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Degradation of BACE by the ubiquitin-proteasome pathway

HONG QING^*, WEIHUI ZHOU^*, MICHELLE A. CHRISTENSEN^*, XIULIAN SUN^,*, YIGANG TONG^* and WEIHONG SONG^,*,1

^* Department of Psychiatry, Brain Research Center,
Graduate Program in Neuroscience, The University of British Columbia, Vancouver, BC, Canada.

¹Correspondence: Department of Psychiatry, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada. E-mail: weihong{at}interchange.ubc.ca

SPECIFIC AIMS

BACE (or BACE1), the major ß-secretase in cleaving ß-amyloid precursor protein (APP) to generate amyloid ß protein (Aß) in Alzheimer’s disease (AD), has been identified as a type 1 membrane-associated aspartyl protease. In this report we demonstrate that BACE protein is ubiquitinated and that the ubiquitin-proteasome pathway mediates degradation of BACE and regulates APP processing to generate Aß.

PRINCIPAL FINDINGS

1. The degradation of BACE protein is mediated by the proteasome pathway
To investigate whether the degradation of BACE protein is regulated by the ubiquitin-proteasome pathway, neuroblastoma SH-SY5Y cells were treated with the highly specific proteasome inhibitor lactacystin and BACE protein levels were analyzed by immunoblotting. Endogenous BACE proteins were detected by 208, a polyclonal antibody raised in a rabbit against BACE COOH terminus. Lactacystin markedly increased endogenous BACE protein levels in SH-SY5Y cells (Fig. 1 A). Lactacystin treatment also significantly increased the level of BACE proteins in BACE cDNA transiently transfected SH-SY5Y cells (Fig. 1B ). To further examine whether the ubiquitin-proteasome pathway mediates BACE degradation, the BACE stably transfected cell line B2 was established by introducing Myc-tagged BACE cDNA plasmid construct pBACE-mycHis into HEK293 cells. B2 cells were treated with lactacystin and BACE protein levels were analyzed. BACE protein levels were markedly increased in cells treated with lactacystin in a dose- (Fig. 1C ) and time-dependent (Fig. 1D ) manner. Lactacystin treatment had no effect on ß-actin expression, suggesting that inhibition of the ubiquitin-proteasome pathway by lactacystin decreased degradation of BACE in neuronal and non-neuronal cells.

View larger version (46K): [in this window] [in a new window]   Figure 1. Proteasome inhibitors increase BACE protein levels. A) Neuroblastoma SY-SY5Y cells were treated with 10 µM lactacystin for 9 h and endogenous BACE protein was detected by antibody 208. B) Plasmid pBACE-mycHis was transiently transfected into SH-SY5Y cells using Lipofectamine 2000. Transfected cells were treated with 10 µM lactacystin for 9 h and harvested at 48 h. BACE proteins were detected by monoclonal anti-myc antibody 9E10. C) BACE cDNA stably transfected B2 cells were treated with vehicle solution control or lactacystin for 9 h at 2, 5, 10, or 20 µM for a dosage-dependent assay or with lactacystin at 10 µM for 0 to 20 h for assay (D). Cells were harvested at the same endpoint of treatment and lysed in RIPA-DOC buffer. Cell lysates were analyzed by 12% Tris-glycine gel with 9E10 antibody to detect BACE. Monoclonal anti ß-actin antibody (AC-15) was used to detect ß-actin. E) B2 cells were treated with vehicle solution control, ALLM (25 µM), ALLN (25 µM), MG132 (5 µM), or lactacystin (10 µM) for 9 h. Western blot analysis was performed with 9E10 antibody to detect BACE and AC-15 antibody to detect ß-actin.

B2 cells were treated with other proteasome inhibitors. N-acetyl-L-leucinyl-L-leucinyl-L-methional (ALLM), N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN), and N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal (MG-132) are a group of peptide aldehydes. Like lactacystin, ALLN and MG-132 specifically inhibited protein degradation by the proteasome. In contrast, ALLM, a nonproteasome-specific protease inhibitor, had little effect on the ubiquitin-proteasome pathway in intact cells. Figure 1E shows that ALLN and MG-132, like lactacystin, significantly increased BACE protein levels relative to control in B2 cells. However, ALLM did not affect BACE proteolysis.

