RESUMO
Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule's poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both (15)N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.
Assuntos
Desenho de Fármacos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeos/química , Peçonhas/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cristalografia por Raios X , Exenatida , Peptídeo 1 Semelhante ao Glucagon/síntese química , Peptídeos/síntese química , Estabilidade Proteica , Estrutura Secundária de Proteína , Ratos , Peçonhas/síntese químicaRESUMO
Trp-cage miniprotein was used to investigate the role of a salt-bridge (Asp(9) -Arg(16) ) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side-chains are also part of a dense interaction network composed of electrostatic, H-bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt-bridge with those of the salt-bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp-cage miniproteins is more complex than a simple two-state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt-bridge stabilization; in the miniprotein variants equipped with salt-bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt-bridge deleted mutants. In this molecular framework Arg(16) stabilizes more than Asp(9) does, because of its higher degree of 3D-fold cooperation. In conclusion, the Xxx(9) leftright arrow Xxx(16) salt-bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network.
Assuntos
Estrutura Secundária de Proteína , Proteínas/química , Sais/química , Triptofano/química , Sequência de Aminoácidos , Arginina/química , Asparagina/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Análise Espectral/métodos , TermodinâmicaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a cerebrovascular dilator and was found neuroprotective in numerous in vitro and in vivo models of cerebral ischemia. However, the mechanism of its cerebrovascular action is poorly known, especially in newborns. Therefore, we tested pial arteriolar responses to the two naturally occurring forms PACAP27 and 38 as well as to shorter sequences (PACAP6-27, 6-38, 1-15, 6-15, 20-31). We also investigated the involvement of nitric oxide synthase (NOS), cyclooxygenase-1 and -2 (COX-1 and -2) activity in PACAP-induced pial arteriolar responses using the NOS inhibitor N-omega-nitro-l-arginine methyl ester (L-NAME 15 mg/kg iv), the non-selective COX inhibitor indomethacin (5 mg/kg iv), and the selective COX-1 and COX-2 inhibitors SC-560 (1 mg/kg iv) and NS-398 (1 mg/kg iv), respectively. Anesthetized, ventilated piglets (n=127) were equipped with closed cranial windows, and pial arteriolar diameters were determined via intravital microscopy. Topical application of both natural PACAPs, but none of the PACAP segments, resulted in prominent, repeatable, dose-dependent vasodilation. Percentage changes ranged 5+/-1-29+/-6 (n=7) and 4+/-1-36+/-7 (n=9) to 10(-)(8) to 10(-)(6) M PACAP27 and 38 (mean+/-SEM), respectively. Vasodilation to both natural PACAPs was significantly reduced by co-application with PACAP6-27 or 6-38, but not by L-NAME. Indomethacin abolished PACAP38 but not PACAP27-induced vasodilation. Arteriolar responses to PACAP38 were also sensitive to SC-560 but not to NS-398 suggesting the unique involvement of COX-1 activity in this response. In summary, PACAP27 and 38 are potent vasodilators in the neonatal cerebral circulation with at least two distinct mechanisms of action: a COX-dependent and a COX-independent pathway.
Assuntos
Arteríolas/efeitos dos fármacos , Pia-Máter/irrigação sanguínea , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Vasodilatadores/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Arteríolas/fisiologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Pia-Máter/efeitos dos fármacos , Suínos , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system including the retina. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently, we have shown that PACAP1-38 is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. Studying the molecular mechanisms of this protection has revealed that PACAP1-38 stimulates anti-apoptotic mechanisms such as phosphorylation of ERK1/2 and inhibits pro-apoptotic signaling molecules such as JNK1/2, p38MAPK, caspase-3 and the translocation of mitochondrial cytochrome c and apoptosis inducing factor in glutamate-treated retinas in vivo. In the present study we investigated the effects of PACAP1-38 on a further signal transduction pathway possibly involved in the protective effect of intravitreal PACAP1-38 administration against apoptotic retinal degeneration induced by neonatal MSG treatment. The focus of the present study was the protein kinase A (PKA)-Bad-14-3-3 transduction pathway. In vivo MSG treatment led to a reduction in the levels of anti-apoptotic molecules (phospho-PKA phospho-Bad, Bcl-xL and 14-3-3 proteins) in the retina. Co-treatment with PACAP1-38 counteracted these effects: the level of phospho-PKA, phospho-Bad, Bcl-xL and 14-3-3 were increased. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP1-38 activates the PKA-Bad-14-3-3 pathway which is inhibited by MSG treatment. Our results also provide new insights into the signaling mechanisms possibly involved in the PACAP-mediated anti-apoptotic effects.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Doenças Retinianas/metabolismo , Transdução de Sinais/fisiologia , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Animais Recém-Nascidos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico , Modelos Biológicos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Ratos , Ratos Wistar , Doenças Retinianas/induzido quimicamente , Transdução de Sinais/efeitos dos fármacosRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.
