Characterization of Alzheimer's β-Secretase Protein BACE

279

134

Processing of the ␤-amyloid precursor protein (␤APP) by ␤-and ␥-secretases generates the amyloidogenic peptide A␤, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the ␤-secretase ␤-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant ␤APP sw ␤-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylationdependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the ␣-secretase product APPs␣. Although there is little increase in the generation of A␤ by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced A␤ levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of ␤APP.Alzheimer's disease is a progressive degenerative disorder of the brain characterized by mental deterioration, memory loss, confusion, and disorientation. Among the cellular mechanisms contributing to this pathology are two types of fibrous protein deposition in the brain, intracellular neurofibrillary tangles consisting of polymerized tau protein, and abundant extracellular fibrils largely composed of ␤-amyloid 1 (for reviews see Refs. 1-3). ␤-Amyloid, also known as A␤, arises from proteolytic processing of the ␤-amyloid precursor protein (␤APP) at the ␤-and ␥-secretase cleavage sites. The cellular toxicity and amyloid-forming capacity of the two major forms of A␤ (A␤ 40 and especially A␤ 42 ) have been well documented (1-3).An alternative, anti-amyloidogenic cleavage carried out by ␣-secretase(s) is located within the A␤ peptide sequence of ␤APP, thus precluding the formation of intact insoluble A␤. This cleavage by ␣-secretase within the (His-His-GlnLys2Leu-Val) sequence of ␤APP is the major physiological route of APP maturation. The products of this reaction are a soluble 100 -120-kDa N-terminal fragment (␤APPs␣) and a C-terminal membrane-bound ϳ9-kDa segment (C83). In several recent reports, metalloproteinases such as ADAM9, -10, and -17 were shown to be involved in the ␣-secretase cleavage

Section: Discussionmentioning

confidence: 89%

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Benjannet

Elagöz

Wickham

et al. 2001

279

134

“…It has been previously shown by immunofluorescence microscopy that ectopically expressed BACE is localized primarily in the TGN and the endosomal system (9,24,25). However, significant quantities of the glycoprotein are also present in the endoplasmic reticulum, at the cell surface (9,26,27), and to a lesser extent in the lysosomes (9,24). Ectopically expressed BACE demonstrated a highly punctuate staining pattern reminiscent of vesicular compartments in H4-B cells (Fig.…”

Section: Treatment With Lysosomal Inhibitors Cause Accumulation Of Bamentioning

confidence: 99%

BACE Is Degraded via the Lysosomal Pathway

Koh

Arnim

Hyman

et al. 2005

155

127

Amyloid plaques are formed by aggregates of amyloid-␤-peptide, a 37-43-amino acid fragment (primarily A␤ 40 and A␤ 42 ) generated by proteolytic processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases. A type I transmembrane aspartyl protease, BACE (␤-site APP cleaving enzyme), has been identified to be the ␤-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH 4 Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosomeassociated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu 499 and Leu 500 in the COOHterminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.

“…BACE appears to localize to the Golgi apparatus (5). Despite about 40% amino acid similarity between BACE and pepsin proteases (9), the cysteine residues in BACE involved in intramolecular disulfide bonds are not conserved with other pepsin family members (10). Such fundamental structural differences may explain why ␤-secretase is insensitive to pepstatin, a specific inhibitor of pepsin proteases (7).…”

mentioning

confidence: 94%

The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

Steinhilb¹,

Turner²,

Gaut³

2001

Amyloid (A␤) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by ␤-and then by ␥-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block A␤ peptide secretion by interfering with ␥-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the ␤-secretasecleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented ␤-secretase cleavage. This block in ␤-secretase cleavage was not observed with clasto-lactacystin ␤-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of ␤-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of ␤-secretase cleavage was immediate and did not affect ␣-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of ␣-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the ␤-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in ␤-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in A␤ peptide secretion.