Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.

Alzheimer's disease is characterized by formation of neurofibrillary tangles and amyloid plaques in the regions of the central nervous system that are involved in learning and memory (1). It is believed that accumulation of A␤ 1 in plaques or as soluble aggregates initiates a pathological cascade leading to synaptic dysfunction and neuronal toxicity, with neurodegeneration and dementia as the final outcome (1, 2). Therefore, strategies to reduce the level of brain A␤ are being aggressively pursued as an approach likely to benefit Alzheimer's disease patients. A␤ is produced as the result of sequential proteolysis of a type I transmembrane protein APP by ␤-and ␥-secretases. ␤-Secretase cleaves APP in its extracellular domain at a site close to the membrane surface, a reaction that generates a membrane-bound APP C-terminal fragment of 99 amino acid residues (C99). A subsequent endoproteolysis within the transmembrane domain of C99 by ␥-secretase produces A␤. Whereas ␤-secretase, an aspartyl protease, has been well characterized (3-6), the identity and structure of ␥-secretase, also thought to be an aspartyl protease (7-12), remains elusive, and its kinetic and catalytic mechanisms are poorly understood. To a large extent, this is due to the highly complicated structural organization of this unusual protease. In contrast to other known proteases, ␥-secretase is composed of a high molecular weight multicomponent complex of transmembrane proteins (13,14). Primarily due to this structural complexity, the catalytic site and mechanism of action of ␥-secretase has not been unequivocally established. Early findings point to presenilin 1 or 2 as the catalytic subunit of ␥-secretase (15, 16). These multipass transmembrane proteins contain two essential aspartate residues in putatively adjacent transmembrane domains (15) and can be cross-linked by high affinity ␥-secretase inhibitors (16 -18). Recent advances have identified three additional proteins, nicastrin (19), aph-1 (20, 21), and pen-2 (20, 22), in the same multicomponent complex, whose co-expression with presenilin appears to be critical for ␥-secretase activity (19 -22). However, the precise roles of these additional protein subunits in the catalytic mechanism of ␥-secretase await further investigation.Associated with the structural complexity of ␥-secretase is the versatility of this protease in cleaving several type I transmembrane proteins. In addition to APP processing, ␥-secretase is required for proteolytic activation of Notch receptor (23-26), a signaling molecule essential for embryonic development of all metazoan species (27). Cleavage of Notch in the transmembrane domain by ␥-secretase generates Notch intracellular domain (NICD), which then translocates into the nucleus, where it regulates gene transcription (28,29). The list of other potential protein substrates for ␥-secretase has recently been expanded to include ErbB4 (30, 31), E-cadherin (32), and CD44 (33). However, the mechanisms by which ␥-secretase reacts with these different substrates remains un...

Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury.