Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells
Bibliographic record
Abstract
N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 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".