Fig. 2: The R4S mutation destabilizes NAA10 and reduces enzymatic function.
From: Dysregulation of N-terminal acetylation causes cardiac arrhythmia and cardiomyopathy
a NAA10 protein sequence alignment across multiple species shows an evolutionarily conserved arginine (arrowhead). b In silico prediction (DynaMut2) suggests that the R4S mutation affects NAA10 stability and potential interactions with NAA15. c HEK293 cells co-transfected with NAA10-FLAG and NAA15 were immunoprecipitated with anti-FLAG antibody. Binding capacity was quantified. (WT = 1.0 ± 0.0 vs R4S = 0.5 ± 0.1; independent transfection experiments, n = 4 (right panel); p = 0.0267; two-tailed paired t-test). d HEK293T cells expressing NAA10WT-FLAG and NAA10R4S-FLAG were treated with cycloheximide for 0, 2, 4, and 6 hours, and lysates were analyzed by western blotting with fluorescent secondary antibodies for vinculin and FLAG. MW: molecular weight. e Quantification of d, with normalization of NAA10/vinculin ratio to initial NAA10 protein levels at multiple time points: 2 h (WT: 0.98 ± 0.08 vs R4S: 0.43 ± 0.02, p < 0.0001), 4 h (WT: 0.65 ± 0.10 vs R4S: 0.17 ± 0.02, p < 0.0001), and 6 h (WT: 0.40 ± 0.03 vs R4S: 0.13 ± 0.01, p = 0.0204); independent transfection experiments, n = 3; p = 0.0001; two-way ANOVA). f Schematic of ThioGlo4 assay to quantify the enzymatic activity of NAA10. Created in BioRender. Yoshinaga, D. (2025) https://BioRender.com/z86v428. g Representative traces of ThioGlo4 reaction over time indicating NAA10 activity. NAA10-WT or NAA10-R4S enzyme concentrations were 100 nM. No enzyme was added to the control sample. h Initial reaction velocity was measured by ThioGlo4 assay at varying NAA10 concentrations, with significant differences at 100 nM (p = 0.0205) and 200 nM (p = 0.0020) (n = 6, duplicates from 3 independent experiments; p < 0.0001; mixed-effects analysis). i The total amount of CoA released 30 minutes after initiation of the enzymatic reaction was significantly reduced at 100 nM (p = 0.0205) and 200 nM (p = 0.0020) (n = 6, duplicates from 3 independent experiments; p < 0.0001; mixed-effects analysis). Data are presented as mean ± SEM. ****p < 0.0001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file.
