Evin Geneviève, Canterford Louise D, Hoke David E, Sharples Robyn A, Culvenor Janetta G, Masters Colin L
Department of Pathology, The University of Melbourne, Parkville 3010, and The Mental Health Research Institute, Parkville 3052, Australia.
Biochemistry. 2005 Mar 22;44(11):4332-41. doi: 10.1021/bi0481702.
Gamma-secretase mediates the final step, which generates Alzheimer's disease Abeta amyloid protein, by cleaving the transmembrane domain of the amyloid-beta protein precursor. Four gene products, presenilin, nicastrin, APH-1, and PEN-2, are required for gamma-secretase activity that is contained within a high molecular mass complex. To further characterize gamma-secretase, we probed membranes from human neuroblastoma SH-SY5Y cells with gamma-secretase inhibitor biotin derivatives of L-685,458, pepstatin A, and the difluoro alcohol 1-Bt. These inhibitor derivatives bound and precipitated PS1 fragments from membrane CHAPSO extracts. Analysis of PS1 complexes by blue native gel electrophoresis and western blotting indicated that the CHAPSO extracts contained complexes of approximately 900, 500, and 400 kDa. With this technique, derivatives of the three inhibitors were detected only in association with the 900 kDa species. Size-exclusion chromatography showed that 13% of PS1 immunoreactivity extracted with CHAPSO was comprised within a >or=900 kDa species with the remaining eluting in fractions of 669-250 kDa but that most enzymatic activity was associated with the 900 kDa fractions. After treatment with L-685,458 inhibitor, 49% PS1 immunoreactivity was eluted in the 900 kDa fraction, supporting evidence that the inhibitor stabilized this complex. Subcellular fractionation of SH-SY5Y cells indicated that the 900 kDa complex was formed as PS1 and NCT matured through the secretory pathway and that enzymatic activity correlated with complex maturation. From these observations, we propose a model for the structure of active gamma-secretase that would consist of dimerization of 400-500 kDa subunits and be consistent with the apparent molecular mass of the complex.
γ-分泌酶介导最后一步反应，该反应通过切割淀粉样β蛋白前体的跨膜结构域来生成阿尔茨海默病的Aβ淀粉样蛋白。γ-分泌酶活性需要四种基因产物，即早老素、尼卡斯特林、APH-1和PEN-2，它们包含在一个高分子量复合物中。为了进一步表征γ-分泌酶，我们用γ-分泌酶抑制剂L-685,458的生物素衍生物、胃蛋白酶抑制剂A和二氟醇1-Bt探测人神经母细胞瘤SH-SY5Y细胞的膜。这些抑制剂衍生物结合并沉淀了膜CHAPSO提取物中的PS1片段。通过蓝色非变性凝胶电泳和蛋白质印迹分析PS1复合物表明，CHAPSO提取物中含有约900、500和400 kDa的复合物。用这种技术，仅在与900 kDa的物质结合时检测到三种抑制剂的衍生物。尺寸排阻色谱显示，用CHAPSO提取的PS1免疫反应性的13%包含在一个≥900 kDa的物质中，其余的在669 - 250 kDa的级分中洗脱，但大多数酶活性与900 kDa的级分相关。用L-685,458抑制剂处理后，49%的PS1免疫反应性在900 kDa级分中洗脱，支持了该抑制剂稳定了这种复合物的证据。SH-SY5Y细胞的亚细胞分级分离表明，900 kDa的复合物是在PS1和NCT通过分泌途径成熟时形成的，并且酶活性与复合物成熟相关。基于这些观察结果，我们提出了一个活性γ-分泌酶结构的模型，该模型由400 - 500 kDa亚基的二聚化组成，并且与复合物的表观分子量一致。
