Purification and characterization of the human γ-secretase complex
Purification and characterization of the human γ-secretase complex
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DOI：
10.1021/bi0494976
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发表时间：
2004-08-03
期刊：
影响因子：
2.9
通讯作者：
Wolfe, MS
中科院分区：
文献类型：
--
作者：
Fraering, PC;Ye, WJ;Wolfe, MS
γ-分泌酶是一类具有膜内催化位点的蛋白酶。这种酶切割许多I型膜蛋白，包括淀粉样β蛋白（A）前体（APP）和Notch受体。生物化学和遗传学研究已经确定了四种膜蛋白作为γ-分泌酶的组分：由其N-和C-末端片段组成的异二聚体早老素（PS）（PS-NTF/CTF），成熟的糖基化形式的nicastrin（NCT），Aph-1和Pen-2。最近在果蝇、哺乳动物和酵母细胞中的研究数据表明，PS、NCT、Aph-1和Pen-2是重建γ-分泌酶活性所必需和充分的。然而，许多未解决的问题，特别是其他结构或调节成分的可能性，将通过实际纯化酶来解决。在这里，我们报告了一个详细的，多步骤的活性γ-分泌酶的纯化程序和纯化的蛋白酶的初步表征。纯化蛋白质的广泛质谱分析强烈表明PS-NTF/CTF、mNCT、Aph-1和Pen-2是活性γ-分泌酶的组分。使用纯化的γ-分泌酶，我们描述了调节特定A β种类产生的因素：（1）磷脂酰胆碱和鞘磷脂显着提高活性而不改变APP底物内的切割特异性;（2）将CHAPSO浓度从0.1%增加到0.25%产生类似于100%的A β 42产生增加;（3）将基于APP的重组底物暴露于0.5%SDS将切割特异性从疾病模拟模式（高A β 42/43）调节为生理模式（高A β 40）;和（4）舒林酸硫化物直接且优先降低纯化复合物内的A β 42切割。两者合计，我们的研究结果定义了一个程序，用于纯化活性γ-分泌酶，并表明，脂质介导的构象的酶和底物调节生产的潜在神经毒性Abeta 42和Abeta 43肽。
gamma-Secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (A) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a similar to100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high A 40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.