Palmitoylated APP Forms Dimers, Cleaved by BACE1

Abstract

A major rate-limiting step for Aβ generation and deposition in Alzheimer's disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8-10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

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 3. DHHC7 equally increases palmitoylation and dimerization of APP.

A. Co-IP assays to detect dimerization of APP_V5 and HA-APP_Y in presence or absence of indicated DHHC proteins. APP_V5 pulled-down HA-APP_Y, indicating APP dimerization. Overexpression of DHHC7 or DHHC21 increased co-IP of APP_V5 and HA-APP_Y, suggesting increased dimerization of APP in presence of these two palmitoylating enzymes. No effect on APP dimerization was observed in presence of DHHC1. EV represents empty vector. B. ABE analysis detected increased level of in palAPP (both palHA-APP_Y and palAPP_V5), as expected. C. Quantitation of dimerization assays (n = 3) detects 2.3 ± 0.17 fold increase of APP dimerization and ~2 fold (not shown) increase in APP palmitoylation in presence of DHHC7. Error bars show the s.e.m.

Fig 4. Palmitoylation inhibitors impair APP dimerization in CHO cells.

A. Co-immunoprecipitation assay of CHO cells expressing HA-APP_Y and APP_V5 in presence of increasing amounts of cerulenin (Cer) (0–100 μg/ml), where 0 μg/ml represents DMSO-treatment. Cerulenin decreased HA-APP_Y/APP_V5 interaction (APP-dimerization) in dose dependent manner similar to cerulenin’s effect on APP-palmitoylation. B. 25 μg/ml cerulenin and 50 μM 2-BP reduced both APP-palmitoylation and APP-dimerization in HA-APP_Y/APP_V5-expressing cells compared to DMSO (Veh)-treated cells. C. Quantitation showed 54 and 58% decrease of APP-APP dimerization by cerulenin (25 μg/ml) and 2-BP (50 μM), respectively. D. Naïve CHO cells were either transiently transfected with an expression plasmid encoding APP_mGFP or co-transfected with expression plasmids encoding APP_mGFP and APP_mCherry. After 24 h, cells were either treated with DMSO or with indicated palmitoylation inhibitors cerulenin or 2-BP for 6 h prior to formalin treatment. FRET/FLIM analysis was employed to measure decay time constant T_m of APP_mEGFP in cells expressing APP_mEGFP (n = 54), and APP_mGFP and APP_mCherry (n = 46). T_m of life-time decay of APP_mEGFP in APP_mEGFP/APP_mCherry co-expressing cells was monitored in absence and in presence of palmitoylation inhibitors (25 μg/ml cerulenin or 50 μM 2-BP). Quantitation revealed that inhibitors increased T_m values by ~1.5 fold, indicating disruption of dimerization between APP_mGFP and APP_mCherry.

Fig 5. Bimolecular Fluorescence Complementation (BiFC) assay detects APP dimers.

A. Fluorescence microscopy of cells transiently expressing APP C-terminal Split GFP 1–10 (APP(1–10)) and APP C-terminal split GFP 11 (APP(11)), FACS sorted for equal intensity. Cells showing green fluorescence represents the APP dimers. These cells were sorted in a fluorescence activated cell sorter (FACS) to obtain equal intensity cells. Next the sorted cells were grown on coverslips for 18 h before treating with 0 (DMSO), 25, 50 and 100μg/ml cerulenin for 6 h showed little or no change in fluorescence intensities. B. Cells expressing APP(1–10) or APP(1–10)+APP(11) were subjected to ABE assay. Probing the samples with anti-GFP detected palmitoylaed APP(1–10) (palAPP(1–10)). Anti-Flotillin antibody detected palmitoylated flotillin (palFlotillin) in ABE assay. C. BiFC intensities of the cells were quantitated using ImageJ software. Intensities of more than 50 cells were measured for each treatment. Average intensities are plotted in percent (%) change in dimerization, using no treatment as 100% describing changes in APP-dimerization percent by increasing amount of cerulenin (solid line). The discontinuous line represents decrease in palAPP levels obtained from ABE analysis upon cerulenin treatment. Error bars show the s.e.m.

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). flAPP_V5 (APP_V5) pulled down fl- as well as the C-terminal fragments of APP_HA (APP_HA and CTF_HA, 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_HA 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.

Fig 7. In vitro BACE-activity assay on palAPP-dimers in detergent resistant membranes (DRMs).

A. In vitro BACE-activity assay on DRMs isolated from CHO cells expressing HA-APP_Y and myc-APP, treated with increasing amounts of cerulenin (Cer) prior to membrane preparation. The in vitro BACE-activity assay was followed by co-IP analysis as described in Materials and Methods. Myc-sAPP_β pulled down HA-sAPP_β in absence of cerulenin. In presence of cerulenin, pull-down of HA-sAPP_β with myc-sAPP_β decreased in a dose dependent manner. 2-bromopalmitate (100μM) dramatically reduced co-IP of HA-sAPP_β and myc-sAPP_β. B. ABE assay of cells co-expressing HA-APP_Y and myc-APP. pal HA-APP_Y and palmyc-APP decreased upon cerulenin-treatment in a dose-dependent manner. C. Representation of dose-dependent decrease in HA-sAPP_β pull-down with myc-sAPP_β in presence of cerulenin.