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    • Although domain organization had been clearly delineated by

    2021-10-19

    Although domain organization had been clearly delineated by the 4.3-Å structure of T4-γ-secretase [], atomic modeling of the side chains had to wait for the 3.4-Å structure []. In total, 598 residues in the transmembrane region and 632 residues in the ECD were modeled for the four components of human γ-secretase (Figure 3a). Despite the high resolution, the EM density for TM2 of PS1 remains obscure and its atomic model was generated on the basis of its sequence homology to PSH. The atomic structure of γ-secretase, the first of its kind, reveals a wealth of information. Proteolytic activity of an aspartate protease requires proper alignment of the two Asp residues, which are hydrogen bonded to each other during catalysis [44]. However, in PS1, the distance between the Cα atoms of Asp257 and Asp385 is approximately 10.6Å, considerably longer than that in an activated aspartate protease. This observation suggests that γ-secretase exists in an inactive conformation that may be activated by substrate binding or other forms of regulation. Consistent with a role in substrate recognition [45, 46, 47], the PAL motif in TM9 is located close to the catalytic Asp residues (Figure 3b). The carboxyl-terminal residues Phe465–Tyr466–Ile467 of PS1 are accommodated by a hydrophobic cavity in APH-1 (Figure 3c). This observation illustrates a critical role of APH-1 in stabilizing pramiracetam mg γ-secretase assembly and explains why protein engineering involving the carboxyl-terminus of PS1 leads to unsatisfactory solution behavior of γ-secretase. Two tightly bound phospholipids are found in γ-secretase, one at the interface between PS1 and APH-1, the other at the interface between APH-1 and nicastrin (Figure 3d,e). The lipids may stabilize the interface between subunits in γ-secretase. Because no exogenous lipids were supplemented during protein purification, these lipids may associate with γ-secretase in pramiracetam mg and are likely required for the assembly, stability, and function of γ-secretase []. Notably, the proteolytic activity of γ-secretase in vitro strictly depends on the presence of phospholipids. It is possible that these exogenous lipids stabilize the γ-secretase assembly by reinforcing the bound phospholipids in case of dissociation. A specific compound, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (known as DAPT), inhibits the proteolytic activity of γ-secretase with an inhibitory constant (IC50) of approximately 115nM in human primary neuronal cells [48]. DAPT binding appears to induce a marked conformational change in PS1, as revealed by the 4.2-Å structure of γ-secretase soaked in DAPT. A lobe of EM density in the active site is attributed to DAPT. TM6 of PS1 displays a kink at Pro264, which is located underneath the catalytic cavity on the cytoplasmic side (Figure 3f). TM6 rotates towards TM7, thus bringing together the two catalytic aspartates (Figure 3f,g) []. Comparison between the inhibitor-bound and -free states reveals insights into the activation mechanism of PS1.
    Functional implications In the cryo-EM studies, the assembled γ-secretase exists as a stable 1:1:1:1 complex among its four components, consistent with the report that purified γ-secretase is a monomeric complex [49]. Importantly, however, purification of γ-secretase requires rigorous manipulation in the presence of detergents, which are known to weaken or even disrupt protein-protein interactions [50]. Therefore, these structural and biochemical characterizations cannot rule out homo-oligomerization of γ-secretase in cells or under physiological conditions, which had been previously reported [51]. Physical interactions between wild-type and pathogenic mutant PS1 proteins form the basis for the proposed dominant negative effect of γ-secretase [52]. PS1, PS2, and APP are targeted by AD-associated mutations, of which greater than 80 percent affect specific amino acids in PS1. Remarkably, the majority of those residues that are mutated to two or more types of amino acids are mapped to two analogous hotspots in PS1, in the axial center of either structural repeat (Figure 3h) []. These residues are supposed to be more reliable in predicting functional disruption. Becuase of their close proximity to the catalytic aspartates, the affected residues in the axial center of TMs 6–9 may potentially affect the proteolytic activity. In contrast, the affected residues in TMs 2–5, far away from the active site, are left with no obvious explanation. Their predominant center-facing locations in these two repeats strongly suggest an activity that is related to substrate transport. AD patients are characterized by deficiency in learning and memory, which is thought to be linked to disrupted calcium leak across membrane as a result of mutations in PS1 [53]. It is unclear whether the observed calcium flow could be mediated by the axial path in any of the two structural repeats.