CLEAVAGE OF FLUOROGENIC SUBSTRATES FOR APP-PROCESSING PROTEASES BY HUMAN BRAIN EXTRACTS

The proteases that cleave amyloid precursor protein (APP) leading to g eneration of amyloid A beta peptide are potential targets for therapeu tical intervention of Alzheimer disease. We have been pursuing the ide ntification and characterization of these proteases using as probes th e fluorogenic substrates encompassing the cleavage sites of APP that w e described recently (Wang, G. T., Krafft, G. A. [1992] Bioorg. Med. C hem. Lett. 2, 1665). This article describes results of experiments des igned to examine the effect of Ca2+ on the cleavage of these substrate s by human brain extracts. Fluorogenic substrates encompassing either the N-terminal amyloidogenic cleavage site or the secretory cleavage s ite were synthesized in five formats with various peripheral residues. Incubation with extracts from normal brain tissue revealed that more negatively charged amyloidogenic substrates were less reactive and exh ibited larger rate enhancement in the presence of Ca2+, The results im ply that Ca2+ stimulation of substrate cleavage by brain proteases occ urs primarily as a result of Ca2+-substrate interactions, and caution against interpretations that invoke the involvement of Ca2+-stimulated proteases in A beta formation.