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).
Kalvodova
Lucie
L
Max-Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany.
Kahya
Nicoletta
N
Schwille
Petra
P
Ehehalt
Robert
R
Verkade
Paul
P
Drechsel
David
D
Simons
Kai
K
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D016367:Baculoviridae / Q000235:genetics; D002554:Cerebrosides / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010718:Phosphatidylserines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016229:Amyloid beta-Peptides; D002554:Cerebrosides; D006028:Glycosphingolipids; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D002784:Cholesterol; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human