EXAMINATION OF THE ROLE OF ENDOPEPTIDASE-3.4.24.15 IN A-BETA SECRETION BY HUMAN TRANSFECTED CELLS

1 We have taken advantage of our recent development of highly potent a nd specific phosphinic inhibitors of endopeptidase 3.4.24.15 to examin e the putative contribution of the enzyme in the secretion of A beta b y HK293 transfected cells overexpressing the wild type and the Swedish (Sw) double mutated form of beta APP(751). 2 First, we showed that HK 293 cells contain a peptidase activity, the inhibition profile of whic h fully matches that of purified endopeptidase 3.4.24.15. Second, we e stablished that the treatment of HK293 cells with specific phosphinic inhibitors leads to about 80% inhibition of intracellular endopeptidas e 3.4.24.15 activity, indicating that these inhibitors penetrate the c ells. 3 Metabolic labelling of wild type and Sw beta APP(751)-expressi ng cells, followed by immunoprecipitation of A beta-containing peptide s, revealed the secretion of A beta and the intracellular formation of an A beta-containing 12 kDa product. 4 A beta secretion by Sw beta AP P(751) transfected cells was drastically enhanced when compared to cel ls expressing wild type beta APP(751). This production was not affecte d by endopeptidase 3.4.24.15 inhibitors in either cell type. This corr elates well with the observation that endopeptidase 3.4.24.15 does not cleave recombinant baculoviral Sw beta APP(751), in vitro. 5 Our prev ious data indicated that endopeptidase 3.4.24.15 activity was reduced in the parietal cortex of Alzheimer's disease affected brains and that the enzyme probably participated, in this brain area, to the cataboli sm of somatostatin 1-14. However, the present work indicates that endo peptidase 3.4.24.15 does not seem to behave as a beta-secretase in HK2 93 transfected cells. Therefore, it is suggested that endopeptidase 3. 4.24.15 could participate in the symptomatology, but probably not in t he aetiology of Alzheimer's disease.