BACE1 palmitoylation occurs mainly at four 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) () 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 (). Next, we evaluated palmitoylation of BACE1 using 3H-PA labeling. As shown in , 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 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 by immunoprecipitation (more ...)
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 high-density membrane fractions (fractions 7–10) (). 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 (). These results indicate that lipid raft distribution of BACE1 depends on its palmitoylation in SH-SY5Y cells.
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 (more ...)
Since the distribution patterns of BACE1 in 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 (), suggesting that palmitoylation specifically at cysteine 474 does not have a major impact on lipid raft targeting of BACE1.
Lipid raft association of BACE1 depends on palmitoylation in primary cortical neurons
We further evaluated lipid raft localization of BACE1 in rat primary cortical neurons infected with recombinant adenoviruses expressing BACE1-WT or BACE1-CA4. Upon sucrose density gradient fractionation, lipid rafts were recovered mainly in fraction 4, where flotillin-1 was predominantly present. A proportion of BACE1 was recovered in fraction 4, while the majority was present in high-density nonraft fractions (fraction 8–10) (). The percentage of BACE1 in the raft fraction was 14% and 3% in neurons expressing BACE1-WT and BACE1-CA4, respectively (), confirming that lipid raft association of BACE1 is dependent on its palmitoylation in primary neurons as well.
Figure 4 Lipid raft distribution of BACE1 depends on palmitoylation in primary rat cerebral cortical neurons. (a) CHAPS extracts of cultured neurons infected with recombinant adenoviruses expressing BACE1-WT or BACE1-CA4 were fractionated via sucrose density gradient (more ...)
A proportion of APP was detected in fraction 4, with the majority recovered from nonraft fractions. The percentages of APP in the raft fraction were similar for SH-BACE1-WT- and SH-BACE1-CA4-expressing cells (), suggesting that raft distribution of BACE1 does not directly affect that of APP.
We additionally evaluated the cellular localization of BACE1 using double immunofluorescence staining. Immunostaining with 1D4 revealed localization of BACE1 in both soma and neurites. There were no appreciable differences in the staining patterns between BACE1-WT- and BACE1-CA4 (). Upon double labeling with 1D4 and anti-flotillin-1, flotillin-1 immunoreactivity was observed as punctate staining that partially overlapped that of 1D4. The extent of the colocalization of 1D4 and flotillin-1 immunoreactivities appeared reduced in neurons expressing BACE1-CA4, relative to those expressing BACE1-WT (), consistent with the results of biochemical fractionation.
Aβ production is not influenced by raft association of BACE1 in neurons
Since lipid rafts appear to represent an important site for amyloidogenic processing of APP by BACE1 (Cordy et al. 2006
; Araki 2010
; Rushworth and Hooper 2010
; Vetrivel and Thinakaran 2010
), we analyzed the secretion of Aβ from primary neurons overexpressing BACE1-WT or BACE1-CA4. On Western blots, neurons expressed comparable levels of BACE1-WT and BACE1-CA4 (). BACE1-WT and BACE1-CA4 enhanced secretion of both Aβ40 and Aβ42 to similar extents (by approximately 80%), compared to control cells infected with empty adenovirus ( and c).
Figure 5 Neuronal Aβ production is not influenced by raft association of BACE1. (a–c) Cultured neurons were infected with recombinant adenoviruses expressing BACE1-WT, BACE1-CA4, or empty adenoviruses (mock). (a) Cell lysates were analyzed by Western (more ...)
To determine the effects of BACE1-WT and BACE1-CA4 on Aβ production from overexpressed APP, neurons were infected with both recombinant wild-type APP and BACE1 adenoviruses. Compared to neurons expressing APP only, mature APP levels were significantly diminished in those expressing APP plus BACE1-WT or APP plus BACE1-CA4, suggesting that a significant proportion of APP is cleaved by BACE1 (). Consistently, neurons coexpressing APP and either BACE1-WT or BACE1-CA4 secreted ~6.5-fold higher amounts of Aβ40 and ~2.5-fold or ~2.9-fold higher amounts of Aβ42, respectively, than those expressing APP alone (). Together, BACE1-WT and BACE1-CA4 exerted similar Aβ-promoting effects, suggesting that β-cleavage of APP does not depend on raft localization of BACE1.
