Identification of new Presenilin-1 phosphosites: implication for [gamma]-secretase activity and A[beta] production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (A[beta]) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease [gamma]-secretase is responsible for the intramembrane proteolysis of the amyloid-[beta] precursor protein (APP), which leads to the production of the toxic A[beta] peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the [gamma]-secretase activity, to reduce A[beta]42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human [gamma]-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the [gamma]-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of [gamma]-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate [gamma]-secretase activity and the production of the Alzheimer's A[beta] peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral A[beta] plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's [gamma]-secretase complex. By combining a mutagenesis approach with cell-based and cell-free [gamma]-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of [gamma]-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral A[beta] plaque formation in Alzheimer's Disease. A[beta], amyloid beta.