Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

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 and the degree of processivity (%) between the APH-1A and APH-1B isoforms in detergent conditions are indicated in grey and pink, respectively. c Size exclusion chromatograms of GSEC1B and GSEC1B D257A -Aβ46 after reconstitution into MSP1D1 lipid nanodiscs. Grey area shows peak fraction used for cryo-EM. d Coomassie stained SDS-PAGE of purified GSEC1B (WT and D257A mutant) solubilised in CHAPSO and reconstituted into lipid nanodiscs. Aβ46 was added to the purified GSEC1B D257A prior to reconstitution. e ELISA-based quantification of de novo Aβ products generated from Aβ46 by GSEC1A or GSEC1B reconstituted into lipid nanodiscs. Aβ profiles show the percentage of Aβ products, relative to total measured Aβ (37, 38, 40, 42 and 43) peptides. Data are presented as mean ± SD, n = 3 for GSEC1A and n = 8 for GSEC1B. The amounts of Aβ products measured are shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

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

Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

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: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

Figure Lengend Snippet: a Structural alignment of GSEC1B-Aβ46 and GSEC1A-APP C83 (PDB: 6IYC; shown in grey) complexes. PSEN1 TMs are indicated with circled numbers. b Closeup of extracellular side of the substrate and loop 1. The GSEC1A-APP C83 complex was stabilised by disulphide cross-link between V7C APP C83 (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. NCT m and NCT i indicate mature glycosylated and immature NCT, respectively. Molecular weights of protein standards are indicated on the left. f GSEC processivity of APP C99 in Psen1 −/− Psen2 −/− MEFs rescued with WT or mutated PSEN1. Data are presented as mean ± SD, n = 6 for the WT and n = 3 for the mutants. Multiple comparison ANOVA was used to determine statistical significance ( P < 0.05); P(WT vs Y115A) < 0.0001, P(WT vs Y115F) < 0.0001, P(WT vs W165F) < 0.0001, P(WT vs S169A) = 0.0001, P (Y115A vs Y115F) = 0.0115. Source data are provided as a Source Data file.

Article Snippet: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

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

Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

Figure Lengend Snippet: a Structural alignment of apo (grey) and Aβ46-bound (colour-coded as in Fig. ) GSEC1B. PSEN1 and APH-1 TMs are indicated with circled numbers. b Structural rearrangement in PSEN1 and APH-1 subunits upon Aβ46 binding. PSEN1 from the apo structure is depicted in light grey, and APH-1 is depicted in dark grey.

Article Snippet: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

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 APP C99 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: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

Techniques: Membrane, Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform

doi: 10.1038/s41467-024-48776-2

Figure Lengend Snippet: Cryo-EM data collection, refinement, and validation statistics

Article Snippet: Assays were carried out with 0.23 μM GSEC1A/GSEC1B/GSEC1B D257A and 2.5 μM Aβ46 for 24 h. Reactions were quenched by placing the assay tubes on ice and adding 10 μM inhibitor L-685,458 (Santa Cruz Biotechnology).

Techniques: Biomarker Discovery