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

No abstract

The cerebral deposition of amyloid ␤-peptide is an early and critical feature of Alzheimer's disease. Amyloid ␤-peptide is released from the amyloid precursor protein by the sequential action of two proteases, ␤-secretase and ␥-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of ␤-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a se- , and Cys 330 -Cys 380 ). Despite the conservation of the active site residues and the 30 -37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.The hallmarks of Alzheimer's disease (AD) 1 pathology are brain plaques and vascular deposits (1) consisting of the 4-kDa amyloid ␤-peptide (A␤) (2). Overproduction of the 42-amino acid form of A␤, A␤42, has been suggested to be the cause of all known cases of familial early onset AD (3), and it is assumed that A␤42 deposition plays an early and critical role in sporadic AD as well. Therefore, A␤ metabolism has attracted considerable interest. In 1987 it was shown (4) that formation of A␤ requires proteolytic cleavage of a large type I transmembrane protein, the ␤-amyloid precursor protein (APP), which is constitutively expressed in most cell types. Over the next decade the proteolytic processing of APP has been studied in great detail in a variety of systems by many groups. Taken together, these studies have shown that A␤ is generated at a low rate by most cells analyzed and that two different proteolytic activities are required for A␤ generation. First, ␤-secretase cleaves APP to generate the N terminus of A␤, and second, ␥-secretase cleaves the C terminus, leading to the release of A␤ (for review see Ref. 5). Studies with intact cells expressing APP and the endogenous secretases have led to conclusions about the properties of the ␤-and ␥-secretases, e.g. their tissue distribution, subcellular localization, substrate requirements (see e.g. Ref. 6) etc., but until recently the identity of both ␤-and ␥-secretase was unknown. This changed when we very recently identified the novel transmembrane aspartic protease BACE as the major ␤-secretase (7). Three subsequently published independent studies (8 -10) have confirmed this conclusion. Here we characterize the BACE protein. We show that BACE is an Nglycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We determine the processing and N-glycosylation sites and the disulfide bonds. Our results demonstrate that BACE is an unusual member of the pepsin family. EXPERIMENTAL PROCEDURESMat...

Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....