Insulysin Cleaves the APP Cytoplasmic Fragment at Multiple Sites

Abstract

The amyloid peptide (Abeta) deposited in Alzheimer's disease (AD) is generated by beta- and gamma-secretase processing of a larger integral membrane protein precursor (APP). Intramembrane processing of APP by gamma-secretase also yields an intracellular fragment, CTFgamma (a.k.a. AICD), which is highly conserved and is believed to regulate the transcription of several genes including KAI-1 and GSK3beta. The intracellular domain of APP is also processed by caspase to a 31 aa fragment that was shown to induce apoptosis by several groups. Although large quantities of CTFgamma are generated continuously by neurons, little if any is normally detected in cell lysates, which suggests that it is very rapidly turned over in vivo. Previous studies demonstrated that insulysin (IDE), an Abeta-degrading enzyme, is responsible for cytosol-mediated CTFgamma degradation in vitro. Consistent with this finding, knockout mice lacking IDE accumulate CTFgamma to detectable levels in the brain, although its levels remain lower than its precursor, suggesting that it continues to be turned over in the brain. Moreover, when we treated cultured cells with IDE inhibitors, we did not observe an increase in CTFgamma in cell lysates, suggesting that pathways other than IDE are also involved in CTFgamma turnover. To understand CTFgamma turnover further, we have mapped the IDE cleavage sites with the intention of mutating them to examine alternative pathways in future studies. Edman degradation revealed that IDE cleaves CTFgamma at multiple sites to small peptides ranging from 5 to 14 aa. The cleavage sites do not reveal the existence of any sequence specificity for IDE cleavage. Understanding the turnover mechanisms of CTFgamma is critical to the understanding of the signaling function of APP mediated by this fragment. The current study presents the interesting specificity of CTFgamma turnover by IDE, which has been previously identified as the major degrading enzyme for Abeta as well as CTFgamma. In addition, the study provides evidence for the presence of alternative CTFgamma-degrading pathways in the cell.