Design and characterization of a novel cellular prion-derived quenched fluorimetric substrate of alpha-secretase.
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.
Cissé
Moustapha Alfa
MA
Institut de Pharmacologie Moléculaire et Cellulaire, UMR6097 CNRS/UNSA, Equipe labélisée Fondation pour la Recherche Médicale, Valbonne, France.
Gandreuil
Céline
C
Hernandez
Jean-François
JF
Martinez
Jean
J
Checler
Frédéric
F
Vincent
Bruno
B
D051722:ADAM Proteins / Q000378:metabolism; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018621:PrPC Proteins / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods
D008565:Membrane Proteins; D018621:PrPC Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D051722:ADAM Proteins; C098339:ADAM9 protein, human; C498316:Adam9 protein, mouse; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; C491234:Adam10 protein, mouse; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse