Transmembrane Aspartic Protease Research Articles

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.

Characterization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Expressed in a Variety of Cell Lines

Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.

The beta-secretase, BACE: a prime drug target for Alzheimer's disease.

Evidence suggests that the beta-amyloid peptide (Abeta) is central to the pathophysiology of Alzheimer's disease (AD). Amyloid plaques, primarily composed of Abeta, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Abeta42 peptide. Given the strong association between Abeta and AD, therapeutic strategies to lower the concentration of Abeta in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Abeta. Abeta is a product of catabolism of the large Typel membrane protein, amyloid precursor protein (APP). Two proteases, called beta- and gamma-secretase, mediate the endoproteolysis of APP to liberate the Abeta peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the gamma-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the beta-secretase has been shown to be the novel transmembrane aspartic protease, beta-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the beta-secretase, and as the key rate-limiting enzyme that initiates the formation of Abeta, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.

A Furin-like Convertase Mediates Propeptide Cleavage of BACE, the Alzheimer's β-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.

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration.

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

β-Secretase revealed: starting gate for race to novel therapies for Alzheimer’s disease

Although remarkable progress has been made in the identification of BACE as β-secretase, there is still much to be done. For example, although BACE-mediated cleavage of APP might be important to the pathogenesis of AD, its role in normal cellular function remains unknown. This issue could be addressed by analysing transgenic mice that overexpress BACE, as well as mice that are deficient in both copies of the gene that encodes BACE. It will also be important to understand how BACE activity is regulated in the cell and how this regulation affects APP processing and Aβ production. For example, we already know that overexpression of BACE leads to decreased processing of APP by the alternative α-secretase pathway10xβ-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Vassar, R. et al. Science. 1999; 286: 735–741Crossref | PubMed | Scopus (2413)See all References10. Similarly, protein kinase C (PKC)-mediated stimulation of α-secretase results in decreased β-secretase processing of APP, which suggests that these two enzymes might compete for cleavage of APP (Ref. 23xProtein kinase C-dependent α-secretase competes with β-secretase for cleavage of amyloid-β precursor protein in the trans-Golgi network. Skovronsky, D.M. et al. J. Biol. Chem. 2000; 275: 2568–2578Crossref | PubMed | Scopus (243)See all ReferencesRef. 23). Whether dysregulation of β-secretase or α-secretase contributes to the pathogenesis of AD has yet to be tested.It is tempting to speculate that polymorphisms in the BACE gene that affect BACE function might be linked to altered risks of AD. However, the BACE gene, which has been mapped to chromosome 11 (2xA pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of β-amyloid. Mullan, M. et al. Nat. Genet. 1992; 1: 345–347Crossref | PubMed | Scopus (813)See all References, 12xMembrane-anchored aspartyl protease with Alzheimer’s disease β-secretase activity. Yan, R. et al. Nature. 1999; 402: 533–537Crossref | PubMed | Scopus (1098)See all References), has not yet been associated genetically with AD. Interestingly, the BACE2 gene, like the gene encoding APP, maps to the obligate Down syndrome region of chromosome 21 (12xMembrane-anchored aspartyl protease with Alzheimer’s disease β-secretase activity. Yan, R. et al. Nature. 1999; 402: 533–537Crossref | PubMed | Scopus (1098)See all References, 22xBACE maps to chromosome 11 and a BACE homolog, BACE2, reside in the obligate down syndrome region of chromosome 21. Saunders, A.J. et al. Science. 1999; 286: 1255aCrossrefSee all References), and Down syndrome patients invariably develop amyloid plaques and AD-like pathology.Finally, the discovery of BACE as β-secretase greatly facilitates the prospects for the development of potential AD therapeutics. With a target protease now in hand, it will be possible to generate small-molecule inhibitors of BACE that could reduce Aβ production. On the basis of the success of protease inhibitors to retard progression of AIDS, the future propects for protease inhibition therapy for AD appear bright. Clinical trials of such BACE inhibitors will provide valuable clues to the pathogenesis of AD. By inhibiting Aβ production and senile plaque formation, these drugs will enable us to examine the link between senile plaques and dementia, thus providing the means for testing the amyloid cascade hypothesis in vivo.

BACE and BACE2 define a novel subfamily of trans-membrane aspartic proteases

BACE and BACE2 define a novel subfamily of trans-membrane aspartic proteases

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the Down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called ‘Down critical region’ in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described β-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase

The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.