Document Detail

Design and characterization of a novel cellular prion-derived quenched fluorimetric substrate of alpha-secretase.
MedLine Citation:
PMID:  16806063     Owner:  NLM     Status:  MEDLINE    
Under normal conditions, the cellular prion protein (PrP(c)) undergoes a proteolytic attack between amino acids 111 and 112 which gives rise to the N-terminal secreted N1 fragment and its C-terminal membrane-tethered counterpart C1. Importantly, this cleavage precludes the integrity of the neurotoxic 106-126 sequence. Here, we describe an original and reliable assay based on a quenched fluorimetric substrate (JMV2770) encompassing the 111/112 sequence of PrP(c). In whole brain homogenate, the JMV2770-hydrolysing activity is optimal at neutral pH and sensitive to the metalloprotease inhibitor BB3103 but not to acidic and serine protease blockers. JMV2770 is efficiently cleaved by intact HEK293 cells and fibroblasts in culture, consistent with an hydrolysis by a typical ectoprotease. Overexpressions of alpha-secretases a disintegrin and metalloprotease-9 (ADAM9), ADAM10 or TACE (ADAM17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM10 and TACE or reduced expression of ADAM9 by an antisense approach significantly reduced its cleavage. Finally, analysis of JMV2770 hydrolysis following transient transfection of ADAM10 or ADAM9 cDNA in ADAM10(-/-) fibroblasts allowed to confirm our previous data establishing that ADAM9 does not behave as a genuine alpha-secretase but rather acts as an important upstream regulator of ADAM10 activity.
Moustapha Alfa Ciss?; C?line Gandreuil; Jean-Fran?ois Hernandez; Jean Martinez; Fr?d?ric Checler; Bruno Vincent
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-06-21
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  347     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2006 Aug 
Date Detail:
Created Date:  2006-07-11     Completed Date:  2006-09-13     Revised Date:  2010-04-12    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  254-60     Citation Subset:  IM    
Institut de Pharmacologie Mol?culaire et Cellulaire, UMR6097 CNRS/UNSA, Equipe lab?lis?e Fondation pour la Recherche M?dicale, Valbonne, France.
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MeSH Terms
ADAM Proteins / metabolism
Amyloid Precursor Protein Secretases
Aspartic Acid Endopeptidases
Cell Line
Endopeptidases / genetics*,  metabolism*
Fibroblasts / metabolism*
Fluorometry / methods*
Kidney / metabolism*
Membrane Proteins / metabolism
PrPC Proteins / metabolism*
Protein Engineering / methods*
Substrate Specificity
Transfection / methods
Reg. No./Substance:
0/Membrane Proteins; 0/PrPC Proteins; EC 3.4.-/Amyloid Precursor Protein Secretases; EC 3.4.-/Endopeptidases; EC 3.4.23.-/Aspartic Acid Endopeptidases; EC protein, human; EC protein, mouse; EC 3.4.24.-/ADAM Proteins; EC 3.4.24.-/ADAM9 protein, human; EC 3.4.24.-/Adam9 protein, mouse; EC 3.4.24.-/tumor necrosis factor-alpha convertase; EC protein, human; EC protein, mouse

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