RESUMO
Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing 31P NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using 13C solid-state NMR spectroscopy. α-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to ß-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k1), and the second step is an autocatalytic heterogeneous fibrillation process (k2).
Assuntos
Glucagon/química , Cinética , Bicamadas Lipídicas/química , Fosfolipídeos/química , Sequência de Aminoácidos/genética , Apoptose/genética , Radioisótopos de Carbono/química , Glucagon/genética , Humanos , Espectroscopia de Ressonância Magnética , Fosfolipídeos/genética , Radioisótopos de Fósforo/química , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Transdução de Sinais/genética , TemperaturaRESUMO
Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. To understand mechanism of fibril formation, we investigated the structure and kinetics of glucagon fibril formation using 13C solid-state NMR spectroscopy. In aqueous acetic acid solution at pH 3.3, distorted α-helical structure appeared around Gly4, Leu14, Ala19 and Leu26 in the monomeric form. In contrast, Gly4 and Ala19 were involved in ß-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k1), and the second step is an autocatalytic heterogeneous fibrillation process (k2). The rate constants k1 and k2 were separately determined in the acetic acid solution. Fibril formation was further investigated in the presence of lipid bilayers to mimic the physiological condition. We used bicelles which form discoidal nano-particles as the bilayer system and observed that the N-terminal α-helix did not change to ß-sheet when fibrils formed in the presence of bicelles. Rate constant k1 became faster and k2 became slower in the presence of bicelles compared to the case in the absence of bicelles. Our findings reveal that the structure and kinetics of fibril formation by glucagon are altered in the presence of lipid bilayers.