métodos , Miócitos Cardíacos/ultraestrutura , Animais , Membrana Celular , Formamidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica/instrumentação , Mitocôndrias/efeitos dos fármacos , Sarcômeros/ultraestrutura , Propriedades de Superfície
9.
Ann Phys Rehabil Med ; 60(1): 27-35, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27542313

RESUMO

Patients with cardiovascular disease show autonomic dysfunction, including sympathetic activation and vagal withdrawal, which leads to fatal events. This review aims to place sympathovagal balance as an essential element to be considered in management for cardiovascular disease patients who benefit from a cardiac rehabilitation program. Many studies showed that exercise training, as non-pharmacologic treatment, plays an important role in enhancing sympathovagal balance and could normalize levels of markers of sympathetic flow measured by microneurography, heart rate variability or plasma catecholamine levels. This alteration positively affects prognosis with cardiovascular disease. In general, cardiac rehabilitation programs include moderate-intensity and continuous aerobic exercise. Other forms of activities such as high-intensity interval training, breathing exercises, relaxation and transcutaneous electrical stimulation can improve sympathovagal balance and should be implemented in cardiac rehabilitation programs. Currently, the exercise training programs in cardiac rehabilitation are individualized to optimize health outcomes. The sports science concept of the heart rate variability (HRV)-vagal index used to manage exercise sessions (for a goal of performance) could be implemented in cardiac rehabilitation to improve cardiovascular fitness and autonomic nervous system function.


Assuntos
Sistema Nervoso Autônomo/fisiopatologia , Reabilitação Cardíaca/métodos , Doenças Cardiovasculares/fisiopatologia , Terapia por Exercício/métodos , Exercício/fisiologia , Frequência Cardíaca/fisiologia , Humanos , Resultado do Tratamento
10.
J Biol Chem ; 292(2): 575-584, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27895119

RESUMO

Biased agonism at G protein-coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12 Binding of J113863 to CCR2 or CCR5 also induced the recruitment of ß-arrestin 2, whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and ß-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist, or full agonist according to the receptor, the enantiomer, and the signaling pathway investigated.


Assuntos
Movimento Celular/efeitos dos fármacos , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xantenos/farmacologia , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Mutação de Sentido Incorreto , Ligação Proteica/efeitos dos fármacos , Receptores CCR2/agonistas , Receptores CCR2/química , Receptores CCR2/genética , Receptores CCR5/agonistas , Receptores CCR5/química , Receptores CCR5/genética , Xantenos/química , beta-Arrestina 2/química , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
11.
PLoS One ; 11(10): e0164179, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27716822

RESUMO

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of ß-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate ß-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and ß-arrestin2 activation but not ß-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.


Assuntos
Receptores CCR/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Linhagem Celular , Quimiocinas/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia/fisiologia , Cricetulus , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , beta-Arrestina 2/metabolismo
12.
Hypertension ; 68(6): 1365-1374, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27698068

RESUMO

Hyperactivity of the renin-angiotensin-aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin-angiotensin-aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1-7) in AT1-R-expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective ß-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1-7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin-angiotensin-aldosterone system peptides act as agonists on the AT1-R/ß-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/ß-arrestin2 intracellular routing. Importantly, we reveal Ang-(1-7) a known Mas receptor-specific ligand, as an AT1-R-biased agonist, selectively promoting ß-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1-7) at AT1-R, similar to that of synthetic AT1-R-biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1-7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1-7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin-angiotensin-aldosterone system.


Assuntos
Angiotensina II/farmacologia , Angiotensina I/metabolismo , Cardiotônicos/metabolismo , Células HEK293/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Músculos , Fenilefrina/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Sensibilidade e Especificidade , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , beta-Arrestinas/metabolismo
13.
Cell ; 166(4): 920-934, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27499022

RESUMO

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.


