Fig. 5.

Neo-Nt-acetylation of nascent polypeptide–associated complex A (NACA) of ORF and neo-N-terminal peptides.A, winnowing of acetylated neo-N-terminal peptides identified in HCT116 cells. B, the N-terminal peptides of nascent polypeptide–associated complex subunit alpha (NACA) identified in HCT116 cells. Cleaved peptide sequences and modifications are shown on the top and the peptide-spectrum match (PSM) numbers in the different experiments are on the bottom. C, the position of NACA cleavage (in yellow and light blue) by caspase-3, before Ser43, lead to the formation of a neo-Nt-acetylated form of NACA. The structural model is based on the structure of the ribosome-nascent chain containing an endoplasmic reticulum signal sequence in a complex with the nascent polypeptide–associated complex (Protein Data Bank: 7QWR). The ribosome and RNA are in gray. The full NACA (in dark pink) and BTF3 (in light pink) structures are based on AlphaFold (86) prediction and were aligned on top of the original partial structures. D, time-course of in vitro NACA cleavage and Nt-acetylation. The upper panel shows NACA cleavage at Ser43 by caspase-3 with (green) or without (orange) lysate dialysis before caspase-3 addition. The lower panel shows NACA neo-acetylation at Ser43 after caspase-3 cleavage with (green) or without (orange) lysate dialysis before caspase-3 addition. E, F and G, time-course N-terminomics of HCT116 cells treated with ABT-199 or DMSO (control). Log₂ abundance ratios for individual peptides are plotted on the y-axis, and the corresponding total peptide mass spectrometry (MS) intensities are shown on the x-axis. The circle colors correspond to the peptide type: ORF Nt-Acetylated (light gray), ORF peptides with Nt-free (dark gray), caspase-generated neo-Nt peptides (pink), and neo-Nt-acetylated peptides (light green). H, the suggested order of events for NACA neo-Nt-acetylation following caspase-3 processing. HYTANE, hydrophobic tagging-assisted N-termini enrichment; LATE, LysN amino terminal enrichment.