Purification and cloning of amyloid precursor protein β-secretase from human brain
Purification and cloning of amyloid precursor protein β-secretase from human brain
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作者：
Sinha, S;Anderson, JP;John, V
淀粉样前体蛋白(APP)的蛋白质分解处理产生淀粉样β蛋白(Aβ)肽，被认为是阿尔茨海默病的病理和随后的认知能力下降的原因。在APP的671和672残基之间，Aβ多肽序列的氨基末端被β-分泌酶切割，导致β-切割的可溶性APP(1)的产生和细胞外释放，以及相应的细胞相关的羧基末端片段。C末端片段被伽马分泌酶(S)切割后形成Aβ。APP(2)的β-分泌酶裂解位点的致病突变K670M671-->；N670L671是在一个家族性阿尔茨海默病瑞典家系中发现的，导致突变底物(3)的β-分泌酶裂解增加。在这里，我们描述了一种膜结合的酶活性，它在β-分泌酶裂解部位裂解全长APP，并发现它是人脑中主要的β-裂解活性。我们用一种新的酶活性底物类似物抑制剂从人脑中纯化了这种酶的活性，并表明纯化的酶具有预测的β-分泌酶的所有性质。该酶的克隆和表达表明，人脑β-分泌酶是一种新的膜结合天冬氨酸蛋白酶。
Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (A beta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the A beta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP(1), and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of A beta. The pathogenic mutation K670M671 --> N670L671 at the beta-secretase cleavage site in APP(2), which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate(3). Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.