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  • Even with all of the subunits

    2021-11-25

    Even with all of the subunits present, the complex must also be correctly assembled for the enzyme to function properly. The complex is first assembled in the endoplasmic reticulum, with NCT and APH-1 binding together. They form the initial scaffold, so full-length PS can attach itself, and finally PEN2 associates and causes the endoproteolysis of PS into the N-terminal fragment (NTF) and C-terminal fragment (CTF). The active complex is then shuttled to the Golgi where it is glycosylated (Takasugi et al., 2003). Only after the assembly of all four subunits and the glycosylation will GS become active, and even then, not all present complexes are active (Beher et al., 2003, Lai et al., 2003). The disconnect between the presenilin level and the activation of γ-secretase complex remain a large area of research for the GS community. To help address these questions about GS structure, much work has focused on generating high-resolution structures for GS. Many groups have recently published structures using electron microscopy techniques, building on previous work giving a GS structure at a lower resolution (Lazarov et al., 2006). The group by Li et al. published a 17Å model, showing a large Pimavanserin with a smaller head, and NCT’s extracellular domain is in the smaller head (Li et al., 2014). This study was followed up by a 3.4Å resolution structure that showed GS in the same shape as the previous report, with APH-1 and Pen-2 holding PS1 under the NCT extracellular domain, while leaving PS1 flexible (Bai et al., 2015, Lu et al., 2014). Other EM papers have also shown that GS exists in multiple conformations (Bai et al., 2015, Elad et al., 2015). In order to measure the distinct conformations, Bai et al. used a GSI, DAPT, to lock the enzyme in place. The sixth transmembrane helix of PS1 can exist in 3 shapes, potentially providing the range of enzyme activities. The group also found a single helix in the cavity of PS1 that does not belong to any of the subunits, and mass spectrometry identified a mixture of proteins. This mixture could include potential regulatory subunits. The structural information from the EM work will serve as a jumping off point for future rational drug design, as well as highlighting the importance of enzyme regulation.
    Gamma-secretase regulation An enzyme complex with such a large range of substrates and functions requires tight regulation. It is important to keep in mind that only a small fraction of GS complexes are active (Beher et al., 2003, Gu et al., 2004, Lai et al., 2003). All active complexes have all four subunits, and previously it was thought that activity cannot be increased by only overexpressing PS (Levitan et al., 2001)-all subunits must be increased (Edbauer et al., 2003). However, when these studies moved into a mouse model, overexpression of PS alone was able to increase GS activity (Li et al., 2011). This discrepancy between cells and animal models show that GS regulation in vivo is much more complicated than originally anticipated. GS is regulated by layers of control, from subunit composition to associated proteins that may regulate the complex in specific tissues or disease situations. There are multiple GS complexes since PS and APH-1 have 2 isoforms, and APH-1 also has two splice variants, APH-1A and APH-1B (Lai et al., 2003, Shirotani et al., 2004). These variants can exist at the same time in the same tissue, and isoforms sometimes compete for substrates (Placanica et al., 2009a, Placanica et al., 2009b). APH-1A regulates Notch during embryogenesis (Ma et al., 2005, Serneels et al., 2005), and APH-1B contributes to the production of longer Aβ fragments. By targeting APH-1B, researchers can reduce aggregates without Notch-related toxicity (Serneels et al., 2009). More work is required to fully understand how GS activity is regulated by its subunits. GS is also regulated by associated proteins. It can form differential complexes with modulatory proteins. One example is GSAP, which complexes with GS and APP, giving preference to APP cleavage over Notch. GSAP knockdown mice reduce Aβ when crossed with an AD model (Chu et al., 2014, He et al., 2010), and there is a GSAP SNP associated with AD (Zhu et al., 2014). However, the precise mechanism is unknown. Recent work has also shown that GS can be regulated by Hif1α, long known as the master regulator of hypoxia (Villa et al., 2014). Hif1α normally acts as a transcription factor, stabilized in low oxygen conditions, and turning on several genes in response. However, Hif1α binds directly to GS and increases its Notch activity by shifting inactive complexes to their active form, independent of Hif1α’s ability to act as a transcription factor.