RESUMEN
The acetylation of protein N-termini is a co- or posttranslational modification that plays important roles in protein homeostasis and stability. N-terminal acetyltransferases (NATs) catalyze the introduction of this modification using acetyl-coenzyme A (acetyl-CoA) as source of the acetyl-group. NATs operate in complex with auxiliary proteins that impact activity and specificity of these enzymes. Proper function of NATs is essential for development in plants and mammals alike. High resolution mass spectrometry (MS) is a powerful tool for investigating NATs and protein complexes in general. However, efficient methods for enriching NAT complexes ex vivo from cellular extracts are needed for the subsequent analysis. Based on bisubstrate analog inhibitors of lysine acetyltransferases, peptide-CoA conjugates have been developed as capture compounds of NATs. The N-terminal residue of these probes, serving as attachment site of the CoA moiety, was shown to impact NAT binding according to the respective amino acid specificity of these enzymes. This chapter reports the detailed protocols for the synthesis of peptide-CoA conjugates, the experimental procedures for NAT enrichment as well as the MS and data analysis. Collectively, these protocols provide a set of tools for profiling NAT complexes in cell lysates of healthy or diseases backgrounds.
Asunto(s)
Acetiltransferasas , Proteómica , Animales , Acetiltransferasas/química , Acetiltransferasas/metabolismo , Péptidos/química , Acetiltransferasas N-Terminal/metabolismo , Proteínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Mamíferos/metabolismoRESUMEN
Acetyl groups are transferred from acetyl-coenzyme A (Ac-CoA) to protein N-termini and lysine side chains by N-terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. Building on lysine-CoA conjugates as KAT probes, we have synthesized peptide probes with CoA conjugated to N-terminal alanine (α-Ala-CoA), proline (α-Pro-CoA) or tri-glutamic acid (α-3Glu-CoA) units for interactome profiling of NAT complexes. The α-Ala-CoA probe enriched the majority of NAT catalytic and auxiliary subunits, while a lysine CoA-conjugate bound only a subset of endogenous KATs. Interactome profiling with the α-Pro-CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins and α-3Glu-CoA steered the interactome towards NAA80 and NatB. These findings agreed with the inherent substrate specificities of the target proteins and showed that N-terminal CoA-conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.