Effect of Metal Chelators onγ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

Michael Ho,Geneviève Evin,Anthony R White,Colin Masters,Janetta G Culvenor,Qiao-Xin Li,David E Hoke,Yee Jia Chua

doi:10.4061/2011/950932

Abstract

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. Mg2+ stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+ and Mg2+ stabilize γ-secretase and enhance its activity.

Highlights

Gamma-secretase is a key protease activity involved in the production of Alzheimer’s disease Aβ amyloid peptides and in regulated intramembrane processing of a subset of membrane receptors, including Notch
Shedding of the large amyloid precursor protein (APP) ectodomain by β-secretase [3] produces a 99 amino acid membrane-tethered stub (β-secretase generated APP Cterminal fragment, βCTF, or C99) that is further processed by γ-secretase to liberate Aβ peptides in the extracellular/luminal space. γ-Secretase cleaves the transmembrane domain of APP at multiple sites
C100 substrate, expressed in E. coli, is based on the human APP C-terminal sequence, which corresponds to the C-terminal fragment produced by β-secretase cleavage of APP (β-CTF, the direct precursor to Aβ) plus N-terminal Met596

Summary

Introduction

Gamma-secretase is a key protease activity involved in the production of Alzheimer’s disease Aβ amyloid peptides and in regulated intramembrane processing of a subset of membrane receptors, including Notch (reviewed in [1]). Shedding of the large APP ectodomain by β-secretase (βAPP cleaving enzyme, BACE1) [3] produces a 99 amino acid membrane-tethered stub (β-secretase generated APP Cterminal fragment, βCTF, or C99) that is further processed by γ-secretase to liberate Aβ peptides in the extracellular/luminal space. APP undergoes ectodomain shedding by cleavage within the Aβ domain by α-secretase, in a nonamyloidogenic cellular pathway, followed by γ-secretase processing of the corresponding membrane stub (C83) to release AICD and a p3 fragment (reviewed in [8])

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