γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

Guojun Zhao,Mei-Zhen Cui,Guozhang Mao,Yunzhou Dong,Jianxin Tan,Longsheng Sun,Xuemin Xu

doi:10.1074/jbc.m507993200

Guojun Zhao, Mei-Zhen Cui + Show 5 more

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https://doi.org/10.1074/jbc.m507993200

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Abstract

beta-Amyloid precursor protein apparently undergoes at least three major cleavages, gamma-, epsilon-, and the newly identified zeta-cleavage, within its transmembrane domain to produce secreted beta-amyloid protein (Abeta). However, the roles of epsilon- and zeta-cleavages in the formation of secreted Abeta and the relationship among these three cleavages, namely epsilon-, zeta-, and gamma-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known gamma-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Abeta(46) is an intermediate precursor of secreted Abeta. Our co-immunoprecipitation data also reveal that, as an intermediate, Abeta(46) is tightly associated with presenilin in intact cells. Furthermore, we identified a long Abeta species that is most likely the long sought after intermediate product, Abeta(49), generated by epsilon-cleavage, and this Abeta(49) is further processed by zeta- and gamma-cleavages to generate Abeta(46) and ultimately the secreted Abeta(40/42). More interestingly, our data demonstrate that gamma-cleavage not only occurs last but also depends on zeta-cleavage occurring prior to it, indicating that zeta-cleavage is crucial for the formation of secreted Abeta. Thus, we conclude that the C terminus of secreted Abeta is most likely generated by a series of sequential cleavages, namely first epsilon-cleavage which is then followed by zeta- and gamma-cleavages, and that Abeta(46) produced by zeta-cleavage is the precursor of secreted Abeta(40/42).

Highlights

In understanding the mechanism by which the C termini of secreted A␤ are generated during the processing of APP, three major intramembranous cleavages have been established
L-685,458 Inhibits the Formation of amyloid protein (A␤)46—In our recent study, we have shown that treatment of cells with nontransition state ␥-secretase inhibitors, such as DAPT, DAPM, and compound E, caused an increase in the accumulation of intracellular A␤46, indicating that these inhibitors have no effect, or little effect, on the newly identified ␨-cleavage
It cannot be ruled out that the absence of A␤46 in cells treated with L-685,458 may be due to the inability of this inhibitor to block the turnover of A␤46

Summary

Introduction

In understanding the mechanism by which the C termini of secreted A␤ are generated during the processing of APP, three major intramembranous cleavages have been established. The second one is the ⑀-cleavage occurring between A␤ residues 49 and 50, which produces the N terminus of most of the APP intracellular domain (AICD) [5,6,7,8] The identification of this ⑀-cleavage site raises a question as to whether this ⑀-cleavage is obligatory for the generation of the C terminus of A␤, and this raises a question as to the relationship between ⑀- and ␥-cleavages, i.e. whether they are independent of each other or sequential. After centrifugation at 20,000 ϫ g at 4 °C for 15 min, the supernatant was diluted with an equal amount of IP buffer, and the A␤49 and other intracellular A␤ species were immunoprecipitated using 6E10 in the presence or absence of DAPT. DAPM was used to cause the accumulation of A␤46 in cells that would be used for determining the turnover of A␤46 either in intact cells or in a cell-free system in which the inhibitor used for causing the accumulation of A␤46 needs to be removed

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