Structure of the Catalytic Pore of -Secretase Probed by the Accessibility of Substituted Cysteines

ArticleinThe Journal of Neuroscience : The Official Journal of the Society for Neuroscience 26(46):12081-8 · December 2006with3 Reads
DOI: 10.1523/JNEUROSCI.3614-06.2006 · Source: PubMed
Abstract
Several single-span membrane proteins are cleaved within their transmembrane domains (TMDs) by intramembrane-cleaving proteases, although the structure of the active site executing intramembrane cleavage remains unknown. Here we use the substituted cysteine accessibility method to examine the structure of presenilin-1, a catalytic subunit of gamma-secretase, involved in amyloid beta protein generation in Alzheimer's disease and Notch signaling. We show that TMD6 and TMD7 of presenilin-1 contribute to the formation of a hydrophilic pore within the membrane. Residues at the luminal portion of TMD6 are predicted to form a subsite for substrate or inhibitor binding on the alpha-helix facing a hydrophilic milieu, whereas those around the GxGD catalytic motif within TMD7 are highly water accessible, suggesting formation of a hydrophilic structure within the pore. Collectively, our data suggest that the active site of gamma-secretase resides in a catalytic pore filled with water within the lipid bilayer and is tapered around the catalytic aspartates.
    • "Aβ49, both of which are thought to be processed to produce the Aβ peptides with various C-terminal lengths in a successive manner (Aβ48-Aβ45-Aβ42-Aβ38 or Aβ49-Aβ46-Aβ43- Aβ40, respectively). For γ-secretase to achieve this atypical endoproteolysis, catalytic aspartates located within transmembrane domains (TMDs) 6 and 7 of PS (Wolfe et al., 1999) act to form a hydrophilic environment called the catalytic pore/cavity within the lipid bilayer (Bai et al., 2015b; Sato et al., 2006; Tomita and Iwatsubo, 2013). This type of structure is common among the intramembrane proteases (Sun et al., 2016). "
    [Show abstract] [Hide abstract] ABSTRACT: γ-Secretase is an intramembrane-cleaving protease that generates various forms of amyloid-β peptides (Aβ) that accumulate in the brains of Alzheimer disease (AD) patients. The intracellular trafficking and subcellular localization of γ-secretase are linked to both qualitative and quantitative changes in Aβ production. However, the precise intracellular localization of γ-secretase as well as its detailed regulatory mechanisms have remained elusive. Recent genetic studies on AD provide ample evidence that alteration of the subcellular localization of γ-secretase contributes to the pathogenesis of AD. Here we review our current understanding of the intracellular membrane trafficking of γ-secretase, the association between its localization and proteolytic activity, and the possibility of γ-secretase as a therapeutic target against AD.
    Article · Jul 2016
    • "The preferred orientation and the difference in insertion efficiency could be due to the charged residues flanking the TMD and the length and the hydrophobicity of the PS1 segment [28,32] . Importantly, the partial integration of TMD7 is consistent with results reporting that only half the TMD7 is integrated as an a-helix [49,50] . Moreover, as a control , glycosylation of TMD7 was confirmed by Endo H-treatment (Fig. 2D).Table 1 Predicted vs. measured DG values for each of the hydrophobic domains in PS1 wild type. "
    [Show abstract] [Hide abstract] ABSTRACT: The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37–43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.
    Full-text · Article · Dec 2014
    • "However, other substrates, including Notch, N-cadherin and ErbB4, link csecretase activities to development, cancer and immunity (De Strooper and Annaert, 2010; Selkoe and Wolfe, 2007). Presenilin 1 (PS1), nicastrin (NCT), presenilin enhancer 2 (PEN-2) and anterior pharynx defective 1A (APH-1A) assemble in a tetrameric complex (Fig. 1A) (De Strooper, 2003) and PS1 autoproteolysis results in an active pentameric c-secretase (Thinakaran et al., 1996), in which the catalytic center is structured at the interface between the N-terminal and the Cterminal fragments of PS1 (NTF and CTF respectively) (Esler et al., 2000; Li et al., 2013; Li et al., 2000; Wolfe et al., 1999) and connected to the intracellular aqueous environment (Sato et al., 2006; Tolia et al., 2006). NCT, a type 1 integral membrane glycoprotein, plays a crucial role in complex maturation and stabilization (Gutiérrez et al., 2008) and might be involved in substrate binding (Shah et al., 2005; Zhang et al., 2012), although this is debated (Chávez-Gutiérrez et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: The structure and function of the γ-secretase proteases are of vast interest because of their critical roles in cellular and disease processes. We established a novel purification protocol for γ-secretase complex that involves a conformation and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase structure and its conformational variability. Under steady state conditions the complex adopts three major conformations, which are different in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors differentially stabilize sub-populations of particles with compact conformations, whereas a Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic population of inter-converting conformations, involving rearrangements at the nanometer scale and high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect Aβ generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely instrumental for highly allosteric behavior of the enzyme.
    Full-text · Article · Dec 2014
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