Correction: Palmitoylated APP Forms Dimers, Cleaved by BACE1

Abstract

[This corrects the article DOI: 10.1371/journal.pone.0166400.].

Fig 1. palAPP dimerizes ~4.5 times more efficiently compared totAPP and in cis-orientiation.

A. Cells expressing APP_V5 or APP_V5 plus HA-APP_Y were subjected to co-immunoprecipitation assays to detect APP_V5/HA-APP_Y interaction or APP-dimerization. APP_V5 was immunoprecipitated with an anti-V5 antibody. Immunoprecipitates were probed with an anti-HA antibody to detect pull-down of HA-APP_Y. Subsequently the immunoprecipitates were subjected to mABE assay to detect palAPP_V5/HA-APP_Y interaction (or palAPP-dimerization). PalAPP_V5 pulled down both palAPP_V5 (M_wt ~102 kD) and palHA-APP_Y (M_wt ~150 kD) from cells expressing APP_V5 plus HA-APP_Y but not from cells expressing only APP_V5. B. TotAPP-dimers (APP_V5/HA-APP_Y) only form in cells expressing both APP_V5 and HA-APP_Y. C. Quantitation of palAPP-dimers (palAPP_V5/palHA-APP_Y) versus totAPP-dimers (APP_V5/HA-APP_Y). Error bars show the s.e.m. (**p<0.01). D. palAPP dimerizes is cis-orientiation. Cells expressing HA-APP_Y and cells expressing mycAPP were co-cultured in absence or presence of 1mM cell-impermeable cross-linker DTSSP. Cell extracts were subjected to a pull-down assay, using an anti-HA antibody to immunoprecipitate HA-APP_Y. To test for APP-dimerization, the precipitates were probed with an anti-myc antibody (panel b, co-culture). Cells co-expressing HA-APP_Y and mycAPP were also subjected to a co-IP assay using the anti-HA antibody to pull-down mycAPP with HA-APP_Y.(panel b, co-expression). To detect palAPP-dimerization, the immunoprecipitates were also subjected to mABE assay to detect co-IP of palHA-APP_Y with pal-mycAPP (panel a). The experiment is a representative of three independent experiments.

Fig 2. Palmitoylation-prone APP mutants exhibit increased APP dimerization compared to wtAPP.

A. Schematic representation of the Cys to Ser mutants of APP used for the following co-immunoprecipitation assays. B. Co-immunoprecipitation assay in cells co-expressing APP_V5 and HA-APP_Y and its mutants containing indicated Cys to Ser substitution. HA-APP_Y pulls down APP_V5, indicating APP-APP dimerization. APP(C¹³³S) and APP(C¹⁵⁸S) show 2 fold increase in dimerization, while APP(C¹⁸⁶S) and APP(C¹⁸⁷S) fail to dimerize. APP(C¹⁸⁶S) and APP(C¹⁸⁷S) generated trace amounts of palmitoylation-independent dimers (* and **). C. ABE assay of cells overexpressing indicated APP mutants show 2 fold increased palmitoylation of APP(C¹³³S) and APP(C¹⁵⁸S), where as APP(C¹⁸⁶S) and APP(C¹⁸⁷S) were defective in palmitoylation.

Fig 6. Palmitoylation inhibitors specifically impair ectodomain-dependent APP dimerization but not APP-CTF-dimerization.

A. Naïve CHO cells co-expressing APP_V5 and APP_HA were subjected to a co-IP assay in presence of DMSO (0 μg/ml) or increasing concentrations of cerulenin (25, 50 and 100 μg/ml). flAPPHA (APP_HA) pulled down fl- as well as the C-terminal fragments of APP_V5 (APP_V5 and CTF_V5, respectively) in DMSO-treated (0 μg/ml cerulenin) cells. In presence of cerulenin, co-IP of flAPP_V5 (APP_V5) with flAPP_HA (APP_HA) decreased in a dose-dependent manner. Little or no co-IP of flAPP observed upon treatment with100 μg/ml cerulenin. In contrast, cerulenin had no effect on CTF_V5 pull-down even at the highest concentration (100 μg/ml). Cerulenin reduced palAPP_HA levels in a dose-dependent manner (ABE assay) reaching complete inhibition at 100 μg/ml concentration. B. co-IP assay using an antibody specific for mGFP (anti-mGFP) to pull-down full-length (fl) APP_mGFP with APP_mCherry from differentiated neuronal cells (RenVM) co-expressing APP_mGFP+APP_mCherry. Anti-mGFP also pulled-down CTF_mCherry with CTF_mGFP. Cerulenin (25 μg/ml) treatment of the cells prior to co-IP assay dramatically decreased flAPP_mGFP-flAPP_mCherry interaction, but not that of CTF_mGFP-CTF_mCherry.