4.G.1. The γ-Secretase (γ-Secretase) Family
γ-secretase is an unusual membrane-embedded protease, which cleaves the transmembrane domains (TMSs) of type I membrane proteins, including amyloid-beta precursor protein and Notch receptor as well as about 80 other proteins. A hydrophilic pore is formed by TMS6 and TMS7 of presenilin 1 (PS1), the catalytic subunit of γ-secretase. TMS8, TMS9 and the C-terminus of PS1, which encompass the conserved PAL motif and the hydrophobic C-terminal tip, are critical for the catalytic activity and formation of the γ-secretase complex. The amino acid residues around the PAL motif and the extracellular/luminal portion of TMS9 are highly water accessible and located in proximity to the catalytic pore (Sato et al., 2008). Furthermore, the region starting from the luminal end of TMS9 toward the C terminus forms an amphipathic alpha-helix-like structure that extends along the interface between the membrane and the extracellular milieu. Competition analysis using γ-secretase inhibitors revealed that TMS9 is involved in the initial binding of substrates. TMS9 for the catalytic pore allowing substrate entry, crucial for intramembrane proteolysis by γ-secretase, Aph-1C which shows sequence similarity with most of putative ABC membrane protein of Thermobifida fusca (Q47P80). This is the NCBI Anterior Pharynx defective (Aph-1) family.
γ-Secretase consists of Presenilin (PS) and three indispensable subunits: Nicastrin, Aph-1 and Pen-2. PS forms a hydrophilic catalytic pore structure within the lipid bilayer. Takeo et al. (2012) showed that the hydrophilic pore with an open conformation is formed by PS within an immature γ-secretase complex. The binding of the subunits induces close proximity between transmembrane domains facing the catalytic pore. Both γ- and β-secretases have been reported to have affinity for inclusion in membrane nanodomains (Sanders and Hutchison 2018). The effect of bilayer lipid composition on the GS structural ensemble and its function have also been studied (Aguayo-Ortiz et al. 2018).
Aberrant cleavage of Notch by γ-secretase leads to several types of cancer. The cryo-electron microscopy structure of human γ-secretase in complex with a Notch fragment at a resolution of 2.7 Å has been determined (Yang et al. 2019). The TMS of Notch is surrounded by three TMSs of PS1, and the carboxyl-terminal β-strand of the Notch fragment forms a β-sheet with two substrate-induced β-strands of PS1 on the intracellular side. Formation of the hybrid β-sheet is essential for substrate cleavage, which occurs at the carboxy-terminal end of the Notch TMSx. PS1 undergoes pronounced conformational rearrangement upon substrate binding (Yang et al. 2019). Moreover, cleavage of amyloid precursor protein (APP) by γ-secretase is linked to Alzheimer's disease (AD). Zhou et al. 2019 reported an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6 Å resolution. The TMS of APP closely interacts with five surrounding TMSs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch (see above in this paragraph, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors (Zhou et al. 2019).
Gamma-secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2 with at least 18 TMSs (Lazarov et al. 2006). EM and single-particle image analyses have been applied to the purified enzyme, which produces physiological ratios of Abeta40 and Abeta42. The 3D EM structure revealed a large, cylindrical interior chamber, approximately 20-40 Å in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, approximately 120-A-long complex within the membrane and revealed approximately 20-Å pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. The reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments (Lazarov et al. 2006).