The Mechanism of γ-Secretase

Abstract

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...

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

“…(31,33). Furthermore, we showed that one of these non-transition state inhibitors, BMS-433796, exhibited noncompetitive displacement of [ 3 H]L-685,458 from SPP, suggesting a distinct binding site, which is potentially equivalent to the allosteric inhibitor binding site proposed for ␥-secretase (17,18). In addition, we found that the NSAID sulindac sulfide could displace the radioligand [ 3 H]L-685,458 from SPP at high concentrations, consistent with its reported modulation of SPP enzyme activity (34).…”

“…If so, the presence of SPP might account for the incomplete displacement of [ 3 H]L-685,458 observed for selective ␥-secretase inhibitors (18), thus calling into question the conclusion that the catalytic active site and the allosteric binding site are present in a 2:1 molar ratio (18). Furthermore, it seems likely that earlier studies of this radioligand in whole cell extracts may have detected predominantly SPP binding rather than ␥-secretase binding (17,37 From the drug discovery perspective, similar pharmacology between ␥-secretase and SPP suggests the potential for off-target liabilities. Additional studies would be needed to understand to what extent inhibitor binding affects enzyme function in vivo and to determine what consequences, if any, would arise from dosing a nonselective drug.…”

“…In addition to the transition state analog inhibitors, there are also non-transition state ␥-secretase inhibitors that target presenilin (15,16). These compounds are proposed to mediate inhibition through a distinct allosteric site on the basis of inhibitor cross-competition kinetics and radioligand displacement studies (17,18). Differences in binding pharmacology between the transition state analogs and non-transition state inhibitors were also inferred from photolabeling studies (16,19).…”

“…To address this question, we utilized an active site-directed ␥-secretase radioligand (17,18,37) and took advantage of our finding that it binds with high affinity to both SPP and ␥-secretase. In the course of this study, we found that SPP family protein binding is abundant in cell homogenates, presenting a challenge to the evaluation of ␥-secretase and SPP binding under these conditions.…”

γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

“…First, these cleavages may be catalyzed by two enzymes, and second, these cleavages may be catalyzed by one enzyme that has two inhibitor binding sites, one for the transition state analogs, such as L-685,458, and the other for the nontransition state inhibitors, such as compound E, as suggested by a recent inhibitor binding kinetic study (23). The sequential relationship of these cleavages and specifically the finding that ␥-cleavage is dependent on ⑀-and -cleavages occurring first, suggest that ␥-, -, and ⑀-cleavages are catalyzed by a single enzyme.…”