Cellular mechanisms of γ-secretase substrate selection, processing and toxicity

Abstract

Presenilins (PSs) are catalytic components of the γ-secretase proteolytic complexes that produce Aβ and cell signaling peptides. γ-Secretase substrates are mostly membrane-bound peptides derived following proteolytic cleavage of the extracellular domain of type I transmembrane proteins. Recent work reveals that γ-secretase substrate processing is regulated by proteins termed γ-secretase substrate recruiting factors (γSSRFs) that bridge substrates to γ-secretase complexes. These factors constitute novel targets for pharmacological control of specific γ-secretase products, such as Aβ and signaling peptides. PS familial Alzheimer's disease (FAD) mutants cause a loss of γ-secretase cleavage function at epsilon sites of substrates thus inhibiting production of cell signaling peptides while promoting accumulation of uncleaved toxic substrates. Importantly, γ-secretase inhibitors may cause toxicity in vivo by similar mechanisms. Here we review novel mechanisms that control γ-secretase substrate selection and cleavage and examine their relevance to AD.

Figure 1. Amyloidogenic and ADAM/γ-secretase signaling processing of APP and other substrates

(A) Extracellular APP is cleaved by β-secretase (BACE) producing soluble N-terminal fragments (sAPPβ) shed to the extracellular space. It is currently believed that the remaining membrane-bound C-terminal fragment (APP-CTFβ) is then cleaved by γ-secretase at several γ sites to produce Aβ peptides with variable c-terminal ends, Aβ 40 and 42 being the most abundant. Cleavage of APP-CTFβ by γ-secretase at the ε site produces AICD released to the cytosol. The temporal relationship of the γ-secretase cleavages however is unclear and available data are also consistent with the suggestion that γ-secretase cleaves only at the ε site of APP-CTFβ releasing AICD (see text). The carboxy terminus of the remaining membrane peptide is then “trimmed” by carboxypeptidases producing Aβ peptides of variable lengths (not shown here). Produced soluble Aβ peptides may then aggregate to form amyloid depositions. (B) Cleavage of APP and other transmembrane proteins by ADAMs near the extracellular face of the plasma membrane, produces a shed ectodomain (N-terminal fragment; NTF) and a membrane bound peptide termed CTF1 (C-Terminal Fragment 1) that is then processed by γ-secretase at the ε site to produce CTF2 peptides (C-terminal Fragment 2) released to the cytosol. CTF2 peptides have been shown to function in cellular signaling (green arrow, see also text). This processing pathway may be stimulated by calcium influx through NMDA receptor and/or by ligand binding to receptors (Fig. 6). Brown arrow indicates an APP-like ε-cleavage that occurs two or three residues inside the membrane while the blue arrow indicates a cadherin-like cleavage that occurs on the membrane/cytoplasm interface (see also text). The red bracket indicates the position of Aβ sequence of APP cleaved in the non-amyloidogenic (α-secretase) processing pathway

Figure 2. Structure of core the γ-secretase proteolytic complex

The core γ-secretase complex is composed of PS (either PS1 or PS2), anterior pharynx-defective 1 (APH-1), nicastrin (NCT) and presenilin enhancer 2 (PEN-2). The latter protein has been reported to stimulate the endoproteolysis of full-length PS zymogen into catalytically active fragments PS-NTF and PS-CTF. The two catalytic aspartates on TMs VI and VII of PS are shown as yellow-red sparks.

Figure 3. γSSRF p120ctn recruits cadherins to γ-secretase by binding PS1-CTF

Catenin protein p120 (p120ctn) known to bind the juxtamembrane region of cadherins (glycine repeat GGG) also binds to the 330–360 amino acid sequence of γ-secretase component PS1-CTF thus recruiting cadherins to γ-secretase complexes for processing (Kouchi et al., 2009). For better clarity, the other core components of γ-secretase are not shown. The cadherin/catenin junction complex binds the actin cytoskeleton via α-catenin. Thus the PS/γ-secretase complex may affect functions of the cellular cytoskeleton (see also Marambaud et al., 2002).

Figure 4. γSSRFs compete for γ-secretase complexes

γSSRFs p120ctn and GSAP bound to cadherins or APP respectively, compete for limited amounts of γ-secretase complexes promoting processing of their respective substrates and production of Cad-CTF2 or Aβ. Thus, overexpression of p120ctn inhibits γ-secretase processing of APP substrates and production of Aβ.

Figure 5. Mechanism of γ-secretase inhibition

Left: γ-secretase catalyzes proteolysis of transmembrane substrates including APP-CTFβ shown here. Processing of this substrate at the active site of the enzyme which includes two aspartate residues shown as yellow-red sparks (one on PS-NTF and the other on PS-CTF) generates AICD when substrate is cleaved at the ε site and Aβ when cleaved at γ sites. Right: γ-secretase is inhibited by GSIs that bind the active site and enhance interactions between γ-secretase subunits thus locking the enzyme in a closed conformation, characteristic of inhibited enzymes (inactivated aspartates are shown in black) (Barthet et al., 2011).

Figure 6. MP/γ-secretase processing of EphB2 receptor can occur at distinct subcellular localizations and is inhibited by FAD mutations

Calcium influx and NMDA receptor agonists promote cell surface (plasma membrane) processing of EphB2 receptor (A) while binding of ephrinB ligands promote a processing pathway that requires endocytosis and takes place in intracellular compartments (B). Both pathways include γ-secretase cleavage at the ε site of substrates producing biologically active CTF2 peptides that contain the cytoplasmic sequence of substrates (Litterst et al., 2007). Importantly, the ε-cleavage of substrates has been shown to be inhibited by PS FAD mutants reducing the levels of biologically active CTF2 products and increasing the amounts of potentially toxic CTF1 precursors. Questionmark indicates the unknown enzyme that cleaves extracellular EphB2 in the endocytic pathway.