Assuntos
Apoptose/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Doenças Retinianas/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Glutamato de Sódio/toxicidade , Animais , Ratos , Ratos WistarRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective in numerous models. Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)-induced neuronal damage. We tested whether PACAP and/or VIP preserve CR to I/R-sensitive dilator responses dependent on endothelial and/or neuronal function. Accordingly, changes in pial arteriolar diameters in response to hypercapnia (5-10% CO(2) ventilation) or topical N-methyl-d-aspartate (NMDA, 10(-4) M) were determined before and after I/R via intravital microscopy in anesthetized/ventilated piglets. Local pretreatment with non-vasoactive doses of PACAP (10(-8) M) and VIP (10(-9) M) prevented the attenuation of postischemic CR to hypercapnia; to 10% CO(2), the CR values were 27+/-8% vs 92+/-5% vs 88+/-13% (vehicle vs PACAP38 vs VIP, CR expressed as a percentage of the response before I/R, mean+/-SEM, n=8-8, p<0.05). PACAP, but not VIP, preserved CR to NMDA after I/R, with CR values of 31+/-10% vs 87+/-8% vs 35+/-12% (vehicle vs PACAP38 vs VIP, n=6-6). Unlike PACAP, VIP-induced vasodilation has not yet been investigated in the piglet. We tested whether VIP-induced arteriolar dilation was sensitive to inhibitors of cyclooxygenase (COX)-1 (SC-560, 1 mg/kg), COX-2 (NS-398, 1 mg/kg), indomethacin (5 mg/kg), and nitric oxide synthase (L-NAME, 15 mg/kg). VIP (10(-8)-10(-7)-10(-6) M, n=8) induced reproducible, dose-dependent vasodilation of 16+/-3%, 33+/-6%, and 70+/-8%. The response was unaffected by all drugs, except that the vasodilation to 10(-8) M VIP was abolished by SC-560 and indomethacin. In conclusion, PACAP and VIP differentially preserve postischemic CR; independent of their vasodilatory effect.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/farmacologia , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Arteríolas/fisiopatologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Artérias Cerebrais/metabolismo , Artérias Cerebrais/fisiopatologia , Circulação Cerebrovascular/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Indometacina/farmacologia , Masculino , Fármacos Neuroprotetores/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Sus scrofa , Peptídeo Intestinal Vasoativo/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
Pituitary adenylate cyclase activating popypeptide (PACAP) is a pleiotropic neuropeptide, exerting neurotrophic and neuroprotective effects in numerous models of in vitro and in vivo nervous injuries. The aim of the present study was to investigate whether PACAP is neuroprotective in ischemic retinal damage. Adult male Wistar rats underwent bilateral carotid occlusion and PACAP was administered unilaterally into the vitreous body immediately following carotid occlusion. Retinas were analyzed three weeks after the injury. It was found that bilateral carotid occlusion led to a severe degeneration of all retinal layers. PACAP treatment significantly ameliorated the carotid occlusion-induced retinal damage: the overall thickness of the retina was significantly more than in control carotid occluded animals and the morphological characteristics of the photoreceptors showed nearly normal appearance. The outer plexiform layer remained discernible and the inner and outer nuclear layers were significantly thicker than in control animals. In summary, our present study provides evidence, for the first time, that PACAP attenuates ischemic retinal degeneration.