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

Abstract

γ-Secretase inhibitors (GSIs) and modulators (GSMs) are at the frontline of cancer and Alzheimer's disease research, respectively. While both are therapeutically promising, not much is known about their interactions with proteins other than γ-secretase. Signal peptide peptidase (SPP), like γ-secretase, is a multispan transmembrane aspartyl protease that catalyzes regulated intramembrane proteolysis. We used active site-directed photophore walking probes to study the effects of different GSIs and GSMs on the active sites of γ-secretase and SPP and found that nontransition state GSIs inhibit labeling of γ-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of GSMs, which have little effect on the labeling of γ-secretase but diminish labeling of SPP. These results demonstrate that GSIs and GSMs are altering the structure of not only γ-secretase but also SPP, leading to potential changes in enzyme activity and specificity that may impact the clinical outcomes of these molecules.

Figure 1

Structural and functional similarities/differences between SPP and γ-secretase. SPP and γ-secretase are similar in that they are both multipass transmembrane enzymes that share the YD/GXGD catalytic motif and cleave their respective substrates in the transmembrane region. SPP and PS, the catalytic subunit of γ-secretase, both transverse the membrane 9 times. The differences between the enzymes include their limited sequence homology, their inverse orientation in the membrane, and their specificity for either type 1 or type 2 substrates. Additionally, SPP functions without protein cofactors while PS requires at least 3 additional proteins (Aph1, Nct, and Pen2) for γ-secretase activity.

Figure 2

L458-based photoreactive probes specifically label PS1 and SPP. (A) Structures of L458 and photoreactive probes JC8, L646, GY4, and L505. L458 side chain residues (P and P′ sites) interact with corresponding subpockets of γ-secretase and SPP (S and S′ sites). Photoreactive probes JC8, L646, GY4, and L505 have an L458 backbone (black), a biotin moiety (green), and a cross-linkable benzophenone (BP). Each probe has a BP incorporated into a different site on the L458 backbone. The location of the BP is illustrated by the color scheme. For example, JC8 has a BP at the P1′ site, in place of the red benzene ring. JC8, L646, GY4, and L505 label S1′, S2, S1, and S3′ subsites of the enzymes, respectively. (B) HeLa membranes were labeled with 20 nM of photoprobes JC8, L646, GY4, or L505 in the presence of 0.25% CHAPSO, and either with (+) or without (−) 2 μM L458. Samples were run on SDS-PAGE and analyzed by Western blot. Anti-SPP and PS1-NTF antibodies were used to detect SPP (upper panel) and PS1-NTF (lower panel). (C) Same as B, except 2 μM (Z-LL)₂-ketone was used to block the labeling of SPP (upper panel) and PS1-NTF (lower panel).

Figure 3

Endogenous, active SPP is a homodimer. (A) Structure of JC10. (B) JC8 and JC10 were used to photolabel SPP. TCEP and/or heat were used to elute photolabeled proteins.

Figure 4

GSIs and GSMs have opposite effects on the photolabeling profiles of γ-secretase and SPP. (A) Structures of E2012, GSM-616, BMS-708163, LY450139, cpd X, and GSI-34. (B) Photoprobes JC8, L646, GY4, and L505 were incubated with HeLa membrane in 0.25% CHAPSO in the presence of 25 μM GSMs E2012/616 (green) or 10 μM GSIs 708163/139/cpd X/GSI-34 (blue). Samples were run on SDS-PAGE and analyzed by Western blot. Anti-SPP and PS1-NTF antibodies were used to detect SPP (upper panel) and PS1-NTF (lower panel). (C) Densitometry quantification of SPP labeling. GSMs are graphed in green, and GSIs are graphed in blue. (D) Same as C, except PS1-NTF. ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 5

Model for the change in active site conformation of γ-secretase and SPP that occurs upon binding by GSIs and GSMs. We propose that the GSIs and GSMs studied here allosterically bind to γ-secretase and SPP, causing a conformational change in the active sites of the enzymes. Surprisingly, the induced conformational change is opposite for the two enzymes, as evidenced by their binding to active site-directed probes. Specifically, GSIs cause decreased binding between γ-secretase and probe while increasing binding between SPP and probe. GSMs cause little change in binding between γ-secretase and probe but reduce binding between SPP and probe. This suggests a model in which GSIs cause the active site of γ-secretase to assume a “closed” conformation but have the reverse impact on the active site structure of SPP.