Distinct Pharmacological Effects of Inhibitors of Signal Peptide Peptidase and γ-Secretase

Abstract

Signal peptide peptidase (SPP) and ␥-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and ␥-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and ␥-secretase, did not affect the labeling of SPP or ␥-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported ␥-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by ␥-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to ␥-secretase, the naphthyl ketone modulators allowed labeling by the transitionstate analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and ␥-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and ␥-secretase.One of the top therapeutic strategies for the prevention and treatment of Alzheimer disease (AD) 2 is suppression of the production of the amyloid ␤-protein (A␤). A␤ is the primary protein component of the hallmark plaques in the AD brain, and aggregated A␤ is widely thought to cause the onset of AD (1). The 4-kDa A␤ is produced from the amyloid ␤-protein precursor (APP), a type I integral membrane protein, through sequential proteolysis by ␤-secretase and ␥-secretase. ␥-Secretase is composed of four essential membrane proteins, including presenilin (PS), Pen-2, nicastrin, and Aph-1 (2), with one of each component being sufficient for proteolytic activity (3). PS is the catalytic component of the enzyme (4), and missense mutations in PS cause early onset familial AD and alter the length of the products, A␤ (5) and the APP intracellular domain (AICD) (6). Although a minor species, the 42-residue A␤42 is initially deposited in the AD brain instead of the more predominant 40-residue A␤40 (7), and A␤42 is especially implicated in the pathogenesis of AD. As the proportion of A␤42 to A␤40 is determined by ␥-secretase, this membrane-embedded aspartyl protease is a major target for the development of AD drugs (8).Signal peptide peptidase (SPP) is an intramembrane aspartyl prot...

γ-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of γ-Secretase and Signal Peptide Peptidase

“…19,20 However, only the monomeric form of SPP was labeled with the active site-directed photoprobe III-63. 33,34 Additionally, endogenous DDM-solubilized human and drosophila SPP formed higher molecular weight complexes around 180−200 kDa. 21 We utilized a photolabeling approach to elucidate whether endogenous active SPP is a homodimer or monomer.…”

Requirement for small side chain residues within the GxGD‐motif of presenilin for γ‐secretase substrate cleavage

“…2005). However, recent data have also selectively identified the NTF (Sato et al. 2008b) as well as the CTF (Sato et al.…”

“…Initially, the site was mapped at the NTF/CTF interface (Kornilova et al 2005). However, recent data have also selectively identified the NTF (Sato et al 2008b) as well as the CTF (Sato et al 2008a). In the latter, a single docking site residue has been identified in the luminal oriented side of TMD9 (Sato et al 2008a), which has been proposed to possibly function as lateral gate for substrate entry to the catalytic active site (Sato et al 2008a;Tolia et al 2008).…”

Three-dimensional Structure of the Signal Peptide Peptidase

“…SPP utilizes two functional sites for the intramembrane cleavage, i.e. the initial substrate binding site and the catalytic site, which are directly targeted by the helical peptide-and the transition state analog-type inhibitors, respectively (8,22,31). A previous report indicated that both sites were present in the recombinant C-terminal fragment of dSPP (43).…”