Complex regulation of γ-secretase: from obligatory to modulatory subunits

Abstract

γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aβ) peptides that form amyloid plaques, which contribute to Alzheimer's disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer.

Keywords: APP; Alzheimer’s disease; Hif-1α; Notch; presenilin; β-amyloid; γ-Secretase.

Figure 1

Proteolytic processing of APP and Notch. Mature Notch receptors are activated by binding to ligands (Jagged-1, -2 and Delta-like -1, -3, and -4) located on adjacent signal-presenting cells. An induced conformational change exposes a cleavage site (S2) for ADAM family metalloproteases that cleave Notch at an extracellular, membrane-proximal region. The membrane-bound Notch segment that results from this cleavage, known as Notch Intracellular Truncation domain (NEXT), is a γ-secretase substrate (Kopan and Ilagan, 2009). γ-Secretase performs the subsequent cleavage at S3 (De Strooper et al., 1999), releasing Notch intracellular domain (NICD) from the membrane and allowing for signal transduction through binding with the CBL-1, Su(H), Lag-1 (CSL; Schroeter et al., ; Struhl and Adachi, 1998) family of DNA binding proteins. APP undergoes sequential proteolytic processing first by β-secretase (BACE1, aspartyl protease) and then by γ-secretase, in the amyloidogenic pathway. The first cleavage results in ectodomain shedding in which the amino-terminal of APP is removed, yielding a soluble APP derivative (sAPPβ) and a carboxy-terminal membrane stub known as βCTF (C99). βCTF is a substrate for γ-secretase, and is cleaved in its transmembrane domain to form AICD and the potentially toxic Aβ. Mutations in presenilin (the catalytic subunit of γ-secretase) and APP can lead to increases in the Aβ42 to Aβ40 ratio, resulting in Aβ deposition and plaque formation.

Figure 2

γ-Secretase complex formation and regulatory roles of individual essential subunits. The γ-Secretase complex is formed by the sequential assembly of Aph1, nicastrin, presenilin, and Pen-2. First, Aph-1 and nicastrin come together to form the scaffold. Next, full length presenilin is incorporated. Last, Pen-2 is recruited and full length presenilin is endoproteolysed into presenilin-NTF/CTF, activating the enzyme. Nicastrin, a heavily glycosylated single-pass transmembrane protein, plays a role in scaffolding, enzyme stabilization, substrate recognition, and trafficking. Nicastrin’s amino acids 312–340 are important for substrate recognition and deletion of these residues reduces γ-secretase activity and nicastrin’s interaction with APP and Notch. Furthermore, mutation of nicastrin’s C213 and C230 leads to different impact on processing of APP and Notch, underscoring nicastrin’s role in substrate selectivity. Aph-1, a 7-pass transmembrane protein with 3 human isoforms, is crucial for scaffolding and stability, and may have an additional role in determining length of Aβ species produced depending on which isoform is incorporated into the γ-secretase complex. The GXXXG motif in Aph-1 is critical for γ-secretase complex assembly. Presenilin, a 9-pass transmembrane protein with 2 isoforms, is the catalytic subunit of γ-secretase, and full length presenilin is a zymogen that must be endoproteolysed into NTF/CTF to be enzymatically active. Mutations in presenilin1 and presenilin2-encoding genes account for the majority of genetic mutations leading to Familial Alzheimer’s disease. Pen-2, a 2-pass transmembrane protein, is required for presenilin endoproteolysis and γ-secretase activation, but also may play an endoproteolysis-independent role in γ-secretase regulation. Active γ-secretase constitutes a small percentage of total γ-secretase and resides primarily in the plasma membrane.

Figure 3

Modulatory-protein-based regulation of γ-secretase. Hif-1α, a master regulator of cellular response to hypoxia, is a γ-secretase interacting partner. In the presence of sufficient oxygen, no Hif-1α is produced. However, in oxygen-deficient conditions, Hif-1α is upregulated. Hif-1α binding to γ-secretase enhances γ-secretase activity by increasing the ratio of active:inactive γ-secretase complexes in the cell, thereby increasing γ-secretase cleavage of its substrates, such as APP and Notch. The implication is twofold: first, inactive γ-secretase complexes are physiologically important, and second, γ-secretase can be temporally regulated.

Figure 4

Substrate-based regulation of γ-Secretase. α or β-secretase cleave amyloid precursor protein to generate αCTF or βCTF, respectively. αCTF and βCTF, which are γ-secretase substrates, can then interact with γ-secretase to form a substrate-enzyme complex. The substrate inhibitory domain (ASID) in αCTF and βCTF interacts with γ-secretase in a location distinct from both the active site and the substrate binding site of the enzyme. Binding of αCTF to γ-secretase results in a low-productivity complex: the ASID in αCTF inhibits γ-secretase activity for Aβ production. ASID in βCTF is a less potent inhibitor of γ-secretase. The implication is that α-secretase cleavage potentiates γ-secretase inhibition by at least two mechanisms: first, α-secretase cleaves APP in the middle of the Aβ region, precluding Aβ formation, and second, α-secretase cleavage results in formation of αCTF, which inhibits γ-secretase activity through its ASID. γ-Secretase cleavage of αCTF produces p3 and cleavage of βCTF produces the amyloidogenic Aβ.