β-CTF is predominantly localized in nonraft membrane domains
To evaluate the β-cleavage of APP in raft and nonraft domains, we performed Western blot analysis of APP CTF. Western blots of RIPA lysates revealed that levels of β-CTF and β′-CTF (derived from alternative BACE1 cleavage of APP between Tyr10 and Glu11 within the Aβ region) were remarkably increased and those of α-CTF (derived from α-secretase cleavage of APP between Lys16 and Leu17 within the Aβ region) decreased in neurons expressing APP plus BACE1-WT or APP plus BACE1-CA4, compared to those expressing APP alone (). Subsequently, we examined the distribution of APP CTF in raft and nonraft fractions following sucrose density gradient fractionation. Immunoprecipitation–Western blot analysis revealed that the majority of β-CTF and β′-CTF was recovered in nonraft fractions (fractions 8–10) of neurons expressing APP plus BACE1-WT or APP plus BACE1-CA4, whereas only low levels were present in the raft fraction (fraction 4). No differences in the localization pattern of CTFs were observed between neurons expressing BACE1-WT and BACE1-CA4 ().
Figure 6 Predominant localization of APP CTF in nonraft domains of neurons coexpressing APP and BACE1. (a) RIPA or CHAPS extracts of neurons coexpressing APP and either mock or BACE1-WT or BACE1-CA4 were subjected to Tris/Tricine SDS-PAGE and immunoblotting with (more ...)
Next, we evaluated the β-cleavage of APP by endogenous BACE1. For this purpose, primary neurons overexpressing Swedish mutant APP, a preferred substrate of BACE1, via recombinant adenoviruses were treated with a γ-secretase inhibitor DAPT (N
-butyl ester) (Dovey et al. 2001
) that augments the levels of APP CTF. The distributions of endogenous BACE1 and APP CTF were then analyzed following sucrose density gradient fractionation. Bands of BACE1 were observed in both raft and nonraft fractions and faint bands probably representing dimeric BACE1 were additionally detected in nonraft fractions (). Higher levels of β-CTF and β′-CTF were obviously recovered in nonraft fractions than in the raft fraction (). These results suggest that β-cleavage of APP by overexpressed as well as endogenous BACE1 occurs mainly in nonraft fractions.
Shedding of BACE1 is regulated by palmitoylation
Next, we focused on the effects of palmitoylation on BACE1 shedding. As reported previously (Murayama et al. 2005
), immunoprecipitation–Western blot analysis of conditioned media of SH-BACE1-WT cells revealed two bands corresponding to soluble BACE1 (sol-BACE1) and full-length BACE1 (FL-BACE1) (). Compared to SH-BACE1-WT cells, the FL-BACE1 level appeared increased, while that of sol-BACE1 was decreased in media of SH-BACE-CA4 cells (). Quantitative analysis disclosed a significant increase in the FL-BACE1/sol-BACE1 ratio in the media of SH-BACE1-CA4 cells relative to SH-BACE1-WT cells ().
Figure 7 BACE1 shedding is regulated by palmitoylation. (a) Conditioned media of SH-BACE1-WT or SH-BACE1-CA4 cells were analyzed by immunoprecipitation–Western blot analysis, as described in section Materials and Methods. Two bands corresponding to FL-BACE1 (more ...)
BACE1 shedding was additionally analyzed in primary neurons expressing BACE1-WT or BACE1-CA4. The FL-BACE1/sol-BACE1 ratio in the conditioned media was significantly increased in BACE1-CA4-expressing neurons, compared to BACE1-WT-expressing neurons (). Altogether, the results suggest that BACE1 shedding is positively regulated by palmitoylation.
BACE1 dimerization is not affected by palmitoylation
Finally, we investigated whether palmitoylation affects the homodimer formation of BACE1. Extracts of the membrane fractions of SH-BACE1-WT, SH-BACE1-CA3, and SH-BACE1-CA4 cells were separated using BN-PAGE, followed by Western blotting. For SH-BACE1-WT cells, a band with mass of ~160 kDa instead of the expected mass of ~70 kDa reacted with the 1D4 antibody, confirming that BACE1 exists as a homodimer under native conditions (). Similarly, only the band representing dimeric BACE1 was observed in SH-BACE1-CA3 and SH-BACE1-CA4 cells (). Therefore, BACE1 dimer formation appeared to be unaffected by palmitoylation.
Figure 8 BACE1 dimerization is not affected by palmitoylation. Extracts of membrane fractions of SH-BACE1-WT, SH-BACE1-CA3, or SH-BACE1-CA4 cells were separated using SDS-PAGE (a) or BN-PAGE (b), and analyzed via Western blotting with 1D4. On BN-PAGE, only a ~160-kDa (more ...)