05. Results BACE1 palmitoylation occurs mainly at four Oligomycin A IC50 cysteine residues in the C-terminal region We investigated S-palmitoylation of BACE1 using human neuroblastoma cells stably expressing BACE1. cDNA of wild-type BACE1 (BACE1-WT) or mutant BACE1 (BACE1-CA3 or BACE1-CA4 with three or four Cys–Ala substitutions, respectively) (Fig. 1) was transfected into SH-SY5Y cells and stable transfectants (designated SH-BACE1-WT, SH-BACE1-CA3, and SH-BACE1-CA4 cells) were established. Western blot analysis revealed that all transfected cells expressed equal levels of BACE1 Inhibitors,research,lifescience,medical (Fig. 2a). Next, we evaluated palmitoylation of BACE1 using 3H-PA labeling. As shown in Figure 2b, the 3H-PA-labeled BACE1 level was reduced to 23% and 2% in BACE1-CA3-
and BACE1-CA4-expressing cells, respectively, compared to BACE1-WT cells. These findings indicate that palmitoylation of BACE1 is abolished in the BACE1-CA4 mutant, confirming that BACE1 is mainly modified at the four cysteine residues in the C-terminal region. Figure 2 Four cysteine residues in BACE1 are palmitoylated. (a) Cell Inhibitors,research,lifescience,medical lysates of SH-BACAE1-WT, SH-BACE1-CA3, and SH-BACE1-CA4 cells were analyzed by immunoblotting with 1D4 antibody. (b) Cells were labeled with 3H-palmitic acid, and lysates analyzed Inhibitors,research,lifescience,medical by immunoprecipitation … Lipid raft distribution of BACE1 depends on palmitoylation in neuroblastoma cells Next, we evaluated the role of palmitoylation in lipid raft
distribution of BACE1 in SH-SY5Y cells via sucrose density gradient centrifugation. Immunoblot
analysis of individual fractions showed that the raft marker, flotillin-1, was present in fraction 4, indicative of the fractionation of lipid raft components. Although a proportion of BACE1 was recovered in fraction 4, the majority of the protein was present in Inhibitors,research,lifescience,medical high-density membrane fractions (fractions 7–10) (Fig. 3a). The percentages of BACE1 distributed in the raft fraction per total BACE1 in all fractions for BACE1-WT, BACE1-CA3, and BACE1-CA4 cells were 15.6%, 11.4%, and 5.8%, respectively (Fig. 3a). These results indicate that lipid raft distribution of BACE1 depends on its palmitoylation in SH-SY5Y cells. Inhibitors,research,lifescience,medical Figure 3 Lipid raft distribution of BACE1 depends on palmitoylation in neuroblastoma cells. (a) SH-BACE1-WT, SH-BACE1-CA3, and SH-BACE1-CA4 cells were subjected to sucrose density gradient fractionation, as described in section Materials and Methods. Each fraction … Since Brefeldin_A the distribution patterns of BACE1 in Palbociclib cell cycle SH-BACE1-CA3 and SH-BACE1-CA4 cells were markedly different, we examined the effect of palmitoylation at cysteine 474 alone. For this purpose, we established SH-SY5Y cells stably expressing BACE1-C474A (designated SH-BACE1-C474A cells). No significant differences were observed in the percentage of BACE1 within the raft fraction between SH-BACE1-WT and SH-BACE1-C474A cells (Fig. 3b), suggesting that palmitoylation specifically at cysteine 474 does not have a major impact on lipid raft targeting of BACE1.