Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a , Schematic representation of GSEC subunits. The catalytic aspartates are indicated, and their respective positions are marked with red stars. b , Sequential processing of APP C99 by GSEC. The difference in processivity between the APH-1 isoforms is indicated. c , Size exclusion chromatograms of GSEC1B and GSEC1B D 257 A -Aβ46 after reconstitution into MSP1D1 lipid nanodiscs. Grey area shows peak fraction used for cryo-EM. d , Coomassie stained SDS-PAGE of purified GSECs (wild-type and D257A mutant) solubilised in CHAPSO and reconstituted into lipid nanodiscs. Aβ46 was added to the purified GSEC D 257 A prior to reconstitution. e , ELISA-based quantification of Aβ40 generated from Aβ46 by GSEC1B solubilised in CHAPSO or reconstituted into lipid nanodiscs. Averages of 4 independent measurements and standard deviation are shown.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Cryo-EM Sample Prep, Staining, SDS Page, Purification, Mutagenesis, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: Schematic representation of cryo-EM data processing procedure used for obtaining reconstruction of GSCE1B-Aβ46.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a , Cryo-EM map of apo GSEC1B coloured by subunit. Resolved glycans in NCT subunit and density corresponding to ordered lipids are coloured in orange. The density corresponding to lipid nanodisc extends ∼ 2 nm around the edge of GSEC and is ∼ 4 nm thick, with the thickest part found next to PEN-2, and the thinnest part close to APH-1B. b , Atomic model of apo GSEC1B. c , Cryo-EM density map of GSEC1B-Aβ46 complex, Aβ46 shown in purple. Regions of PSEN1 resolved in the complex with Aβ46 but not in apo state are shown in dark blue. Density of Aβ46 N-terminus proximal to Glu650 NCT and of the density of Aβ46 TM domain are shown in the inset. The maps shown in panels a and c were filtered using Gaussian filter for better visualisation. d , Atomic model of the GSEC1B-Aβ46 complex.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a, Representative motion-corrected micrograph from the GSEC1B-Aβ46 dataset. b, Example 2D class averages calculated in RELION 3.1. c, Fourier shell correlation curves for masked half-maps and between model and map. The resolution cut-offs at FSC of 0.143 and 0.5 are indicated for half-map and model-map FSCs. d, EM density map coloured by local resolution. e, Distribution of particle orientations. f , Examples of EM density maps (shown as mesh surfaces) around atomic models (shown as cartoons and sticks).

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques:

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a , Structural alignment of apo and Aβ46-bound GSEC1B. b, Structural rearrangement in PSEN1 and APH-1 subunits upon Aβ46 binding.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a , Structural alignment of GSEC1B-Aβ46 and GSEC1A-APPC83 (PDB: 6IYC; shown in grey) complexes. b , Closeup of extracellular side of the substrate and loop 1. The GSEC1A-APPC83 complex was stabilised by disulphide crosslink between V7C APPC83 (unresolved) and Q112C PSEN1. c , Closeup view on intracellular side of substrate binding site. d , Details of PSEN1-Aβ46 interactions in the trans-membrane region. Potential hydrogen bond interactions between the substrates and W165, S169 and G384 are indicated. e , Western blot analysis of solubilised membranes from Psen1 -/- /Psen2 -/- (dKO) mouse embryonic fibroblast cell lines rescued with WT or mutant PSEN1. NCTm and NCTi indicate mature glycosylated and immature NCT, respectively. Molecular weights of protein standards are indicated on the left. f , GSEC processivity of APPC99 in Psen1 -/- Psen2 -/- MEFs rescued with WT or mutated PSEN1. Data are presented as mean ± SD, N ≥ 3. Multiple comparison ANOVA was used to determine statistical significance (P < 0.05); ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Binding Assay, Membrane, Western Blot, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

doi: 10.1101/2023.09.09.556900

Figure Lengend Snippet: a , Surface representation of GSEC coloured by electrostatic potential and Aβ46 shown as cartoon. Fenestration in the intracellular membrane leaflet region of PSEN1 partially exposes Aβ46 to the membrane environment. b , Structure of substrate-binding channel of PSEN1 is identical with three different substrates and the conformations of the three substrates are very similar. c , Sequence alignment of Notch and the APPC99 downstream products along the Aβ49 pathway. The initial endopeptidase cleavage site is indicated with the arrow and different colours indicate the tripeptides sequentially cleaved in the Aβ40 product line. d , A model of sequential catalysis. The structures of substrates in positions corresponding to the cuts producing known Aβ peptides are shown. Panels 1 and 3 counted from the left side are experimental structures, the remaining panels are models.

Article Snippet: To form GSEC1B-Aβ46 complex, 5 μM Aβ46 (rPeptide) resuspended in dimethyl sulfoxide (DMSO) was added to purified GSEC1B D 257 A (1.25 × fold excess), followed by a 1 h incubation at 37°C.

Techniques: Membrane, Binding Assay, Sequencing