Aβ42 Overproduction Associated with Structural Changes in the Catalytic Pore of γ-Secretase: COMMON EFFECTS OF PEN-2 N-TERMINAL ELONGATION AND FENOFIBRATE

Noriko Isoo,Chihiro Sato,Hiroyuki Miyashita,Mitsuru Shinohara,Nobumasa Takasugi,Yuichi Morohashi,Shoji Tsuji,Taisuke Tomita,Takeshi Iwatsubo

doi:10.1074/jbc.m611549200

Noriko Isoo, Chihiro Sato + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m611549200

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Abstract

gamma-Secretase is an atypical aspartyl protease that cleaves amyloid beta-precursor protein to generate Abeta peptides that are causative for Alzheimer disease. gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on gamma-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of Abeta42, the longer and more aggregable species of Abeta. The effect of the N-terminal elongation of Pen-2 on Abeta42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro gamma-secretase assay revealed that Pen-2 directly affects the Abeta42-generating activity of gamma-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an Abeta42-raising gamma-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of Abeta42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.

Highlights

Amyloid ␤ peptide (A␤)4 deposited in the brains of patients with Alzheimer disease (AD), is derived from amyloid ␤-pre
Molecular function of Aph-1, a putative multipass membrane protein that forms a subcomplex with Nct in early methylammonio]-2-hydroxy-1-propanesulfonate; NTF, N-terminal fragment; CTF, carboxyl-terminal fragment; DAPT, N-[N-(3,5-difluorophenacetyl)L-alanyl]-(S)-phenylglycine t-butyl ester; DKO cell, Psen1/Psen2 double knockout cell line; familial AD (FAD), familial Alzheimer disease; GSM, ␥-secretase modulator; Nct, nicastrin; PS, presenilin; SC100, C-terminal 99-amino acid fragment of human APP with start methionine and signal peptide; TMD, transmembrane domain; Bicine, N,N-bis(2-hydroxyethyl)glycine; EGFP, enhanced green fluorescent protein; UTR, untranslated region; RNAi, RNA interference; dsRNA, double strand RNA; HA, hemagglutinin; MTSEA, methanethiosulfonate ethylammonium
We found that the N-terminal extension of Pen-2 with various tag sequences directly affected the enzymatic property of the ␥-secretase complex in a way that increases at position 42 (A␤42) production both in insect and mammalian cell systems

Summary

Introduction

Amyloid ␤ peptide (A␤) deposited in the brains of patients with Alzheimer disease (AD), is derived from amyloid ␤-pre-. Nct is a single pass transmembrane protein harboring a large extracellular region that captures the N-terminal tip of substrates [9] This binding is independent of the formation of an active ␥-secretase complex, suggesting that Nct functions as a substrate binding site that is distinct from the active site (i.e. exosite) of ␥-secretase. Molecular function of Aph-1, a putative multipass membrane protein that forms a subcomplex with Nct in early methylammonio]-2-hydroxy-1-propanesulfonate; NTF, N-terminal fragment; CTF, carboxyl-terminal fragment; DAPT, N-[N-(3,5-difluorophenacetyl)L-alanyl]-(S)-phenylglycine t-butyl ester; DKO cell, Psen1/Psen double knockout cell line; FAD, familial Alzheimer disease; GSM, ␥-secretase modulator; Nct, nicastrin; PS, presenilin; SC100, C-terminal 99-amino acid fragment of human APP with start methionine and signal peptide; TMD, transmembrane domain; Bicine, N,N-bis(2-hydroxyethyl)glycine; EGFP, enhanced green fluorescent protein; UTR, untranslated region; RNAi, RNA interference; dsRNA, double strand RNA; HA, hemagglutinin; MTSEA, methanethiosulfonate ethylammonium. We report on an unexpected observation that the length of the N terminus of Pen-2 affects the structure of the active site of ␥-secretase in a way to increase A␤42 generation, in a similar fashion to fenofibrate, an A␤42-raising ␥-secretase modulator

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