We recently cloned endopeptidase‐24.16 (neurolysin; EC 3.4.24.16), a neurotensin‐degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3‐λ7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase‐24.16 immunoreactivity and exhibited QFS‐ and neurotensin‐hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane‐associated form of endopeptidase‐24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase‐24.16. The membrane‐associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the “neuronal” phenotype exhibited by the enzyme in transfected cells.

Alzheimer's disease is characterized by the extracellular deposition of the amyloid beta-peptide that derives from its precursor betaAPP by sequential actions of beta- and gamma- secretases, respectively. Recent studies aimed at identifying these enzymes have been reported as it is thougth that their inhibition should hopefully lead to reduce Abeta load in the AD brains. beta-secretase seems to be due to BACE1, a novel membrane-bound aspartyl protease. gamma-secretase identification is still a matter of controversy. Invalidation of presenilin genes was reported to impair both gamma-secretase-mediated Abeta production and Notch cleavage leading to NICD production. This observation together with another biochemical and pharmacological evidences led to suggest that presenilins could be the genuine long-searched gamma-secretase that would be responsible for both APP and Notch cleavages. We have designed novel non peptidic potential inhibitors of gamma-secretase (referred to as JLK inhibitors) and examined their ability to prevent Abeta40 and Abeta42 secretions as well as NICD production. Three out of a series of these agents drastically lower the recoveries of both Abeta40 and Abeta42 produced by betaAPP-expressing cell lines and concomitantly protect intracellular C99 and C83 recoveries. These inhibitors also prevent Abeta40/42 productions by C99-expressing cells. Interestingly, these inhibitors were totally unable to affect the DeltaENotch cleavage leading to NICD generation. Here, we also further characterize the pharmacological properties and specificity of these JLK inhibitors.