Cryo-EM structures of human γ-secretase
Introduction
A hallmark of AD is the accumulation of β-amyloid plaque in patient brain [1, 2]. APP is first cleaved by β-secretase, generating a 99-residue transmembrane fragment known as C99; C99 is then trimmed successively by γ-secretase to produce a number of β-amyloid peptides (Aβ). Longer forms of Aβ such as Aβ42 and Aβ43 are prone to aggregation [3, 4, 5]. The four-component intramembrane γ-secretase, comprising presenilin 1/2 (PS1/2), presenilin enhancer protein 2 (PEN-2), anterior pharynx defective protein 1 (APH-1a/b), and nicastrin, is thought to play a central role in the development of AD by producing Aβ [6, 7, 8].
Genetic screening of AD patients led to the identification of presenilin [9]. An autocatalytic cleavage of the cytoplasmic loop between transmembrane segment (TM) 6 and TM7 of presenilin is required for γ-secretase maturation, generating an amino-terminal fragment (NTF) comprising TMs 1–6, and a carboxyl-terminal fragment (CTF) comprising TMs 7–9 [10]. Two transmembrane aspartate residues are essential for presenilin maturation and substrate cleavage [8, 11, 12, 13]. PEN-2 facilitates the autocatalytic cleavage of presenilin [14]. APH-1 may serve as a scaffold to stabilize γ-secretase [15, 16]. Nicastrin, with a single TM and a large extracellular domain (ECD), is thought to be responsible for substrate recruitment [17, 18].
For much of the past two decades, structural characterization of γ-secretase was lagging behind rapid advances in functional understanding. Until the beginning of 2014, there were only several low-resolution EM structures [19, 20, 21, 22, 23], a nuclear magnetic resonance (NMR) structure of PS1 CTF [24], and a crystal structure of a presenilin archaeal homologue (PSH) [25•]. Breakthroughs in recombinant γ-secretase expression [26••], together with the application of direct electron detector and improvement of image analysis software [27, 28, 29], allowed successful determination of four near-atomic resolution structures: γ-secretase at 4.5 Å [26••], γ-secretase fused with T4 lysozyme at 4.32 Å [30••], and γ-secretase at 3.4 Å [31••] and soaked with a specific inhibitor DAPT at 4.2 Å [32••].
Section snippets
Expression and purification of human γ-secretase
The acquisition of a homogenous membrane protein is always a major bottleneck for structure determination. This problem had been particularly worse for γ-secretase than for most other mammalian membrane proteins, because γ-secretase contains four components and as a protease poses a threat to the host cells upon overexpression. Earlier effort to obtain decent amounts of recombinant γ-secretase relied on stable transfection of CHO and HEK293S cells. Three CHO cell lines, named γ-30, S1 and S20,
Electron microscopy
Procedures of electron microscopy reviewed here only pertain to the four structures of γ-secretase at near-atomic resolutions [26••, 30••, 31••, 32••]. Getting recombinant human γ-secretase into vitreous ice proved to be tricky. Among many detergents tested, only digitonin and amphipol work well. Preparation of the cryo-EM specimens using an FEI Vitrobot follows a standard protocol, with an aliquot of 3-μl concentrated γ-secretase on a glow-discharged holey carbon grid blotted for 4 s and flash
Structures of human γ-secretase
Before the near-atomic resolution structures, the highest resolutions achieved for γ-secretase were 15 and 12 Å [19, 21]. The 15-Å structure revealed a hydrophilic chamber in the transmembrane domain and two pores at the top and bottom of γ-secretase, which were speculated to provide exit for the cleavage product [19]. The subsequent 12-Å structure contained several extracellular domains, a potential substrate binding groove and three solvent accessible cavities [21]. Another study on
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
Perspective
The structures at near-atomic resolutions differ markedly from the other reported EM structures of γ-secretase that were determined at lower resolutions. At resolutions lower than 10 Å, separation of the detergent micelles away from the transmembrane domain of γ-secretase becomes quite challenging. Consequently, the structures determined in these resolution ranges usually contain information of the detergent micelles. In our case, 3D classifications at resolutions lower than 10 Å always yield a
Conflicts of interest
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We apologize to those colleagues whose important contributions are not cited in this article due to space limitation. This work was supported by funds from the Ministry of Science and Technology and the National Natural Science Foundation of China.
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