Differential Effects of Inhibitors on the γ-Secretase Complex

Abstract

␥-Secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid ␤-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse ␥-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most ␥-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.Cerebral accumulation of the amyloid-␤ protein (A␤) 1 is considered a central event in the pathogenesis of Alzheimer's disease (AD). A␤ is produced via ␤-and ␥-secretases, proteases that have become important therapeutic targets for AD (1). ␥-Secretase plays a crucial role in determining the proportion of two forms of A␤, A␤ 40 and A␤ 42 . The 42-residue A␤ 42 is more prone to fibril formation and is disproportionately present in the plaques characteristic of the AD brain. Accumulating evidence (2-4) strongly suggests that ␥-secretase is an intramembrane-cleaving aspartyl protease with presenilin (PS) as the catalytic component. Three other multipass membrane proteins, nicastrin, Aph-1, and Pen-2, are genetically linked to ␥-secretase activity (5-7), and biochemical isolation has provided evidence that these proteins are indeed necessary members of the protease complex (8 -10). Despite the remarkable progress in uncovering the identity of ␥-secretase, its mechanism of action remains unclear.A body of work (11) suggests that ␥-secretase cleaves amide bonds within the transmembrane regions of its substrates, a poorly understood process of hydrolysis within a hydrophobic environment. Elucidating the molecular interaction between an inhibitor and its enzyme target can help identify the enzyme and provide insight into the catalytic mechanism. The study of peptidomimetic inhibitors of ␥-secretase that contain classic aspartyl protease transition state-mimicking moieties led to the suggestion that ␥-secretase is an aspartyl protease and that two conserved aspartates in presenilins are catalytic residues (12). PS is processed into N-terminal (NTF) and C-terminal (CTF) fragments. These fragments are metabolically stable, remain associated, and their formation is tightly regulated, suggesting that together they are the bioactive form of PS (12). Th...

Deducing the Transmembrane Domain Organization of Presenilin-1 in γ-Secretase by Cysteine Disulfide Cross-Linking

“…4). This is consistent with our previous findings that Compound E and D-13 bind PS1 in a mode partial overlapping with the active site of γ-secretase (23,45). Most importantly, the ability of the active-site directed III-31-C to interfere with PS1 oxidative crosslinking suggests that native Cys92, Cys410 and/or Cys419 are close to or within the active site of γ-secretase with the inhibitor binding preventing their interaction, or that the occupation of the active site results in a substantial conformational change in the PS1 molecule which in turn translates into a shift in distance between cysteine pairs.…”

A Signal Peptide Peptidase (SPP) Reporter Activity Assay Based on the Cleavage of Type II Membrane Protein Substrates Provides Further Evidence for an Inverted Orientation of the SPP Active Site Relative to Presenilin

“…3c). III-31-C, a transition state ␥-secretase inhibitor (30), inhibited reporter activity with an IC 50 of ϳ3 M. This compound is the parent compound to III-63, which we have shown binds the SPP homodimer (3). Consistent with studies using in vitro SPP cleavage assays (4), two other ␥-secretase inhibitors, DAPT and Compound E, did not inhibit reporter activity (Fig.…”

Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1

“…To address the importance of Notch 1 in the role of Ovol2 in regulating differentiation competence, we repeated the calcium induction experiment, this time including DAPT, a ␥-secretase inhibitor that blocks Notch signaling (53,54). The induction in K1 expression by the loss of Ovol2 was completely abolished when DAPT was added (Fig.…”