Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.     

Enzymic properties of recombinant BACE2.

BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.     

β-Site Amyloid Precursor Protein Cleaving Enzyme 1(BACE1) Regulates Notch Signaling by Controlling the Cleavage of Jagged 1 (Jag1) and Jagged 2 (Jag2) Proteins

BACE1 is a type I transmembrane aspartyl protease that cleaves amyloid precursor protein at the β-secretase site to initiate the release of β-amyloid peptide. As a secretase, BACE1 also cleaves additional membrane-bound molecules by exerting various cellular functions. In this study, we showed that BACE1 can effectively shed the membrane-anchored signaling molecule Jagged 1 (Jag1). We also mapped the cleavage sites of Jag1 by ADAM10 and ADAM17. Although Jag1 shares a high degree of homology with Jag2 in the ectodomain region, BACE1 fails to cleave Jag2 effectively, indicating a selective cleavage of Jag1. Abolished cleavage of Jag1 in BACE1-null mice leads to enhanced astrogenesis and, concomitantly, reduced neurogenesis. This characterization provides biochemical evidence that the Jag1-Notch pathway is under the control of BACE1 activity.     

The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.     

Crystal structure of an active form of BACE1, an enzyme responsible for amyloid beta protein production

BACE1 (beta-secretase) is a transmembrane aspartic protease that cleaves the beta-amyloid precursor protein and generates the amyloid beta peptide (Abeta). BACE1 cycles between the cell surface and the endosomal system many times and becomes activated interconvertibly during its cellular trafficking, leading to the production of Abeta. Here we report the crystal structure of the catalytically active form of BACE1. The active form has novel structural features involving the conformation of the flap and subsites that promote substrate binding. The functionally essential residues and water molecules are well defined and play a key role in the iterative activation of BACE1. We further describe the crystal structure of the dehydrated form of BACE1, showing that BACE1 activity is dependent on the dynamics of a catalytically required Asp-bound water molecule, which directly affects its catalytic properties. These findings provide insight into a novel regulation of BACE1 activity and elucidate how BACE1 modulates its activity during cellular trafficking.     

The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1

The aspartyl protease BACE1 cleaves the amyloid precursor protein and the sialyltransferase ST6Gal I and is important in the pathogenesis of Alzheimer's disease. The normal function of BACE1 and additional physiological substrates have not been identified. Here we show that BACE1 acts on the P-selectin glycoprotein ligand 1 (PSGL-1), which mediates leukocyte adhesion in inflammatory reactions. In human monocytic U937 and human embryonic kidney 293 cells expressing endogenous or transfected BACE1, PSGL-1 was cleaved by BACE1 to generate a soluble ectodomain and a C-terminal transmembrane fragment. No evidence of the cleavage fragment was seen in primary cells derived from mice deficient in BACE1. By using deletion constructs and enzymatic deglycosylation of the C-terminal PSGL-1 fragments, the cleavage site in PSGL-1 was mapped to the juxtamembrane region within the ectodomain. In an in vitro assay BACE1 catalyzed the formation of the PSGL-1 products seen in vivo. The cleavage occurred at a Leu-Ser peptide bond as identified by mass spectrometry using a synthetic peptide. We conclude that PSGL-1 is an additional substrate for BACE1.     

Crystallization of glycosylated human BACE protease domain expressed in Trichoplusia ni

Human beta-amyloid precursor protein cleaving enzyme (beta-secretase, or BACE) belongs to the aspartyl protease family, and is responsible for generating the N-terminus of beta-amyloid peptide (Abeta). BACE is a type I transmembrane glycoprotein with pre-, pro- and catalytic domains, a short transmembrane helix and a cytoplasmic region. In this study, a truncated form was engineered to produce the authentic catalytic domain of BACE in Trichoplusia ni (High 5) cells. The glycosylated BACE zymogen (proBACE) was secreted into the conditioned medium for facile purification by metal chelate and gel filtration chromatographies. The mature catalytic domain was obtained by a trans cleavage event under acidic conditions and crystallized in the absence of a bound inhibitor. A complete 3.4 A data set was collected on a single orthorhombic crystal with unit cell parameters a=74 A, b=130 A, c=134A. Successful molecular replacement shows two BACE molecules in the asymmetric unit.     

Modeling of substrate specificity of the Alzheimer's disease amyloid precursor protein beta-secretase

The enzyme BACE (beta-site APP-cleaving enzyme) has recently been identified as the beta-secretase that cleaves the amyloid precursor protein (APP) to produce the N terminus of the Abeta peptide found in plaques in the brains of Alzheimer's disease patients. BACE is an aspartic protease similar to pepsin and renin. Comparative modeling of the three-dimensional structure of BACE in complex with its substrate shows that several residues confer specificity of the enzyme for APP. In particular, Arg296 forms a salt-bridge with the P1' Asp of the APP substrate, explaining the unusual preference of BACE among aspartic proteases for a P1' residue that is negatively charged. Several hydrophobic residues in the enzyme form a pocket for the P1 hydrophobic residue (Met in wild-type APP and Leu in APP with the "Swedish mutation" associated with early-onset of Alzheimer's disease). Inhibitors that can bind to the BACE active site may prove useful for drugs to treat and prevent Alzheimer's disease.     

