Τίτλος:
Novel processing of Notch 1 within its intracellular domain by a cysteine protease
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
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.
Συγγραφείς:
Fassa, A.
Parisiadou, L.
Robakis, N.K.
Efthimiopoulos, S.
Περιοδικό:
Neurodegenerative Diseases
Λέξεις-κλειδιά:
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
