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  • Human GS is a multi subunit complex consisting of four

    2021-09-02

    Human GS is a multi-subunit complex consisting of four components: presenilin 1 (PS1), presenilin enhancer 2 (PEN-2), anterior pharynx-defective 1A (APH-1A) and nicastrin (NCT) (Fig. 1a) [5,6]. PS1 comprise nine transmembrane helices (TM 1–9) and contains the catalytic aspartate residues in its TM6 (Asp257) and TM7 (Asp385). This subunit consists in a N-terminal (NTF, TM1-6) and C-terminal (CTF, TM7-9) fragments obtained as a result of the autoproteolytic process that occurs during GS maturation [7]. These fragments interact with PEN-2 (3 TMs) and APH-1A (7 TMs) subunits, which have important roles in the maturation and assembly of the GS complex [8]. Finally, NCT is a type-1 transmembrane glycoprotein formed by a single TM in its C-terminal region and a large N-terminal extracellular domain (ECD). It has been proposed that the NCT ECD has a key role in preventing the entry of bulky substrates into the active site [9]. Currently, six cryo-electron microscopy (cryo-EM) structures of human GS have been deposited in the Protein Data Bank (PDB): 4UI2, 5A63, 5FN2, 5FN3, 5FN4 and 5FN5 [[10], [11], [12], [13]]. The fragment comprising the large PS1 region between TM6 and TM7 was not assigned in any of these structures. And PS TM2 could only be resolved in 4UI2, 5FN2 and 5FN3. Moreover, 5FN3 and 5FN4 were resolved with two unidentified external Baricitinib sale at the active site, while the density map of DAPT inhibitor was reported at the same site for the 5FN2 structure. Structural and conformational differences of these structures were reviewed elsewhere [14]. Experimental studies of GS have provided valuable information related to its structure and biological functions [15,16]; however, details of its mechanisms of activation, substrate recruitment and cleavage remain unclear. Employing the available GS structures and experimental data, different research groups have focused on studying key dynamic features of the PS1 component and the whole GS complex, such as its structural ensemble, activation mechanism, and the substrate recognition and internalization mechanisms. Current GS molecular dynamics (MD) simulation studies employ multiscale modeling approaches at atomistic and coarse-grained (CG) resolutions (Fig. 1b) to address these features. Atomistic simulations are useful to obtain a full detail of intra- and intermolecular interactions and explore subtle conformational changes on the nanosecond timescale. However, the spatiotemporal and computational limitations of atomistic simulations bound conformational movements that occur over longer periods of time [17,18]. To overcome this concern, some authors performed CG replica simulations of GS at microsecond scales to characterize the wide and low frequency movements of NCT ECD and address the substrate internalization process.
    Towards C99-APP internalization by PS1 A key step in the APP cleavage by GS is the substrate internalization mechanism into the active site. Several experimental studies have been performed to understand this process [19]. To date, two possible substrate entry sites have been proposed: (1) between TM6 and TM9 (ES1), or (2) between TM2 and TM6 (ES2) (see Fig. 1a). In the first, Sato and coworkers [20] employed the substituted-cysteine accessibility method (SCAM) and GS inhibitor competition assays to identify the PS1 regions involved in this mechanism. Their results showed that the substrate was transferred from the CT region of the PAL motif in TM9 and TM6 to the region comprising the TM6 and TM7 interface and TM9 NT end. Their second hypothesis proposes that the TMs flexibility observed in the cryo-EM structures should be related to the substrate entry, suggesting that TM2 could serve as a gateway to the active site [21]. The first computational study that attempt to identify the substrate entry site was performed by Kong et al. [22] employing an isolated PS1 component. Since this study was performed before the publication of the GS structure, the authors constructed a PS1 homology model using PDB IDs: 4HYG [23,24] (Methanoculleus marisnigri) and 2KR6 [25] (CTF of human PS1) structures as templates. Their model was employed to study the dynamic properties of the isolated human PS1 subunit in POPC and POPC:POPE (25:75) bilayer systems through 100 ns of atomistic MD simulations from a 100 ns pre-equilibrated GS-POPC system using NAMD 2.9 [26] with the CHARMM36 force field [27]. It is noteworthy that their root-mean square deviation (RMSD) did not converge, suggesting that a longer simulation time was required to reach equilibrium of their PS1 model. With these simulations, the authors examined the PS1 stability, the conformational distribution, and the possible “gate-open” movement for substrate entry. For the first, they identified that the PALP motif in TM9 has a critical role inducing PS1 conformational rearrangements to stabilize its open state during substrate binding and internalization. Moreover, their MD simulations showed a “gate-open” movement between TM2 and TM6 as a result of rearrangements induced by the PAL motif. Kong et al. also measured the distance between catalytic aspartic residues to evaluate the effect of their bilayer lipid composition in the PS1 conformational states. The authors identified an inactive state in their POPC bilayer system and a smaller distance between the catalytic aspartic residues in the POPC:POPE bilayer due to interactions between a POPE lipid headgroup and the carboxylic groups of the aspartic residues. They conclude that this interaction could inhibit the substrate binding, in agreement with results obtained by Osenkowski et al. [28].