Structure, mechanism and inhibition of gamma-secretase and presenilin-like proteases

Abstract

Presenilin is the catalytic component of gamma-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. gamma-Secretase is involved in the pathogenesis of Alzheimer's disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, gamma-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials. Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the gamma-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the gamma-secretase complex. SPP biology, structure, and inhibition will also be discussed.

Figure 1

Presenilin, the γ-secretase complex, and the proteolysis of APP and Notch. A, Presenilin is endoproteolytically cleaved into two polypeptides, an N-terminal fragment (NTF) and a C-terminal fragment (CTF) that remain associated. Each fragment contributes one aspartate to the γ-secretase active site (denoted by D’s). APP is first cleaved in the extracellular domain by β-secretase, and the membrane-bound remnant is cleaved at least twice (at the γ and ε sites) within the membrane by γ-secretase to produce Aβ and the APP intracellular domain (AICD). The Notch extracellular is shed via proteolysis at the S2 site by ADAM17 and then at the S3 and S4 sites by γ-secretase to produce Nβ and the Notch intracellular domain (NICD). Stars indicate general locations of missense mutations in Presenilin and APP that are associated with autosomal dominant, early-onset familial Alzheimer’s disease. B, Other substrates known to be cleaved by the γ-secretase complex. C, Components of the γ-secretase complex. Presenilin assembles with Nicastrin (NCT), Aph1, and Pen-2, whereupon Presenilin is endoproteolytically cleaved to NTF and CTF. Only one of each component is required for γ-secretase activity.

Figure 2

Inhibitors and modulators of γ-secretase. Transition-state analogue inhibitors such as the peptidomimetic 1 include hydroxyl-containing moieties that interact with the catalytic aspartates of aspartyl proteases. Helical peptide inhibitors include α-aminoisobutyric acid (Aib)-containing substrate mimics such as 2 (*denotes that the threonine residue contains an O-benzyl group). These helical peptides mimic the APP transmembrane domain and interact with the substrate docking site on the protease. The potent benzodiazepine inhibitor 3 (LY-450,139) is in late-stage clinical trials for the treatment of Alzheimer’s disease. NSAID-like modulator 4 (R-Flurbiprofen or tarenflurbil), which recently failed in late-stage clinical trials for Alzheimer’s disease, shifts where γ-secretase cuts APP, reducing the aggregation-prone Aβ42 and elevating more soluble Aβ38. Naphthyl ketone 5 inhibits total Aβ production without interfering with the ability of γ-secretase to cleave Notch receptor substrates.

Figure 3

A 12 Å resolution structure of γ-secretase by cryo-electron microscopy. A, Surface-rendered side view of the γ-secretase structure. The thick curved line indicates an apparent membrane surface groove that is hypothesized to represent the approximate position at which a transmembrane substrate might bind. Horizontal dashed lines represent the boundaries of the cell membrane. B, Cut-open view of the structure shown in A. The dotted lines indicate the potential water-accessible spaces.

Figure 4

The presenilin homolog signal peptide peptidase (SPP). Signal peptides are removed from membrane proteins by signal peptidase (SP), and the remnant peptides are released from the membrane by SPP-mediated intramembrane proteolysis. SPP, like presenilin, contains two aspartates essential for protease activity, but the conserved aspartate-containing motifs are in the opposite orientation compared with their presenilin counterparts. Consistent with the flipped orientation of SPP vis-à-vis presenilin, the substrates of these two proteases also run in the opposite direction. Unlike presenilin, SPP apparently does not require other protein cofactors or cleavage into two subunits for proteolytic activity.