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

Abstract

␤-Amyloid precursor protein apparently undergoes at least three major cleavages, ␥-, ⑀-, and the newly identified -cleavage, within its transmembrane domain to produce secreted ␤-amyloid protein (A␤). However, the roles of ⑀-and -cleavages in the formation of secreted A␤ and the relationship among these three cleavages, namely ⑀-, -, and ␥-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 ␥-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that A␤ 46 is an intermediate precursor of secreted A␤. Our co-immunoprecipitation data also reveal that, as an intermediate, A␤ 46 is tightly associated with presenilin in intact cells. Furthermore, we identified a long A␤ species that is most likely the long sought after intermediate product, A␤ 49 , generated by ⑀-cleavage, and this A␤ 49 is further processed by -and ␥-cleavages to generate A␤ 46 and ultimately the secreted A␤ 40/42 . More interestingly, our data demonstrate that ␥-cleavage not only occurs last but also depends on -cleavage occurring prior to it, indicating that -cleavage is crucial for the formation of secreted A␤. Thus, we conclude that the C terminus of secreted A␤ is most likely generated by a series of sequential cleavages, namely first ⑀-cleavage which is then followed by -and ␥-cleavages, and that A␤ 46 produced by -cleavage is the precursor of secreted A␤ 40/42 .The mechanism of the formation of the ␤-amyloid protein (A␤) 2 is the central issue in Alzheimer disease research, not only because A␤ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease, but also because A␤ formation may be a causative event in the disease (1). A␤ is proteolytically derived from a large single transmembrane protein, the ␤-amyloid precursor protein (APP), as a result of sequential cleavages by ␤-and ␥-secretases (1). ␤-Secretase has been identified as a type I membrane aspartyl protease (2, 3). Although the exact nature of ␥-secretase is still a matter of debate, accumulating evidence supports the idea that ␥-secretase is a multiple molecular complex composed of, at least, presenilins, nicastrin, Aph-1, and Pen-2 and that presenilin may function as the catalytic subunit (4).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 first one is the cleavage now specifically referred to as ␥-cleavage (5), which produces the C termini of most of the secreted A␤ species that end at amino acids 40 (A␤40) or 42 (A␤42) of the A␤ sequence. 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) (5-8). The identification of this ⑀-cleavage site raises a question as to whether this ⑀-cleavage is obligatory for the generation of the...

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