Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from c-secretase William A. Campbell, Megan L. O. Reed, Jennifer Strahle, Michael S. Wolfe and Weiming Xia Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA Abstract Presenilin (PS)-dependent c-secretase cleavage is the final proteolytic step in generating amyloid b protein (Ab), a key peptide involved in the pathogenesis of Alzheimer’s disease. PS undergoes endoproteolysis by an unidentified ‘presenilin- ase’ to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the c-secretase active site. To better understand the relationship between presenilinase and c-secretase, we characterized the biochemical properties of presenilinase and compared them with those of c-secretase. Similar to c-secretase, presenilin- ase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC 50 of  1 lM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC 50 <1 lM) over c-secretase (IC 50 )16 lM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and c-secretase inhibition, sug- gesting that presenilinase, like c-secretase, is an aspartyl protease. Interestingly, several of the most potent c-secretase inhibitors (IC 50 ¼ 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of Ab in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permit- ted normal de novo generation of Ab and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from c-secretase. Keywords: amyloid, c-secretase, presenilin, presenilinase. J. Neurochem. (2003) 85, 1563–1574. Genetic and neuropathological studies suggest that process- ing of amyloid precursor protein (APP) to yield amyloid b protein (Ab) plays an important role in the pathogenesis of Alzheimer’s disease (AD) (Selkoe 2001). The final proteo- lytic event in generating Ab is accomplished through presenilin (PS)-dependent c-secretase cleavage (Selkoe 2001). While absolute identification of the catalytic compo- nent of c-secretase activity has been elusive, accumulating evidence points to a role for PS1 and PS2. Evidence for a requirement of PS for c-secretase cleavage of APP for Ab generation comes from numerous studies: Ab generation is impaired when two critical aspartate residues in transmem- brane (TM) domains 6 and 7 of PS1 or PS2 are mutated in cultured cells (Steiner et al. 1999b; Wolfe et al. 1999a; Kimberly et al. 2000) and in transgenic mice (Xia et al. 2002), and there is no Ab production in PS knockout neurons (De Strooper et al. 1998; Herreman et al. 2000). We have demonstrated that PS1 and PS2 bind to C99/C83 at the major sites of Ab generation, i.e. Golgi/trans-Golgi network (TGN)-type vesicles (Xia et al. 2000b). Moreover, aspartyl protease transition state analogue inhibitors of c-secretase bind directly to PS N-terminal and C-terminal fragment (NTF and CTF), suggesting that the protease active site lies between these two PS subunits (Esler et al. 2000; Li et al. 2000b). PS and PS homologues have non-classic protease motifs conserved from bacteria to human (Steiner et al. 2000; Ponting et al. 2002), and the sequence motifs of PS are similar to a recently identified signal peptide peptidase (Weihofen et al. 2002). PS NTF/CTF are derived from endoproteolysis of full- length (FL) PS between the sixth and seventh of the eight Received February 4, 2003; revised manuscript received March 11, 2003; accepted March 12, 2003. Address correspondence and reprint requests to Dr Weiming Xia, Center for Neurologic Diseases, Harvard Institutes of Medicine, HIM 616, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. E-mail: wxia@rics.bwh.harvard.edu Abbreviations used:Ab, amyloid-b protein; AD, Alzheimer’s disease; AICD, amyloid intracellular domain; APP, b-amyloid precursor protein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CTF, C-terminal fragment; DMSO, dimethyl sulfoxide; EDTA, ethylenedi- amine-tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FAD, familial Alzheimer’s disease; FL, full- length; NTF, N-terminal fragment; PS, presenilin; SDS, sodium dodecyl sulfate; TGN, trans-Golgi network; TM, transmembrane; WT, wild-type. Journal of Neurochemistry , 2003, 85, 1563–1574 doi:10.1046/j.1471-4159.2003.01799.x Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1563–1574 1563