Three-dimensional structure of the γ-secretase complex☆
Section snippets
Materials and methods
Construction of expression plasmid and recombinant baculovirus production. N-terminally His/FLAG-tagged PS1 was generated from PS1 inserted in pBlueBacHis2A (Invitrogen) by the long PCR mutagenesis. A cDNA encoding PEN-2 without tag was generated by deletion mutagenesis of His/Xpress-PEN-2 in pBlueBacHis2A [13]. NCT-V5/His and APH-1a-myc/His were inserted in pBlueBac4.5, as described previously [13]. Recombinant baculovirus construction, the culture of Sf9 cells, and the isolation of membrane
Purification of the recombinant active γ-secretase complex from infected Sf9 cells
A deletion of N terminus of PS was tolerant for γ-secretase activity, although placement of a C-terminal tag diminishes complex formation and activity [5], [21], [30]. Thus, we fused His/FLAG tag at the N terminus of PS1 for effective purification of the functional enzyme complex (Fig. 1). Sf9 cells were infected with recombinant baculoviruses encoding His/FLAG-PS1, NCT-V5/His, APH-1-myc/His, and PEN-2. Like wild-type PS1, a majority of the recombinant His/FLAG-PS1 polypeptides were
Discussion
Growing body of evidence demonstrates that several transmembrane proteins are endoproteolyzed within their transmembrane domains. The proteases responsible for this unusual activity are termed intramembrane-cleaving proteases (I-CliPs) [32]. The mechanism of this endoproteolysis, a process that requires ionized water to locate within the hydrophobic lipid bilayer, remains unknown. To date, no structural information is available for any I-CliPs including γ-secretase complex, motivating us to
Acknowledgments
We thank Takeda Pharmaceutical Company for Aβ ELISA, Dr. Yasuko Takahashi for making G1Nr3 antibody, and our lab members for helpful discussions and technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports for the 21st Century Center of Excellence Program (for T.I., T.T.), by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) (for T.I., T.T.), by
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Abbreviations: PS, presenilin; NCT, nicastrin; APP, amyloid-β precursor protein; Aβ, amyloid-β peptides; NICD, Notch intracellular domain; SEC, size exclusion chromatography; 3D, three-dimensional; EM, electron microscopy; MRA, multi-reference alignment; GNG, growing neural gas network; NN, neural network; PAGE, polyacrylamide gel electrophoresis; SA, simulated annealing; SDS, sodium dodecyl sulfate; TMD, transmembrane domain; I-CliPs, intramembrane-cleaving proteases.