P1‐125: DETERMINING THE OLIGOMERIC STATE OF THE BETA‐SITE APP‐CLEAVING ENZYME 1 (BACE1) IN NATURAL MEMBRANES AND DETERGENTS
Bibliographic record
Abstract
The beta-site APP-cleaving enzyme 1 (BACE1) has a transmembrane sequence (TMS), which is necessary for effective BACE1 cleavage of the amyloid precursor protein (APP). An uncommon sulfur-rich motif, MxxxCxxxMxxxCxMxC, spans the entire TMS of BACE1. The sequence is highly conserved among homologues and is reminiscent of a high-affinity binding site for Cu(I) found in other copper-transporting proteins. We designed model peptides of the BACE1-TMS to investigate metal-ion binding and oligomerization uncoupled from the cytoplasmic and the ectodomain. A set of biophysical and colorimetric methods was used to investigate peptide-metal ion complex formation. The role of the metal-ion binding motif in potentially pre-existing oligomers of full-length BACE1 was assessed by Förster resonance energy transfer, automated single-molecule fluorescence counting in living cells, as well as by blue-native and SDS-gel electrophoresis. We found that the sulfur-rich core motif MxxxCxxxM is involved in metal-ion coordination and oligomerization of BACE1. Addition of Cu(II) facilitated the formation of dimers and trimers of the BACE1 TMS. Importantly, the peptide undergoes a redox reaction with copper ions, resulting in a disulfide bridge involving Cys466 in the center of the conserved MxxxCxxxM motif as the key amino acid. Further, we find peptide trimerization to depend on (i) the presence of monovalent copper ions and (ii) the sulfhydryl group of Cys466.For the full-length protein, FLIM-FRET experiments revealed that BACE1 oligomers are naturally present in living cells, as the oligomeric state of BACE1 remained unchanged in the absence or presence of metal-ions.We determined a stable trimeric assembly of BACE1 in the plasma membrane by accurate single-molecule fluorescence counting. Although the oligomeric state of full-length BACE1 was not altered by the addition of copper in living cells, the addition of monovalent metal-ions was required to visualize di- and oligomers by Western blot analysis. We propose that BACE1 acts as a bona fide metalloprotein in an oligomeric form in vivo. Additionally, our results demonstrate a novel metal-ion controlled stabilization mediated by the TMS of the BACE1.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".