Assuntos
Fibroblastos/metabolismo , Mutação de Sentido Incorreto , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Actinas/química , Actinas/metabolismo , Animais , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Difusão , Endocitose , Ativação Enzimática , Glicosilação , Humanos , Interferon gama/metabolismo , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Receptores de Interferon/química
14.
Int J Biochem Cell Biol ; 77(Pt B): 251-63, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27107932

RESUMO

During the last 10 years, the concept of "biased agonism" also called "functional selectivity" swamped the pharmacology of 7 transmembrane receptors and paved the way for developing signaling pathway-selective drugs with increased efficacy and less adverse effects. Initially thought to select the activation of only a subset of the signaling pathways by the reference agonist, bias ligands revealed higher complexity as they have been shown to stabilize variable receptor conformations that associate with distinct signaling events from the reference. Today, one major challenge relies on the in vitro determination of the bias and classification of these ligands, as a prerequisite for future in vivo and clinical translation. In this review, current experimental considerations for the bias evaluation related to choice of the cellular model, of the signaling pathway as well as of the assays are presented and discussed.


Assuntos
Bioensaio/métodos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Humanos , Ligantes , Receptores Acoplados a Proteínas-G/agonistas , Transdução de Sinais
15.
Nat Commun ; 6: 10156, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26658454

RESUMO

Despite the discovery of heterotrimeric αßγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.


Assuntos
Depsipeptídeos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Animais , Ardisia/química , Linhagem Celular Tumoral , Depsipeptídeos/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Melanoma/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Isoformas de Proteínas , Transdução de Sinais , Cauda/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos
16.
Front Pharmacol ; 6: 203, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26483685

RESUMO

Cyclic adenosine 3',5'-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for ß-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability.

17.
J Biol Chem ; 290(45): 27021-39, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26363071

RESUMO

The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.


Assuntos
Receptores de Grelina/agonistas , Receptores de Grelina/antagonistas & inibidores , Arrestinas/metabolismo , Desenho de Drogas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Fosfatos de Inositol/biossíntese , Cinética , Ligantes , Sistema de Sinalização das MAP Quinases , Receptores de Grelina/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , beta-Arrestinas
18.
Nat Chem Biol ; 11(4): 271-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25706338

RESUMO

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR-AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP-PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Tipo 1 de Angiotensina/agonistas , Transdução de Sinais , Agonistas alfa-Adrenérgicos/química , Animais , Biofísica , Doenças Cardiovasculares/metabolismo , AMP Cíclico/metabolismo , Dimerização , Desenho de Drogas , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Norepinefrina/química , Células PC12 , Fosforilação , Conformação Proteica , Ratos , Receptores Adrenérgicos alfa 2/química , Sistema Nervoso Simpático/efeitos dos fármacos
19.
J Immunol ; 194(5): 2128-39, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25617475

RESUMO

Chemokines engage B lymphocyte surface receptors, triggering heterotrimeric G protein Gαi subunit guanine nucleotide exchange. RGS proteins limit the duration that Gαi subunits remain GTP bound, and the loss of an individual RGS protein typically enhances chemokine receptor signaling. In this study, we show that B cells carrying a Gαi2 (G184S/G184S) mutation that disables all RGS protein/Gαi2 interactions exhibit an unexpectedly severe reduction in chemokine receptor signaling. The Gαi2 (G184S/G184S) B cells have markedly elevated basal calcium levels, but poor chemokine-induced increases, enhanced nonspecific migration, but extremely poor chemotaxis. In striking contrast, the Gαi2 (G184S/G184S) B cells exhibited enhanced sensitivity to sphingosine 1-phosphate (S1P). S1P elicited heightened intracellular calcium responses and enhanced S1P-triggered cell migration. Mice with the Gαi2 (G184S/G184S) mutation displayed excessive numbers of germinal center-like structures; abnormal serum Ig profiles; and aberrant B lymphocyte trafficking. These findings establish an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Baço/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/imunologia , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Cálcio/imunologia , Cálcio/metabolismo , Quimiocinas/farmacologia , Feminino , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/imunologia , Regulação da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Cultura Primária de Células , Ligação Proteica , Proteínas RGS/genética , Proteínas RGS/imunologia , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia
20.
J Biol Chem ; 290(15): 9542-54, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25614627

RESUMO

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as ß-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gαi subtypes (Gαi1, Gαi2, and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα12, but not Gα13. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.


Assuntos
Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Receptores CCR7/metabolismo , Transdução de Sinais , Animais , Arrestinas/genética , Arrestinas/metabolismo , Técnicas Biossensoriais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Medições Luminescentes , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores CCR2/genética , Receptores CCR5/genética , Receptores CCR7/genética , beta-Arrestina 2 , beta-Arrestinas
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