Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: (A) Schematic illustration of APP C99 cleavage by PSH. PSH cleaves C99 and releases an AICD fragment and Aβ peptides. The epitope of the PSH specific antibody 6F4 in the loop between TMD6 and TMD7 is indicated. (B) Analysis of PSH activity in DDM micelles after incubation with C100-His 6 substrate overnight at 37 °C by immunoblotting for AICD (Y188) and Aβ (2D8). Specificity of substrate cleavage by PSH in the assay was controlled by sampl incubation at 4 °C or 37 °C in presence of the GSI L-685,458 (20 µM). Immunoblotting of PSH (6F4) was performed to control for PSH levels. (C) Aliquot of samples from (B) separated by Tris-Bicine urea SDS-PAGE for identifying Aβ species produced by PSH in DDM micelles and analysis by immunoblotting (2D8). In (B) and (C), representative immunoblots from 3 to 6 independent biological replicates (i.e. independent protease preparations) are shown. (D) Representative MALDI-TOF MS spectrum of Aβ profile generated by PSH in DDM micelles from 4 independent biological replicates. The intensity of the highest peak was set to 100%. A GSI control is shown in Figure 1 – figure supplement 1 and observed masses for identified Aβ species are shown in Figure 1 – source data 1.

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques: Activity Assay, Incubation, Western Blot, SDS Page, Produced, Generated

Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: MS specificity control for Aβ cleavage products generated from APP C99 by PSH. Representative MALDI-TOF MS spectrum of PSH cleavage assay in DDM micelles in the presence of L-685,458 from 4 independent biological replicates. The intensity of the highest peak was set to 100%.

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques: Generated, Cleavage Assay

Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: (A, B) MALDI-TOF MS analysis of Aβ (A) and AICD (B) species from PSH cleavage assays in DDM micelles and POPC vesicles in the presence of L-685-458 at pH 7.0. Representative mass spectra from 4 independent biological replicates are shown. The intensity of the highest peak was set to 100%.

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques:

Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: (A, B) Interactions between TMD6a of PS1 and substrate residues near the scissile bond(s) in the C83-bound (A) and Notch1 (N100)-bound (B) γ-secretase cryo-EM structures (PDB 6IYC and PDB 6IDF, respectively). The van der Waals radii o TMD6a and directly surrounding residues (R268 to R278) of PS1 are shown by the gray surface. (C, D) Interactions between TMD6a of PS1 and GSI in the Semagacestat-bound (C) and L-685,458 bound (D) γ-secretase cryo-EM structures (PDB 6LR4 and PDB 7C9I, respectively). The van der Waals radii of TMD6a and directly surrounding residues (R268 to R278) of PS1 are shown by the gray surface.

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: (A) Histograms of the Cγ-Cγ distances between the D162 and D220 of PSH measured in DDM micelle (red) and POPC bilayer (blue) environments. The dashed line indicates the distance of 7 Å. The measured distances over time are shown in Figure 8 – figure supplement 1C. (B) Snapshot of the catalytic cavity in DDM (left panel) and POPC (right panel) environment. The Cγ-Cγ distance between the two catalytic aspartates D162 and D220 is larger in DDM micelles and more water molecules enter the catalytic cavity between D162 and the substrate. Detailed geometries of these two active site conformations are depicted in Figure 8 – figure supplement figure 2. (C) Immunoblot analysis of TSA-inhibitor binding to PSH in DDM micelles or POPC vesicles. PSH was affinity-precipitated by Merck C (a biotinylated derivative of L- 685,458; 20 µM). To control for background binding and binding specificity, the affinity precipitation was assessed in the absence of Merck C as well as in the presence of excess amounts of the parental compound L-685,458 (2 mM) as competitor. The input represents 2.5% of the total sample used for the affinity precipitation. A representative immunoblot from 4 independent biological replicates is shown. (D) Quantitation of PSH binding by Merck C. Specific binding was defined as difference of PSH signals in the absence or presence of L-685,458 after additional subtraction of unspecific background binding signals. Quantitative data are represented as mean ± SD (n = 4 biological replicates). The source data are shown in Figure 8 – source data 1. (E, F) Inhibition assay of PSH in DDM micelles and POPC vesicles with increasing concentrations of L-685,458 (E) or Merck C (F), respectively. PSH activity was analyzed by immunoblotting for AICD (Y188) and Aβ (2D8) following incubation with C100-His 6 substrate at 37 °C overnight. Representative immunoblots from 3 independent biological replicates are shown. The asterisks mark two substrate degradation bands, which are independent of PSH cleavage. (G) Inhibition assay of PSH reconstituted in POPC vesicles in the presence of 20 µM TSA and non-TSA γ-secretase inhibitors. PSH activity was analyzed by immunoblotting for AICD (Y188) and Aβ (2D8) following incubation with C100-His 6 substrate at 37 °C overnight. Representative immunoblots from 3 independent biological replicates are shown.

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques: Western Blot, Binding Assay, Affinity Precipitation, Quantitation Assay, Inhibition, Activity Assay, Incubation

Journal: bioRxiv

Article Title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

doi: 10.1101/2022.01.10.475655

Figure Lengend Snippet: (A, B) Structures of TSA GSIs L-685,458 (A) and III-31C (B). (C - F) Structures of non-TSA GSIs DAPT (C), LY411575 (D), Begacestat (E) and MRK-560 (F).

Article Snippet: Despite the demonstration of direct binding of the L-685,458 lead structure to PSH using the Merck C affinity ligand, L-685,458 inhibited PSH much less efficiently than γ-secretase, i.e. micromolar concentrations were needed to inhibit PSH compared to nanomolar concentrations known to inhibit γ-secretase.

Techniques: