Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels

Abstract

We have recently identified a novel form of post-translational regulation of BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid beta-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid beta-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia.

FIGURE 1.

Schematic view of ATase1 and ATase2. A, alignment of amino acid sequences of ATase1 and ATase2. Alignment was performed by using the Megalign component of the DNAstar (Lasergene version 6) software on a basis of ClustalW algorithm. Identical amino acids are highlighted in yellow. B, hydrophilicity profile (Kyte-Doolittle algorithm) of ATase1 and ATase2 was calculated using the protean component of the DNAstar (Lasergene version 6) software. TM indicates the transmembrane (hydrophobic) domain.

FIGURE 2.

ATase1 is localized in the early secretory pathway and has acetyl-CoA:lysine acetyltransferase activity. A, Western blot analysis of control (nontransfected) and ATase1-expressing CHO cells. ATase1 migrates with the expected mass on SDS-PAGE. B, total membrane extract from control and ATase1-expressing cells was incubated with [³H]acetyl-CoA and affinity-purified BACE1 for 1 h at 30 °C. Reaction was stopped by lowering the temperature; BACE1 was then purified again and analyzed on a scintillation liquid counter. As control, BACE1 was also incubated with [³H]acetyl-CoA in the absence of membrane extracts and with a membrane extract that had been boiled for 10 min prior to the reaction. Results are the average (n = 6) ± S.D. Asterisk indicates statistical significance (p < 0.005). C, subcellular distribution of ATase1 was analyzed by SDS-PAGE and immunoblotting after separation of intracellular membranes on a 10-24% discontinuous Nycodenz gradient. The appropriate subcellular markers are indicated. D, fractions shown in C were assayed for acetyl-CoA:lysine acetyltransferase activity as described in B.[³H]Acetyl-CoA served as donor of the acetyl group, whereas affinity-purified BACE1 served as acceptor substrate. Results are the average (n = 3) ± S.D. The migration pattern of ATase1 shown in C is shown here again to allow comparison with the biochemical activity of the single fractions. E, fraction 16 and 18 of a subcellular fractionation gradient (see C) from control and ATase1-expressing cells were compared for acetyl-CoA:lysine acetyltransferase activity. The assay was run as in D. Results are the average (n = 3) ± S.D. Asterisk indicates statistical significance (p < 0.005).

FIGURE 3.

ATase1 acetylates BACE1 in vitro. A, affinity-purified ATase1 and BACE1 were co-incubated in the presence of [³H]Acetyl-CoA for 1 h at 30 °C. Reaction was stopped by lowering the temperature; BACE1 was then purified again and analyzed on a scintillation liquid counter. The same assay was repeated with BACE1_Arg, a mutant form of BACE1 where the lysine residues that serve as acceptors of the acetyl groups have been mutated to arginine (5). Results are the average (n = 6) ± S.D. Asterisk indicates statistical significance (p < 0.005). B, affinity-purified ATase1 and BACE1 were co-incubated in the presence of acetyl-CoA (50 μm) for 6 h at 30 °C. Reaction was stopped by lowering the temperature. BACE1 was then immunoprecipitated with a bio-bead cross-linked antibody specific for the N-terminal domain of BACE1 and analyzed by Western blotting with the indicated antibodies (Ac. Lys., anti-acetylated lysine; BACE1 C-term., anti-BACE1 C-terminal domain; Myc, anti-Myc). Numbers indicate the different species of BACE1 (see supplemental Fig. 1 for detailed explanation). Asterisk indicates a band that could be recognized with an antibody against the N-terminal domain of BACE1 and migrated with the expected molecular mass of the cleaved ectodomain of BACE1. < indicates ATase1.

FIGURE 4.

ATase2 is localized in the early secretory pathway and has acetyl-CoA:lysine acetyltransferase activity. A, Western blot analysis of ATase1, ATase2, and ATase1 + 2 expressing CHO cells. B, total membrane extract from control (nontransfected) and ATase2-expressing cells was assayed for acetyltransferase activity as in Fig. 2B. Results are expressed as percent of control and are the average (n = 6) ± S.D. Asterisk indicates statistical significance (p < 0.005). C, subcellular distribution of ATase2 was analyzed by SDS-PAGE and immunoblotting after separation of intracellular membranes on a 10-24% discontinuous Nycodenz gradient. The appropriate subcellular markers are indicated. D, fractions 16, 18, and 20 of a subcellular fractionation gradient (see C) from control and ATase2-expressing cells were compared for acetyl-CoA:lysine acetyltransferase activity. The assay was performed as in Fig. 2E. Results are the average (n = 6) ± S.D. Asterisk indicates statistical significance (p < 0.005).

FIGURE 5.

ATase2 acetylates BACE1 in vitro. A, affinity-purified ATase2 and BACE1 were co-incubated in the presence of acetyl-CoA (50 μm) for 6 h at 30 °C. This experiment was performed as in Fig. 3B. Following immunoprecipitation with a bio-bead cross-linked antibody specific for the N-terminal domain, BACE1 was analyzed by SDS-PAGE and Western blotting. The following antibodies were used: Ac. Lys., anti-acetylated lysine; BACE1 C-term., anti-BACE1 C-terminal domain; Myc, anti-Myc. Numbers indicate the fully mature and biosynthetic intermediates of BACE1. Asterisk indicates a band that could be recognized with an antibody against the N-terminal domain of BACE1 and migrated with the expected molecular mass of the cleaved ectodomain of BACE1. < indicates ATase2. B, ATase2 was incubated with BACE1 in the presence of [³H]acetyl-CoA for 1 h at 30 °C a sin Fig. 3A. Both the enzyme (ATase2) and the acceptor substrate (BACE1) were purified by affinity chromatography prior to the incubation. BACE1_Arg was used as control because the lysine residues that serve as acceptors of the acetyl groups have been mutated to arginine (5). Results are the average (n = 6) ± S.D. Asterisk indicates statistical significance (p < 0.005). C, acetyl-CoA:lysine acetyltransferase activity of ATase1 and ATase2 was assayed with a commercially available colorimetric assay employing a pool of histone-tail-peptides as acceptor of the acetyl group. Both ATase1 and ATase2 were purified by affinity chromatography prior to the reaction. The acetyl-CoA:lysine acetyltransferase activity of a highly purified pool of nuclear HAT is also shown. Results are the average (n = 3) ± S.D.

