The angiotensin II AT(1), receptor structure-activity correlations in the light of rhodopsin structure

The most prevalent physiological effects of ANG II, the main product of the renin-angiotensin system, are mediated by the AT(1) receptor, a rhodopsin-like AGPCR. Numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation, which were complemented by site-directed mutagenesis studies on the AT(1) receptor to investigate the role of its structure in ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. the knowledge of the high-resolution structure of rhodopsin allowed homology modeling of the AT(1) receptor. the models thus built and mutagenesis data indicate that physiological (agonist binding) or constitutive (mutated receptor) activation may involve different degrees of expansion of the receptor's central cavity. Residues in ANG II structure seem to control these conformational changes and to dictate the type of cytosolic event elicited during the activation. 1) Agonist aromatic residues (Phe(8) and Tyr(4)) favor the coupling to G protein, and 2) absence of these residues can favor a mechanism leading directly to receptor internalization via phosphorylation by specific kinases of the receptor's COOH-terminal Ser and Thr residues, arrestin binding, and clathrin-dependent coated-pit vesicles. On the other hand, the NH2-terminal residues of the agonists ANG II and [Sar(1)]-ANG II were found to bind by two distinct modes to the AT(1) receptor extracellular site flanked by the COOH-terminal segments of the EC-3 loop and the NH2-terminal domain. Since the [Sar(1)]-ligand is the most potent molecule to trigger tachyphylaxis in AT(1) receptors, it was suggested that its corresponding binding mode might be associated with this special condition of receptors.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilUniv São Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, BR-14049 Ribeirao Preto, BrazilUniv São Paulo, Inst Chem, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilWeb of Scienc

A Novel Cellular Model to Study Angiotensin II AT2 Receptor Function in Breast Cancer Cells

Recent studies have highlighted the AT1 receptor as a potential therapeutic target in breast cancer, while the role of the AT2 subtype in this disease has remained largely neglected. The present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (D3H2LN-AT2) stably expressing high levels of Flag-tagged human AT2 receptor (Flag-hAT2). These cells exhibit high-affinity binding sites for AngII, and total binding can be displaced by the AT2-selective antagonist PD123319 but not by the AT1-selective antagonist losartan. Of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. We provide here a novel tool to investigate the AT2 receptor functions in breast cancer cells, independently of AT1 receptor activation

Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

<div><p>GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance <i>in vivo</i>. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.</p> </div

Morte súbita e angina vasoespástica

Variant angina is defined by chest pain occurring at rest associated with transitory ST segment elevation on ECG, and is caused by a spasm of a coronary artery. Frequently, variant angina is associated with atherosclerotic coronary obstruction and patients with normal coronary arteries are rare. Patients with variant angina and normal coronary arteries have good prognosis, and the development of ventricular arrhythmias or sudden death is rare. The authors present two cases of sudden cardiac death in patients with variant angina and normal coronary arteries

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: Effect of catalase overexpression

AbstractThe 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 β-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 β-oxidation of fatty acids

Citocalasinas produzidas por Xylaria sp., um fungo endofítico de Piper aduncum (piperaceae)

A chemical study on the EtOAc extract produced by Xylaria sp., an endophytic fungus from Piper aduncum, resulted in the isolation of a new cytochalasin 1, along with five known 19,20-epoxycytochalasin D (2), C (3), N (4), Q (5), and R (6). The 1-6 were evaluated against the fungi C. cladosporioides and C. sphaerospermum and only 5 showed weak activity. The cytotoxicity in vitro against HeLA and CHO cells lines were investigated and the cytochalasins 2-4, and 6 showed a strong activity against HeLA. The DNAdamaging activity of 1-6 were also investigated against mutant strains of S. cerevisiae

Cytochalasins produced by Xylaria sp., an endophytic fungus from Piper aduncum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)A chemical study on the EtOAc extract produced by Xylaria sp., an endophytic fungus from Piper aduncum, resulted in the isolation of a new cytochalasin 1, along with five known 19,20-epoxycytochalasin D (2), C (3), N (4), Q (5), and R (6). The 1-6 were evaluated against the fungi C. cladosporioides and C. sphaerospermum and only 5 showed weak activity. The cytotoxicity in vitro against HeLA and CHO cells lines were investigated and the cytochalasins 2-4, and 6 showed a strong activity against HeLA. The DNAdamaging activity of 1-6 were also investigated against mutant strains of S. cerevisiae.331020382041Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP_BrasilCNPq_BrasilCAPES_Brasi

Cytochalasins produced by xylaria sp., an endophytic fungus from piper aduncum

A chemical study on the EtOAc extract produced by Xylaria sp., an endophytic fungus from Piper aduncum, resulted in the isolation of a new cytochalasin 1, along with five known 19,20-epoxycytochalasin D (2), C (3), N (4), Q (5), and R (6). The 1-6 were evaluated against the fungi C. cladosporioides and C. sphaerospermum and only 5 showed weak activity. The cytotoxicity in vitro against HeLA and CHO cells lines were investigated and the cytochalasins 2-4, and 6 showed a strong activity against HeLA. The DNAdamaging activity of 1-6 were also investigated against mutant strains of S. cerevisiae331020382041CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informaçãoSem informaçã