Abstract
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).
Highlights
The -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 -amyloid peptide (A)
Recovery and Specific Activity of BACE Reconstituted in Total Brain Lipid Vesicles—The detergent-mediated reconstitution procedure presented here relies on incorporation of protein into preformed liposomes [23] and yields BACE reconstituted directionally (Ͼ80% ectodomain outside, supplemental Fig. S2) in large unilamellar vesicles; the yield was typically 15–30% of protein input by weight
Being an integral membrane protein, the aspartic protease BACE is likely to respond to its lipid environment on a molecular level in a way that could eventually affect its activity
Summary
The -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 -amyloid peptide (A). In this case, APP is first cleaved by the -secretase APP is first cleaved by the -secretase Even though it is not the -cleavage itself that would per se lead to the plaque formation, but rather the misfolding and aggregation of the generated peptide, up-regulation of the -secretase activity is an issue in amyloidogenesis. It is necessary to establish how access of the secretases to APP is regulated, and how are the proteolytic activities of the individual secretases modulated
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