Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins A beta(1-40) and A beta(1-42), respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid P-protein ( AP) may be generated by the action of at least two different proteases term ed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavag e specificity of the two proteases, we treated amyloid precursor protein (A PP)-transfected cell cultures with several dipeptidyl aldehydes including N -benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benz yloxycarbonyl-Val-leucinal (Z-VL-CHO). Ail dipeptidyl aldehydes tested inhi bited production of both A beta(1-40) and A beta(1-42). Changes in the P-1 and P-2 residues of these aldehydes, however, indicated that the amino acid s occupying these positions are important for the efficient inhibition of g amma-secretases. Peptidyl aldehydes inhibit both cysteine and serine protea ses, suggesting that the two gamma-secretases belong to one of these mechan istic classes. To differentiate between the two classes of proteases, we tr eated our cultures with the specific cysteine protease inhibitor E-64d. Thi s agent inhibited production of secreted A beta(1-40), with a concomitant a ccumulation of its cellular precursor indicating that gamma( 40)-secretase is a cysteine protease, in contrast, this treatment increased production of secreted A beta(1-42). No inhibition of A beta production was observed wit h the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting t hat calpain is not involved. Together, these results indicate that gamma(40 )-secretase is a cysteine protease distinct from calpain, whereas gamma(42) -secretase may be a serine protease, In addition, the two secretases may co mpete for the same substrate. Dipeptidyl aldehyde treatment of cultures tra nsfected with APP carrying the Swedish mutation resulted in the accumulatio n of the beta-secretase C-terminal APP fragment and a decrease of the alpha -secretase C-terminal APP fragment, indicating that this mutation shifts AP P cleavage from the alpha-secretase site to the alpha-secretase site.