RESUMO
We have demonstrated in previous studies that a single amino acid change can alter the activity of the recombinant disintegrin r-Moj. In this study, four r-Moj recombinants containing single mutations (r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-MN) and two containing double mutations (r-Moj-MP and r-Moj-NM) at the binding loop were produced, purified, and tested. All r-Moj-W_, r-Moj-M_, and r-Moj-NM mutant peptides inhibited platelet aggregation at higher potency than r-Moj-D_ mutants. Five of the seven r-Moj peptides inhibited angiogenesis at different levels. Two of the mutant peptides with a methionine at the second position carboxyl of the RGD (r-Moj-WM and r-Moj-NM) were the strongest angiogenesis inhibitors, with r-Moj-WM being the most potent. Recombinant r-Moj-MP and r-Moj-WN failed to inhibit angiogenesis. Only the r-Moj-MP mutant peptide induced apoptosis of SK-Mel-28 cells significantly (p = 0.001). This was confirmed by chromatin condensation. Proliferation of SK-Mel-28 cells was inhibited at high levels (>70%) by all r-Moj mutant peptides. Recombinant r-Moj-MN and r-Moj-WN failed to inhibit cell migration significantly (p > 0.5). Recombinant r-Moj-NM was the strongest cell migration inhibitor (98% ± 0.69), followed by r-Moj-MP (80% ± 2.87), and r-Moj-WM (61.8% ± 5.45). The lowest inhibitor was r-Moj-WL (50% ± 12.16). Our functional data suggest that the most potent r-Moj disintegrins contain a methionine in the first or the second position carboxyl to the RGD.
Assuntos
Desintegrinas/toxicidade , Metionina/metabolismo , Mutação , Proteínas Recombinantes/toxicidade , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/genética , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69%-100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desintegrinas/farmacologia , Desenho de Fármacos , Melanoma/tratamento farmacológico , Proteínas Mutantes/farmacologia , Motivos de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Ácido Aspártico/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desintegrinas/genética , Desintegrinas/metabolismo , Repressão Enzimática/efeitos dos fármacos , Humanos , Cadeias alfa de Integrinas/antagonistas & inibidores , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Integrina alfaV/química , Integrina alfaV/genética , Integrina alfaV/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Proteínas de Répteis/farmacologia , Venenos de Víboras/química , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
AIMS: During ß-adrenergic receptor (ß-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during ß-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during ß-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS: Confocal imaging revealed that ß-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS: ß-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Diástole , Ativação Enzimática , Cobaias , Miócitos Cardíacos/enzimologia , Doadores de Óxido Nítrico/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
Disintegrins are low molecular weight peptides isolated from viper venom. These peptides bind to integrin receptors using a conserved binding motif sequence containing an RGD or similar motif. As a consequence, disintegrins can inhibit platelet aggregation and inhibit cell migration, proliferation, and initiate apoptosis in cancer cell lines. Rubistatin is a MVD disintegrin cloned from a Crotalus ruber ruber venom gland. The biological activity of MVD disintegrins is poorly understood. Recombinant rubistatin (r-Rub) was cloned into a pET32b plasmid and expressed in reductase-deficient Escherichia coli. Expression was induced with IPTG and the resulting fusion peptide was affinity purified, followed by thrombin cleavage, and removal of vector coded sequences. r-Rub peptide inhibited ADP-induced platelet aggregation by 54% ± 6.38 in whole blood. We assessed the ability of r-Rub to initiate apoptosis in three human cancer cell lines. Cultures of SK-Mel-28, HeLA, and T24 cells were grown for 24 h with 2.5 µM r-Rub followed by Hoechst staining. Chromatin fragmentation was observed in treated SK-Mel-28, but not in T24 or HeLA cells. A TUNEL assay revealed that 51.55% ± 5.28 of SK-Mel-28 cells were apoptotic after 18 h of treatment with 3.5 µM of r-Rub. Cell migration and proliferation assays were performed in order to further characterize the biological effects of r-Rub on SK-Mel-28 cells. At 3 µM, r-Rub inhibited cell migration by 44.4% ± 0.5, while at 3.5 µM it was able to inhibit cell proliferation by 83% ± 6.0.