RESUMO
The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2 µM and Ki2=26 µM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca(2+)-dependent pNPPase was activated up to â¼5 µM chelerythrine with inhibition thereafter. Ca(2+)-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 µM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 µM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5-1.5 µM resulted in an increase of intracellular Ca(2+) on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca(2+) homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid's effects on SERCA.
Assuntos
Benzofenantridinas/química , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/química , Animais , Benzofenantridinas/metabolismo , Sítios de Ligação , Glutaral/química , Humanos , Hidrólise , Concentração Inibidora 50 , Leucócitos Mononucleares/citologia , Monócitos/metabolismo , Músculo Esquelético/enzimologia , Fosforilação , Ligação Proteica , Conformação Proteica , Coelhos , Tripsina/químicaRESUMO
Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.
Assuntos
Proteínas Sanguíneas/química , Inibidores de Fosfolipase A2/química , Fosfolipases A2/química , Proteínas de Répteis/química , Venenos de Serpentes/química , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/biossíntese , Bothrops , Brasil , Linhagem Celular , Espectrometria de Massas , Camundongos , Gambás , Pichia , Proteínas Recombinantes/biossínteseRESUMO
Thrombin, a highly specific protease of blood coagulation, has two exosites that modulate its specificity. We designed two sets of synthetic substrate FRET peptides with 25- or 11- amino acids (aa) each, based on the PAR 1 sequence, to characterize the effect of exosite 1 engagement on substrate catalysis and preference. The 25-aa set encompassed a sequence binding to exosite 1, and structural modeling showed that binding to thrombin did not differ significantly from that of PAR 1 peptide. Modification at the P3´position of the 25 or 11-aa peptides resulted in small effect on kinetic parameters. Ionic strength higher than physiologic depressed thrombin action on the 25-aa peptides. Addition of ligands of the exosite 1 negatively modulated the catalysis of 25-aa substrates. In conclusion, we succeeded to mimic and study in real time, using these synthetic peptides, the influence of ligand binding to exosite 1 on thrombin activity.