科研之友
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摘要
gamma-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the gamma-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks gamma-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. gamma-Secretase activity was measured based on yeast growth on selective media and beta-galactosidase activity. PS1 gamma-secretase was similar to 24-fold more active than PS2 gamma-secretase in the beta-galactosidase assay. Using yeast microsomes containing gamma-secretase and C55, we compared the concentration of A beta generated by PS1 or PS2 gamma-secretase. PS1 gamma-secretase produced similar to 24-fold more A beta than PS2 gamma-secretase. We found the optimal pH of A beta production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one gamma-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the gamma-secretase complex were similar to 28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in A beta production.
- 出版日期2011-12-30
