Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretas es generates the amino and carboxy termini, respectively, of the A beta amy loidogenic peptides A beta 40 and A beta 42-the major constituents of the a myloid plaques in the brain parenchyma of Alzheimer's disease patients(1). There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activ ity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease(2,3) increase the production of A beta 42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neur onal cultures derived from PS1-deficient mouse embryos(7); and directed mut agenesis of two conserved aspartates in transmembrane segments of PS1 inact ivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain(8). It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmem brane aspartyl protease or a gamma-secretase cofactor, or helps to colocali ze gamma-secretase and APP. Here we report photoaffinity labelling of PS1 ( and PS2) by potent gamma-secretase inhibitors that were designed to functio n as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may conta in the active site of gamma-secretase. Interestingly, the intact, single-ch ain form of wild-type PS1 is not labelled by an active-site-directed photoa ffinity probe, suggesting that intact wild-type PS1 may be an aspartyl prot ease zymogen.