Lipids as modulators of proteolytic activity of BACE - Involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro
Lipids as modulators of proteolytic activity of BACE - Involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro
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DOI：
10.1074/jbc.m504484200
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发表时间：
2005-11-04
影响因子：
4.8
通讯作者：
Simons, K
中科院分区：
文献类型：
--
作者：
Kalvodova, L;Kahya, N;Simons, K
β-分泌酶BACE是一种跨膜天冬氨酸蛋白酶，在蛋白水解加工的第一步中切割淀粉样前体蛋白（APP），导致形成神经毒性β-淀粉样肽（A β）。以前的研究结果表明，β-分泌酶和BACE进入APP的调节是脂质依赖性的，并涉及脂筏。使用杆状病毒表达系统，我们已经在昆虫细胞中表达了重组人全长BACE，并纯化毫克量至均一。我们已经研究了在巨大的（10 - 150 μ m）单层囊泡的液体有序和无序相之间的荧光团共轭BACE的分区，并发现类似20%与筏状，液体有序相关联;与液体有序相关联的分数增加筏脂质交联后。为了检查参与调节BACE活性的个别脂质种类，我们已经重建了纯化的BACE在大（类似于100 nm）单层囊泡，并确定其在各种脂质组合物的囊泡的比活性。我们已经确定了3组刺激BACE蛋白水解活性的脂质：1）中性鞘糖脂（糖苷），2）阴离子甘油磷脂和3）甾醇（胆固醇）。
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 (A beta). 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 mu m) unilamellar vesicles, and found similar to 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 ( similar to 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).