To confirm that the turnover of BACE protein is regulated by the ubiquitin-proteasome pathway, a pulse-chase experiment was performed. B2 cells were metabolically radiolabeled with media containing ³⁵S-methionine and ³⁵S-cysteine, then incubated with fresh media containing an excessive amount of nonradioactive methionine and cysteine. The radiolabeled BACE proteins were immunoprecipitated with 9E10 antibody and analyzed on 420% gradient Tris/glycine gels. Without proteasome inhibitor treatment, the newly synthesized BACE proteins were degraded; the turnover rate was 53.63 ± 5.49% for 9 h chasing time (P<0.001 relative to control). In contrast, addition of lactacystin in the chasing media increased BACE protein levels to 109.77 ± 2.92% from 53.63 ± 5.49% in the nonlactacystin treatment in the chasing media (P<0.001); levels of BACE protein in cells treated with chasing media containing lactacystin for 9 h were similar to those in cells without chasing and lactacystin treatment (P>0.05), indicating that turnover of the newly synthesized radiolabeled BACE protein was inhibited. Furthermore, addition of lactacystin in labeling media increased the radiolabeled BACE protein level to 124.37 ± 4.43% (P<0.05 relative to control). These data demonstrated that the degradation of BACE was inhibited by lactacystin through blockage of the proteasome pathway.

2. Ubiquitination of BACE protein
To investigate whether BACE protein is ubiquitinated, we first performed an immunostaining assay. BACE cDNA was cloned into pEGFP-N2 vector to generate BACE-EGFP fusion protein expression plasmid. pCW-7 plasmid contains myc-tagged ubiquitin cDNA. HEK293 cells were transfected with pBACE-EGFP and myc-tagged ubiquitin plasmid pCW-7 (Fig. 2 A). BACE-EGFP fusion protein was detected by green fluorescent signal. The myc-tagged ubiquitin was immunostained with 9E10 antibody and detected as the red fluorescent signal under a fluorescent microscope. The merged image showed that the green fluorescent BACE proteins colocalized with the red fluorescent ubiquitin proteins.

View larger version (31K): [in this window] [in a new window]   Figure 2. Ubiquitination of BACE protein. A) Colocalization of BACE and ubiquitin. HEK293 cells were transfected with pBACE-EGFP and myc-tagged ubiquitin plasmid pCW-7. Expression of BACE-EGFP fusion protein is indicated by green fluorescent signal (upper left panel) and myc-tagged ubiquitin was immunostained with 9E10 antibody (upper right panel, red). Hoechst staining was used to detect nucleus (lower left panel, blue). Lower right panel: superimposed images showing colocalization of BACE and ubiquitin. Arrows point to examples of the colocalized proteins. B) B2 cells were treated with lactacystin or transfected with ubiquitin protein plasmid pHis-Ubi. BACE protein was detected by 9E10. Lactacystin treatment and ubiquitin plasmid transfection generated high molecular weight (HMW) BACE complex. C) HEK293 cells were transfected with pBACE-EGFP, pCW-7, or a combination of both. Cell lysates were immunoprecipitated with anti-EGFP antibody to pull down BACE-EGFP fusion protein, followed by Western blot analysis with 9E10 to detect myc-tagged ubiquitin in BACE-EGFP-containing immunoprecipitates. Cell lysates from HEK 293 cells and myc-tagged BACE stably expressing B2 cells were immunoprecipitated with anti-ubiquitin antibody, followed by immunoblotting with anti-BACE antibody 208 (D) or anti-myc antibody 9E10 (E) to detect BACE protein.

