Antagonistic Effects of β-Site Amyloid Precursor Protein-cleaving Enzymes 1 and 2 on β-Amyloid Peptide Production in Cells

Abstract

The deposition of extracellular ␤-amyloid peptide (A␤) in the brain is a pathologic feature of Alzheimer's disease. The ␤-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the ␤-site, promotes A␤ production. A second integral membrane aspartyl protease related to BACE1, referred to as ␤-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the ␤-cleavage site in transfected cells. The role of endogenous BACE2 in A␤ production remains unresolved. We investigated the role of endogenous BACE2 in A␤ production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88 -94% in cells. The production and deposition of insoluble A␤ 1 peptide in the brain results in the hallmark pathological feature of Alzheimer's disease (1). The cellular enzymes responsible for production of A␤ peptide are molecular targets for therapeutic intervention in Alzheimer's disease (2, 3). BACE1 (␤-site APP cleaving enzyme, ASP2, Memapsin 2, ␤-secretase), the enzyme responsible for cleavage of the amyloid precursor protein (APP) resulting in the amino terminus of A␤ peptide, is a novel integral membrane aspartyl protease (4 -8). Cellular antisense (6, 7) and knock-out mouse models (9 -11) have unambiguously confirmed the role of BACE1 in promoting A␤ production. A second integral membrane aspartyl protease related to BACE1, referred to as BACE2 (ASP1, Memapsin 1) has also been demonstrated to cleave APP at the ␤-cleavage site (7,8,(12)(13)(14)(15)(16). Both enzymes also cleave APP at a second site within the A␤ region. This second cleavage site for BACE1 is between A␤ residues 10 and 11 and between A␤ residues 19 and 20 for BACE2. Cleavage of APP by BACE1 at either site is amyloidogenic, whereas the second cleavage site for BACE2 on APP precludes formation of A␤.Overexpression of BACE2 in transfected cells produces intracellular carboxyl-terminal fragments (CTFs), as well as release of ␤-cleaved secreted APP (sAPP␤) from cells, consistent with cleavage of APP at the ␤-site by this enzyme (14, 16 -19). We have also observed decreased A␤ production from cells transfected with BACE2 (not shown), consistent with published observations (14, 16 -19). Hence, the reduction in secreted A␤ occurs in spite of the concomitant increased secretion of sAPP␤ from cells overexpressing BACE2. The decrease in A␤ production from BACE2 transfected cells has been attributed to the second cleavage site for BACE2 on APP (14, 16 -19). The observations on the cleavage specificity of BACE2 on APP substrate are derived from transfected cells that overexpress the enzyme. The role of BACE2 in A␤ production in cells expressing endogenous levels of the enzyme remains an unanswered question. Many tissues (including brain) and cell types co-express BACE1 and BACE2 mRNA (8,13,14,16). The therapeutic rele...

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

“…On a cellular level BACE is found mainly in acidic compartments such as the trans-Golgi network and endosomes (9,10,13) where it is associated with rafts by palmitoylation (15,16). BACE is highly homologous to BACE-2, but different cleavage sites were identified within the APP substrate for both enzymes (17)(18)(19). Besides APP, additional substrates for BACE have been described, like the sialyltransferase ST6Gal I (20,21), the adhesion protein P-selectin glycoprotein ligand-1 (PSGL-1) (22), and APP-like proteins (23).…”

Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice

“…Moreover, BACE2-transfected cells produce reduced levels of A␤ (2,8,13,18), and selective knockdown of endogenous BACE2 in human embryonic kidney 293 cells by RNA interference elevates A␤ secretion (19). These observations led to the suggestion that BACE2 does not function as a ␤-secretase, but rather as an ␣-like secretase that precludes A␤ formation (17)(18)(19)(20). However, these in vitro observations cannot rule out a possible contribution of BACE2 to the * This work was supported in part by a pioneer award from the Alzheimer Association (to B. D. S.); an Alzheimer Forschungsinititative…”

Linking vascular disorders and Alzheimer's disease: Potential involvement of BACE1

“…Tg mice deficient in BACE1 do not produce any form of Aβ, and lack the Aβ-associated pathologies, neuronal loss and memory deficits observed in age-matched Tg mice which express BACE1 (reviewed in Cole and Vassar, 2007). The homolog BACE2 shares 64% amino acid similarity with BACE1, but does not have the correct expression pattern for β-secretase (Bennett et al, 2000) and exhibits α-secretase-like activity (Yan et al, 2001;Basi et al, 2003). As reviewed elsewhere, the characteristics of BACE1 match one-to-one with the previously established properties of β-secretase activity in cells and tissues .…”