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首页> 外文期刊>Biochemistry >Transition-State Analogue y-Secretase Inhibitors Stabilize a 900 kDa Presenilin/Nicastrin Complex
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Transition-State Analogue y-Secretase Inhibitors Stabilize a 900 kDa Presenilin/Nicastrin Complex

机译：过渡态类似物y-秘密酶抑制剂可稳定900 kDa早老素/尼卡斯特林复合物

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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 ~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 >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.
机译：γ-分泌酶介导最后一步，该过程通过裂解淀粉样β蛋白前体的跨膜结构域来生成阿尔茨海默氏病Aβ淀粉样蛋白。γ需要4种基因产物，早老素，尼古斯汀，APH-1和PEN-2分子复合物中包含的β-分泌酶活性。为进一步表征γ-分泌酶，我们用γ-分泌酶抑制剂生物素衍生物L-685,458，胃蛋白酶抑制素A和二氟醇1探测了人类神经母细胞瘤SH-SY5Y细胞的膜。这些抑制剂衍生物从膜CHAPSO提取物中结合并沉淀出PS1片段。通过蓝色天然凝胶电泳和Western印迹分析PS1复合物，表明CHAPSO提取物含有约900,500和400 kDa的复合物。仅检测到与900 kDa物种相关的3种抑制剂。大小排阻色谱法显示CHAPSO提取的PS1免疫反应性为13％在一个大于900 kDa的物种中有价值，其余的以669-250 kDa的级分洗脱，但大多数酶活性与900 kDa的级分有关。用L-685,458抑制剂处理后，在900 kDa的级分中洗脱了49％的PS1免疫反应性这些证据表明，SH-SY5Y细胞的亚细胞分馏表明，随着PS1和NCT通过分泌途径的成熟，形成了900 kDa的复合物，并且酶活性与复合物的成熟有关。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，并且与该复合物的表观分子量相符。

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