To examine the interaction between BACE and ubiquitin, B2 cells were transiently transfected with ubiquitin expression plasmid pHis-UBi. Figure 2B shows that the high molecular weight (HMW) BACE complex was generated in cells treated with lactacystin or transfected with ubiquitin plasmid pHis-Ubi, indicating that BACE protein was polyubiquitinated. To confirm the ubiquitination, co-immunoprecipitation assay was performed. HEK293 cells were transfected with pBACE-EGFP, pCW-7, or a combination of both. Cell lysates were immunoprecipitated with anti-EGFP antibody to pull down BACE-EGFP fusion protein. The immunoprecipitates were immunoblotted with 9E10 to detect myc-tagged ubiquitin. Anti-EGFP antibody immunoprecipitating samples from pBACE-EGFP-transfected cells or myc-ubiquitin pCW-7-transfected cells contained no myc-ubiquitin. However, HMW BACE-ubi molecules were detected by 9E10 in the immunoprecipitating samples from the cells transfected with both BACE-EGFP and myc-ubiquitin cDNA, indicating co-immunoprecipitation of BACE and ubiquitin (Fig. 2C ). A reverse co-immunoprecipitation assay was performed for further confirmation. HEK 293 cells and myc-tagged BACE stably expressing B2 cells were lysed with NP-40 lysis buffer and the cell lysates were immunoprecipitated with anti-ubiquitin antibody. The samples were then analyzed by immunoblotting with anti-BACE antibody 208 (Fig. 2D ) or anti-myc antibody 9E10 (Fig. 2E ). BACE was detected only in cells transfected with myc-tagged BACE plasmid, not in control HEK293 nontransfected cells. These results clearly demonstrated that BACE was ubiquitinated.

3. Regulation of ß-secretase cleavage of APP and Aß generation by lactacystin
BACE cleaves APP at the ß-secretase site in vivo. We have shown that BACE degradation was mediated by the proteasome pathway. To further examine whether proteasomal degradation of BACE had affected APP proteolytic processing at the ß-secretase site and Aß production, ß-secretase cleavage product, APP C99, in cell lysates and Aß levels in conditioned media were analyzed. In 2EB2, APP-BACE double stable cells, the major ß-secretase cleavage product APP C99 and minor Glu-11 ß-secretase cleavage product APP C89 were markedly increased by treatment with lactacystin compared with control. Aß ELISA analysis was performed to examine whether this proteasomal inhibition of BACE degradation affected Aß generation. Aß40 production was increased by 156.08 ± 0.70% (P<0.01) in lactacystin-treated neuroblastoma SH-SY5Y cells, and Aß42 production was doubled in the treated cells (38.08 ± 0.86%) relative to control cells (19.06±0.76%) (P<0.001). Thus, the proteasomal inhibitor lactacystin not only regulates the ß-secretase cleavage of APP, but also affects the Aß production.

CONCLUSIONS AND SIGNIFICANCE

Senile neuritic plaques in the brain are the hallmark of AD pathology. A central component of the plaque consists of the 40-42/43 amino acid residue amyloid ß protein Aß. Aß is generated from APP sequentially by the ß and then secretase. Several studies have reported that the proteasome pathway might affect Aß production in the AD pathogenesis. However, the mechanism by which the proteasome pathway regulates APP processing needs to be further defined. Since BACE is the essential enzyme to cleave APP to generate Aß, understanding the post-translational modification and degradation of BACE will be fundamentally important for AD drug development.

In this study we generated cell lines B2 and 2EB2, which stably overexpress BACE or BACE and the Swedish mutant APP, respectively. We found that BACE was ubiquitinated and that blocking the ubiquitin-proteasomal pathway inhibited BACE degradation (Fig. 3 ). As APP cleavage enzymes, ß and -secretase have been the major pharmaceutical targets for development of protease inhibitors on Aß secretion. We found that proteasomal inhibition of BACE degradation led to increased BACE enzymatic activity, more ß-cleavage product C99, and increased Aß40 and Aß42 production in both neuronal and non-neuronal cells. More study is needed to investigate whether Aß degradation may be regulated by the proteasome pathway, but our data indicate that Aß generation is regulated by the proteasomal pathway via BACE degradation control. Our finding that BACE degradation is mediated by a proteasome pathway suggests that the ubiquitin-proteasome pathway dysregulation may constitute an important event in the pathogenesis of certain AD cases.

View larger version (27K): [in this window] [in a new window]   Figure 3. Proteasomal degradation of BACE protein. APP undergoes a complex set of proteolytic processing by 3 enzymes: , ß, and -secretase. BACE, the major APP ß secretase in vivo, is essential to cleave APP to generate Aß. Degradation of BACE is mediated by the ubiquitin-proteasome pathway; proteasomal degradation of BACE regulates APP processing and Aß generation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1994fje;

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