Insights from modeling the tertiary structure of human BACE2

BACE1, or beta-secretase, is a putative prime therapeutic target for the treatment of Alzheimer's disease. Mapping to the Down syndrome critical region (chromosome 21) and identified as a homologue of BACE1, BACE2 also cleaves amyloid precursor protein at the beta-site. Thus, BACE2, named also as Asp1 or Memapsin1, represents a second beta-secretase candidate. In this paper, the tertiary structure of the protease domain of BACE2 was developed. Although the overall structural topology between BACE1 and BACE2 protease domains is quite similar, the former contains 3 disulfide bonds but the latter only two. Particularly, a subtle structural difference around the DTG/DSG active site between the two structures has been observed that is useful for the in-depth selectivity study of BACE1 and BACE2 inhibitors, stimulating new therapeutic strategies for the treatment of Alzheimer's disease and Down syndrome as well.     

BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper

The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.     

Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.     

Beta-galactoside alpha2,6-sialyltransferase I cleavage by BACE1 enhances the sialylation of soluble glycoproteins. A novel regulatory mechanism for alpha2,6-sialylation

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.     

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1

BACE (β-site amyloid precursor protein cleaving enzyme, β-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of β-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans -Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.     

In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer beta-secretase

beta-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Abeta, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH(2) terminus of ST6Gal I secreted from the cells (E41 form) was Glu(41). Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACE1-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form.     

Mutations at the P1' position of Notch1 decrease intracellular domain stability rather than cleavage by gamma-secretase

Gamma-secretase is a unique protease which cleaves within the transmembrane domain of several substrate proteins. Among gamma-secretase substrates are members of the Notch family of receptors and the amyloid precursor protein. In this study we used a cell-free Notch-cleavage assay and specific gamma-secretase inhibitors to study the cleavage of Notch by gamma-secretase. Using this assay, we found that, in contrast to previous reports, the presence of valine at the P1(') position of Notch1 is not required for gamma-secretase cleavage. Our results suggest that the presence of valine at the N-terminus of the Notch intracellular domain cleavage product is important for its stability. Thus it appears that Notch cleavage is very similar to APP cleavage with respect to the lack of sequence specificity.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.     

Alzheimer's beta-secretase cleaves a glycosyltransferase as a physiological substrate

Alzheimer's beta-secretase (BACE1) is a membrane-bound protease that cleaves the amyloid precursor protein (APP) in the trans-Golgi network, an initial step in the pathogenesis of Alzheimer's disease. Although BACE1 is distributed among various tissues including brain, its physiological substrate other than APP have not been identified. We have recently found that when BACE1 was overexpressed in COS cells together with alpha2,6-sialyltransferase (ST6Gal I), the secretion of ST6Gal I markedly increased, suggesting that BACE1 cleaves ST6Gal I as a physiological substrate. Thus BACE1 is the first identified protease that is responsible for the cleavage and secretion of glycosyltransferases.     

BACE2 functions as an alternative alpha-secretase in cells.

BACE1 and BACE2 define a new subfamily of membrane-anchored aspartyl proteases. Both endoproteases share similar structural organization including a prodomain, a catalytic domain formed via DTG and DSG active site motifs, a single transmembrane domain, and a short C-terminal tail. BACE1 has been identified as the Alzheimer's beta-secretase, whereas BACE2 was mapped to the Down's critical region of human chromosome 21. Herein we show that purified BACE2 can be autoactivated in vitro. Purified BACE2 cleaves human amyloid precursor protein (APP) sequences at the beta-secretase site, and near the alpha-secretase site, mainly at A beta-Phe(20)--Ala(21) and also at A beta-Phe(19)--Phe(20). Alternatively, in cells BACE2 has a limited effect on the beta-secretase site but efficiently cleaves the sequences near the alpha-secretase site. The in vitro specificity of APP processing by BACE2 is distinct from that observed in cells. BACE2 localizes in the endoplasmic reticulum, Golgi, trans-Golgi network, endosomes, and plasma membrane, and its cellular localization patterns depend on the presence of its transmembrane domain. BACE2 chimeras that increase localization of BACE2 in the trans-Golgi network do not change its APP processing patterns. Thus, BACE2 can be distinguished from BACE1 on the basis of autoprocessing of the prosegment, APP processing specificity, and subcellular localization patterns.     

A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.     

Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro

The beta-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic beta-amyloid peptide (Abeta). Previous results have suggested that the regulation of beta-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10-150 mum) unilamellar vesicles, and found approximately 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( approximately 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).