Design and characterization of a novel cellular prion-derived quenched fluorimetric substrate of α-secretase

Under normal conditions, the cellular prion protein (PrPc) undergoes a proteolytic attack between amino acids I I I and 112 which gives rise to the N-terminal secreted NI fragment and its C-terminal membrane-tethered counterpart Cl. 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 PrPc. 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), ADAM 10 or TACE (ADAM 17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM 10 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 a-secretase but rather acts as an important upstream regulator of ADAM10 activity. (c) 2006 Elsevier Inc. All rights reserved.