TY - JOUR
TI - Novel processing of Notch 1 within its intracellular domain by a cysteine protease
AU - Fassa, A.
AU - Parisiadou, L.
AU - Robakis, N.K.
AU - Efthimiopoulos, S.
JO - Neurodegenerative Diseases
PY - 2007
VL - 4
TODO - 2-3
SP - 148-155
PB - 
SN - 1660-2854, 1660-2862
TODO - 10.1159/000101839
TODO - aloxistatin;  brefeldin A;  calpain;  caspase;  caspase 3;  caspase inhibitor;  cell protein;  cysteine proteinase;  gamma secretase inhibitor;  l 685458;  Notch1 receptor;  serine;  unclassified drug;  uvomorulin, article;  carboxy terminal sequence;  cell strain HEK293;  cell surface;  controlled study;  embryo;  enzyme active site;  enzyme inhibition;  fragmentation reaction;  human;  human cell;  incubation time;  priority journal;  protein degradation;  protein domain;  protein expression;  protein function;  protein processing;  protein transport;  Western blotting, Carbamates;  Cell Line, Transformed;  Cysteine Endopeptidases;  Cytoplasm;  Dipeptides;  Enzyme Inhibitors;  Gene Expression Regulation;  Humans;  Immunoprecipitation;  Mutation;  Protein Structure, Tertiary;  Protein Transport;  Receptor, Notch1;  Transfection
TODO - In order to study N1 processing, we expressed human N1 (hN1) in HEK293 cells (293-hN1). Following Western blot analysis of 293-hN1 extracts, we detected, in addition to full-length hN1 and the N1 extracellular domain truncated form (N1-TM), a novel extracellular domain truncated form of hN1 with a COOH-terminal deletion, designated hN1-TMΔCT. Treatment of cells with the γ-secretase inhibitor L-685,458 resulted in an accumulation of hN1-TMΔCT suggesting that this fragment is a γ-secretase substrate. To identify the proteolytic activity(ies) that generates hN1-TMΔCT, we treated 293-hN1 cells with inhibitors of proteasome, calpains, caspases, serine and cysteine proteases. Despite the presence of a caspase-3 cleavage site within hN1 intracellular domain, none of the caspase inhibitors inhibited hN1-TMΔCT production. The proteasomal inhibitors used had also no effect. Incubation of cells with the cysteine protease inhibitor E64d resulted in the accumulation of hN1-TM and the inhibition of hN1-TMΔCT production suggesting a precursor-product relationship and that a cysteine protease is involved. Similarly, treatment of cells expressing amyloid precursor protein or E-cadherin with E-64d resulted in the accumulation of COOH-terminal fragments suggesting that these proteins are also processed within their intracellular domain by a cysteine protease. Processing towards hN1-TMΔCT requires maturation and transport of hN1 to the cell surface since treatment with brefeldin A inhibited its production and resulted in accumulation of hN1. Processing of hN1 within its intracellular domain could generate fragments that can exert novel functions and/or interfere with the function of hN1 intracellular domain. Copyright © 2007 S. Karger AG.
ER -