Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices—A Methodological Approach
<p>Comparing both approaches used here to investigate substrate/γ secretase TMD−TMD interactions. (<b>a</b>) In Approach 1, we manually tested four substrate TMDs against most γ secretase TMDs. LD<sub>50</sub> values represent relative affinities. (<b>b</b>) In Approach 2, 28 substrate TMDs were run simultaneously against presenilin TMD2, which corresponds to the γ secretase TMD implied by other studies in substrate recognition, using a highly efficient screening technique. Sequence abundance under selective conditions is equivalent to affinity and encoded by the colors of the heatmap (yellow: lowest affinity; dark green: highest affinity). WT = wild type; NC = negative control. Soluble domains extending into the bacterial periplasmic space represent β-lactamase domains; GFP and ToxR domains pointing to the cytoplasm are annotated.</p> "> Figure 2
<p>Overview of γ-secretase and substrate TMDs. (<b>a</b>) Transmembrane topologies of the γ-secretase subunits presenilin (blue), PEN-2 (yellow), nicastrin (red), APH-1 (purple), and a substrate (green). Arrows correspond to the direction of the sequences. (<b>b</b>) Top view onto the γ-secretase TMDs. The two catalytic aspartates in TMD6 and TMD7 of presenilin are represented by arrowheads and the TMDs are numbered from N- to C-terminus.</p> "> Figure 3
<p>TMD−TMD interactions of PS1 with substrates and the non-substrate ITGB1. (<b>a</b>) Strength of parallel heterotypic interactions normalized to the homodimerization signal of GpA used as a reference. Three different variants of PS1 TMD2 were tested in combination with APP. (<b>b</b>) Antiparallel heterotypic interactions normalized to the homodimerization signal of EmrE. The shown data correspond to the combination of TMD frames showing the strongest interactions in any given case (see <a href="#app1-ijms-24-14396" class="html-app">Figure S2</a> A-T, where the GFP expression controls are also given). Means ± SEM, n > 3. The positive and negative controls (grey bars) were included in every single round of experiments.</p> "> Figure 4
<p>TMD−TMD interactions of nicastrin or PEN-2 with substrates and the non-substrate ITGB1. (<b>a</b>) Strength of parallel heterotypic interactions to the nicastrin TMD normalized to the homodimerization signal of GpA. (<b>b</b>) Strength of antiparallel heterotypic interactions to the PEN-2 TMD normalized to the homodimerization signal of EmrE TMD4. The shown data correspond to the TMD pairs showing the strongest interactions, as shown in <a href="#app1-ijms-24-14396" class="html-app">Figure S2</a>, which also contains the GFP expression controls. Means ± SEM, n > 3. The positive and negative controls (grey bars) were included in every single round of experiments. The color coding given in the inset of panel (<b>b</b>) also applies to panel (<b>a</b>).</p> "> Figure 5
<p>Mutational analysis of the APP TMD in its pairwise interactions with different PS1 TMDs or itself. (<b>a</b>) Strength of the interaction of APP TMD (frame 2) mutants vs. PS1 TMD4 (frame 3) normalized to the signal of wt APP TMD. Residues whose mutation reduced the signal to <50% of wt are colored. (<b>b</b>) Strength of interaction of APP TMD (frame 2) mutants vs. PS1 <sup>125–146</sup>TMD2 (frame 0) normalized to the signal of wt. We note that mutant V46A more than tripled the LD<sub>50</sub> in this case, for reasons that are unclear. For technical reasons, a lower LD<sub>50</sub> limits the potential impact of mutations. (<b>c</b>) Strength of homodimerization of APP TMD (frame 2) mutants normalized to the signal of the wt APP TMD. The same residues as in (<b>a</b>) are highlighted in (<b>b</b>,<b>c</b>). (<b>d</b>) Mapping the mutation-sensitive residue positions onto a helical wheel or the NMR structure of the helix (pdb: 6hyf) model suggests that the amino acids colored in yellow or green, respectively, may correspond to two separate helix−helix interfaces formed by the APP TMD. Single, double, or triple asterisks denote statistical significance at the 0.05, 0.01, or 0.001 confidence levels (relative to wt APP). Means ± SEM, n = 4–5. The pairs used as references (dark grey bars) were included in every single round of the respective experiments.</p> "> Figure 6
<p>TMD−TMD interactions of various parts of PS1 TMD2 with substrate TMDs, as identified by the BLaTM library screening approach. Candidate pairs (shown in yellow) were identified by next generation sequencing and the resulting full dataset of 1344 pairs (see <a href="#app1-ijms-24-14396" class="html-app">Figure S7</a>) was filtered for abundances exceeding 40% of the signal of GpA wt. Homotypic interactions of positive and negative controls are given by grey bars; heterotypic TMD pairs also covered by approach 1 (<a href="#ijms-24-14396-f003" class="html-fig">Figure 3</a>) are shown in dark green and orange. The TMD sequences of the pair A4_HUMAN_2|PS1_TMD2_0 (identified by approach 2) are equivalent to those of the APP | PS1_TMD2_0 pair (approach 1) (both colored in green).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Determining TMD−TMD Interactions by Manual Testing of Candidate Pairs
2.2. TMD−TMD Interactions Determined by Library Screening
3. Discussion
4. Materials and Methods
4.1. Plasmid Design and Construction
4.2. Determining Ampicillin LD50 Values
4.3. Determining GFP Expression
4.4. Design and Screening of Combinatorial TMD Libraries
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Pauli, T.M.; Julius, A.; Costa, F.; Eschrig, S.; Moosmüller, J.; Fischer, L.; Schanzenbach, C.; Schmidt, F.C.; Ortner, M.; Langosch, D. Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices—A Methodological Approach. Int. J. Mol. Sci. 2023, 24, 14396. https://doi.org/10.3390/ijms241814396
Pauli TM, Julius A, Costa F, Eschrig S, Moosmüller J, Fischer L, Schanzenbach C, Schmidt FC, Ortner M, Langosch D. Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices—A Methodological Approach. International Journal of Molecular Sciences. 2023; 24(18):14396. https://doi.org/10.3390/ijms241814396
Chicago/Turabian StylePauli, Theresa M., Ayse Julius, Francesco Costa, Sabine Eschrig, Judith Moosmüller, Lea Fischer, Christoph Schanzenbach, Fabian C. Schmidt, Martin Ortner, and Dieter Langosch. 2023. "Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices—A Methodological Approach" International Journal of Molecular Sciences 24, no. 18: 14396. https://doi.org/10.3390/ijms241814396