FIGURE 6.

The C-terminal and catalytically active domains of ATase1 and ATase2 face the lumen of the ER/ERGIC. A, schematic view of the rationale of the experiments reported here (described under “Results”). The N terminus of either ATase1 or ATase2 is indicated. The C-terminal Myc tag is in red. B, ER and ERGIC vesicles from ATase1- and ATase2-expressing cells were incubated with affinity-purified BACE1 and [³H]acetyl-CoA for 1 h at 30 °C. Reaction was stopped by lowering the temperature; BACE1 was then purified again and analyzed on a scintillation liquid counter. Vesicles were used under the sealed (no detergent; black bars) or opened (+ detergent; white bars) condition. Results are expressed as percent of BACE1 acetylation obtained in the presence of detergent (opened condition) and are the average (n = 6) ± S.D. Numbers on top of the black bars indicate the percent of total acetyl-CoA: lysine acetyltransferase activity recovered in the absence of detergent (sealed condition). C, sealed and opened vesicles shown in A were incubated with an anti-Myc antibody covalently attached to aldehyde-activated agarose beads (ProFound system) for immunoprecipitation. After extensive washing, bound proteins were eluted by lowering the pH and analyzed by SDS-PAGE and immunoblotting.

FIGURE 7.

Overexpression of ATase1 and ATase2 increases the levels of BACE1 and the generation of Aβ. A, ATase1 and ATase2 were purified from stable-transfected cells with an anti-Myc antibody covalently attached to aldehyde-activated agarose beads (ProFound system) and then analyzed by Western blotting (Web.). The blot with anti-Myc (upper panel) demonstrates the presence of ATase1 and ATase2 in the immunoprecipitate, whereas the blot with anti-BACE1 (middle panel) shows the presence of endogenous BACE1. Finally, the blot with anti-acetylated lysine (Ac. Lys.; lower panel) shows the acetylation pattern of co-immunoprecipitated BACE1. The bands visible with the anti-BACE1 antibody correspond to biosynthetic intermediates of nascent BACE1 (see also supplemental Fig. 1A). The migration of the different forms of affinity-purified BACE1 on a similar SDS-PAGE system is shown in Figs. 3B and 5A. B, steady-state levels of endogenous BACE1 in ATase1-, ATase2-, and ATase1 + 2-expressing cells were analyzed by immunoblotting of total cell lysates. C, Aβ levels in the conditioned media were determined by standard sandwich enzyme-linked immunosorbent assay. Results are the average (n = 3) ± S.D. Symbols indicate statistical significance (^*, p < 0.05; catalog number, p < 0.005). D, endogenous BACE1 was immunoprecipitated (I.P.) from total cell lysates of ATase1-, ATase2-, and ATase1 + 2-expressing cells prior to Western blot analysis with an anti-acetylated lysine antibody. As expected (5), only the ∼52-55-kDa band corresponding to immature BACE1 could be visualized with an anti-acetylated lysine antibody. The migration of the different forms of BACE1 on a similar SDS-PAGE system is also shown for comparison (E). F, experiment described in D was repeated after immunoprecipitation of endogenous BACE1 from a pool of ER/ERGIC fractions (representative gradients are shown in Fig. 2C, Fig. 4C. and in supplemental Fig. 1).

FIGURE 8.

Down-regulation of ATase1 and ATase2 decreases the levels of BACE1 and the generation of Aβ. A-C, H4 (human neuroglioma) cells were treated with two different siRNAs targeting ATase1 prior to real time quantification of ATase1 mRNA levels (A) and Western blot analysis of BACE1 and C99 levels (B). The quantification of BACE1 protein changes after siRNA is shown in C. As control, cells were also treated with scrambled (nonsilencing) siRNA. D-F, H4 cells were treated with siRNA targeting ATase2 prior to real time quantification of ATase2 mRNA levels (D) and Western blot analysis of BACE1 and C99 levels (E). The quantification of BACE1 protein changes after siRNA is shown in F. As control, cells were also treated with scrambled (nonsilencing) siRNA. G, Aβ levels in the conditioned media of siRNA-treated cells were determined by standard sandwich enzyme-linked immunosorbent assay. Results are expressed as percent of control (no treatment) and are the average (n = 6) ± S.D. Symbols indicate statistical significance (^*, p < 0.05; catalog number, p < 0.005).

FIGURE 9.

Ceramide treatment increases the levels of endogenous ATase 1and ATase2.SH-SY5Y cells were treated with ceramide (10 μm) for the indicated period of time prior to real time quantitative PCR. The 4-h treatment was performed in the presence of 0.5% fetal bovine serum, whereas the 4-day treatment was performed in the presence of 10% fetal bovine serum, as described previously (5, 7, 8). Results are expressed as fold (n = 4) ± S.D. of control (no treatment). Symbols indicate statistical significance (^*, p < 0.05; catalog number, p < 0.005). No difference was observed in the mRNA levels of GAPDH, which was used as internal control.