Premium
P1‐219: AMYLOID‐β DEGRADATION IN INDUCED PLURIPOTENT STEM CELL (IPSC)‐DERIVED NEURONS
Author(s) -
Rowland Helen A.,
Corbett Nicola J.,
Fisher Kate,
Jones Alys C.,
Kellett Katherine A.,
Hooper Nigel M.
Publication year - 2018
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2018.06.224
Subject(s) - induced pluripotent stem cell , proteases , chemistry , cell culture , neprilysin , biochemistry , microbiology and biotechnology , biotin , embryonic stem cell , biology , enzyme , genetics , gene
C-terminal end of Ng. The present work was aimed at identifying enzymes generating this cleavage pattern. Methods:Two quenched fluorogenic peptides based on sequences of the main cleavage regions were used to test enzymes known to be upregulated in AD as well as probe fractionated mouse brain extracts for Ng-cleaving activity. The Ng fragments from in vitro cleavage were determined by MALDI-TOF MS and LC-ESI-MS. Results:We identified calpain I as cleaving Ng in its central region, at the C-terminal end of amino acids 37, 42 and 65. Fragments starting at amino acid 43 and smaller were among the dominant fragments seen by IPMS in CSF.We also identified the intracellular enzyme prolyl endopeptidase as being able to generate fragments lacking the very Cterminal three amino acids, which are also seen in CSF. While shorter Ng fragments were readily cleaved in vitro by prolyl endopeptidase, the efficiency of cleavage on larger Ng fragments was much lower. Conclusions: Calpain I and prolyl endopeptidase were identified as candidate enzymes involved in the formation of endogenous Ng peptides found in CSF, cleaving in the central region near the IQ domain and after Ng75. Whereas full length Ng is able to bind to calmodulin in the absence of Ca ions, most of the calpain fragments of Ng in CSF are lacking the IQ domain and are thus unable to do so. While the action of prolyl endopeptidase can explain the appearance of fragments lacking the last three C-terminal amino acids, the functional significance of that activity is still unclear. However, calpainand prolyl endopeptidase-specific fragments of Ng may give clues to increased activities of these enzymes during progression of AD.