Ho Michael, Hoke David E, Chua Yee Jia, Li Qiao-Xin, Culvenor Janetta G, Masters Colin, White Anthony R, Evin Geneviève
Department of Pathology, The University of Melbourne, Parkville, VIC 3010, Australia.
Int J Alzheimers Dis. 2010 Dec 28;2011:950932. doi: 10.4061/2011/950932.
Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg(2+) stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca(2+) and Mg(2+) stabilize γ-secretase and enhance its activity.
γ-分泌酶参与β淀粉样肽（Aβ）的生成。它在不同位点切割淀粉样前体蛋白（APP）的跨膜结构域，从而产生Aβ和APP细胞内结构域（AICD）。金属离子在Aβ的聚集和代谢中起重要作用，因此金属螯合剂和配体是治疗阿尔茨海默病（AD）的潜在治疗药物。金属螯合剂对γ-分泌酶的直接作用尚未得到研究。作者采用了一种体外γ-分泌酶检测方法，该方法包括用来自啮齿动物脑和人神经母细胞瘤SH-SY5Y的内源性γ-分泌酶切割APP C100-3XFLAG，并通过蛋白质印迹法检测AICD的产生。向反应中添加金属蛋白酶抑制剂表明，氯碘羟喹、磷酰胺脒和锌金属蛋白酶抑制剂对γ-分泌酶活性没有显著影响。相反，菲咯啉、乙二胺四乙酸（EDTA）和乙二醇双四乙酸（EGTA）显著降低了γ-分泌酶活性，而添加钙和镁离子后可恢复该活性。通过蓝色非变性凝胶电泳和尺寸排阻色谱法，镁离子（Mg²⁺）稳定了一种1000 kDa的早老素1复合物。数据表明，钙离子（Ca²⁺）和镁离子（Mg²⁺）稳定γ-分泌酶并增强其活性。
