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

Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 12/2006; 26(46):12081-8. DOI: 10.1523/JNEUROSCI.3614-06.2006
Source: PubMed


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.

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    • "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 (Chávez-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). "
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    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.
    Journal of Cell Science 12/2014; 128(3). DOI:10.1242/jcs.164384 · 5.43 Impact Factor
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    • "Eight kDa N-terminal fragment of PS1 NTF generated by thrombin cleavage of PS1-Th1 after Fen-B crosslinking was specifically precipitated in a similar fashion to that by phenylpiperidine-type photoprobes, suggesting that Fen-B also targets to the most N-terminal region of PS1, including TMD1 (Figure  2B and C). We showed that the cytosolic side of TMD1 participates in the catalytic hydrophilic pore [7,11]. To analyze the relationship between the fenofibrate binding site and the catalytic site within TMD1, we employed the cross-competition analysis in Fen-B labeling using different classes of compounds. "
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    ABSTRACT: Amyloid-beta peptide ending at 42nd residue (Abeta42) is believed as a pathogenic peptide for Alzheimer disease. Although gamma-secretase is a responsible protease to generate Abeta through a processive cleavage, the proteolytic mechanism of gamma-secretase at molecular level is poorly understood. We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the gamma-secretase, as a key modulatory domain for Abeta42 production. Abeta42-lowering and -raising gamma-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Abeta species including Abeta45 that are the precursor of Abeta42. We further found that the helical surface of TMD1 is involved in the binding of Abeta45/48 and that the binding was altered by GSMs as well as TMD1 mutation. Binding between PS1 TMD1 and longer Abeta is critical for Abeta42 production.
    Molecular Neurodegeneration 01/2014; 9(1):7. DOI:10.1186/1750-1326-9-7 · 6.56 Impact Factor
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    • "Electronic microscopic analysis and single particle imaging revealed the existence of intramembrane water-accessible cylindrical chamber in gamma-secretase with a low-density cavity from extracellular side [78,79]. Parallel substituted cysteine accessible method (SCAM) and cross-link experiment confirmed that TM6, TM7 and TM9 of PS formed the intramembrane chamber with two catalytic aspartates residing oppositely on TM6 and TM7, respectively [80-84]. The constitutive autoendoproteolysis of PS removes the inhibitory allosteric effect of the large hydrophobic loop from the catalytic chamber structure in PS [64,65]. "
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    ABSTRACT: Presenilins (PSs) are the catalytic core of gamma-secretase complex. However, the mechanism of FAD-associated PS mutations in AD pathogenesis still remains elusive. Here we review the general biology and mechanism of gamma-secretase and focus on the catalytic components -- presenilins and their biological functions and contributions to the AD pathogenesis. The functions of presenilins are divided into gamma-secretase dependent and gamma-secretase independent ones. The gamma-secretase dependent functions of presenilins are exemplified by the sequential cleavages in the processing of APP and Notch; the gamma-secretase independent functions of presenilins include stabilizing beta-catenin in Wnt signaling pathway, regulating calcium homeostasis and their interaction with synaptic transmission.
    Translational Neurodegeneration 07/2013; 2(1):15. DOI:10.1186/2047-9158-2-15
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