RESUMO
Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Insulisina/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hidrólise , Insulisina/química , Insulisina/genética , Insulisina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Placa Amiloide/química , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid-beta (Abeta) appears central to Alzheimer's disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Abeta toxicity. We report that tPA alone had no effect on Abeta toxicity. However, in combination with plgn, tPA reduced Abeta toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Abeta accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Abeta resulted in a marked reduction of Abeta degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Abeta.
