PEN-2 and APH-1 Coordinately Regulate Proteolytic Processing of Presenilin 1

Wen-Jie Luo,Hong Wang,Hongqiao Li,Benny S Kim,Sanjiv Shah,Hahn-Jun Lee,Gopal Thinakaran,Tae-Wan Kim,Gang Yu,Huaxi Xu

doi:10.1074/jbc.c200648200

Abstract

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH(2)- and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-gamma-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the gamma-secretase activity. However, their precise function in regulating the PS complex and gamma-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.

Highlights

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous ␥-secretase cleavage of the ␤-amyloid precursor protein and signaling receptor Notch
A␤ peptides are proteolytically derived from amyloid precursor protein (APP), a type 1 transmembrane glycoprotein that predominantly resides in the trans-Golgi network (TGN), by two distinct enzymatic activities known as ␤-secretase and ␥-secretase [2]
Given the fact that markedly lower levels of PS1 COOH-terminal fragment (CTF) are detected in PEN-2 small interfering RNA (siRNA) transfected cells as compared with controls, and the remaining PS fragments were quite stable during the length of cycloheximide treatment (8 h) (Fig. 3B), we conclude that accumulation of stabilized PS1 holoprotein and the concomitant diminution in the levels of PS1 fragment is a direct consequence of deficiency in endoproteolytic processing of PS1, further supporting our model that PEN-2 plays an indispensable role in the proteolytic cleavage of PS1

Summary

Introduction

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous ␥-secretase cleavage of the ␤-amyloid precursor protein and signaling receptor Notch. We characterize endogenous PEN-2 in mammalian cells and demonstrate for the first time the function of PEN-2, with coordination of APH-1, in mediating the proteolytic processing/ biogenesis of PS1 fragments.

Results

Conclusion