Lipids as Modulators of Proteolytic Activity of BACE (original) (raw)

2005, Journal of Biological Chemistry

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␤). Previous results have suggested that the regulation of ␤-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 m) unilamellar vesicles, and found ϳ20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquidordered 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 (ϳ100 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). Amyloid precursor protein (APP) 2 is an abundant type I membrane protein with homology to glycosylated cell surface receptors (1) found in various mammalian tissues. Proteolytic processing of APP in human brain may give rise to the A␤ peptide, which is the major constituent of amyloid plaques in brains of patients suffering from Alzheimer disease (2, 3). APP is a substrate for at least 3 proteolytic ("secretase") activities (4) designated ␣, ␤, and ␥. The major proteolytic pathway, undertaken by ϳ95% of the APP in neurons, is ␣-␥, i.e. APP is first cleaved by a ␣-secretase within the A␤ region, and consequently by the ␥-secretase. The second proteolytic pathway, which leads to the formation of A␤, is the