Gamma-secretase is involved in the production of A β amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce A β and the APP intracellular domain (AICD). Metal ions play an important role in A β 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.
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