Abstract
Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. N-acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S-acylation, as proximity leads to rapid S → N-transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N-transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N-acetylation in vitro. Furthermore, by generating modelled structures of proteins N-acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N-acetylation and identify where nucleotide binding could enhance N-acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N-malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N-acetylation and N-succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N-malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N-acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N-acylation in vivo.
Highlights
Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively
This could occur if acetyl-CoA binds weakly to the surface of proteins before transferring its acetyl moiety. We reasoned that such a CoA-protein association would enhance N-acetylation of nearby lysine residues (Fig. 1D) via a mechanism that could be antagonised by co-incubation with structural mimetics of CoA (Fig. 2A)
When fragments of bovine heart mitochondrial membranes were incubated with 2 mM acetyl-CoA and 10 mM of various nucleotides, the metabolic cofactors ATP, NADPH and FAD, all significantly decreased protein lysine N-acetylation (Fig. 2C)
Summary
Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. Some reassessment was required after observing several thousand sites of lysine N-acetylation in vivo[4,5,6], the vast majority of which have a very low (~ 0.1%) stoichiometry of acetylation[5,7,8,9], and that many other N-linked acyl modifications on lysine residues are generated in vivo without known transferase e nzymes[10,11,12] This was reinforced by the realisation that protein lysines can be non-enzymatically N-acylated by acyl-CoAs7,13,14. While some lysine residues may be intentionally targeted, enhanced N-acylation on its own is not necessarily proof of biological regulation This is because non-enzymatic N-acylation occurs when the amine group ( pKa ~ 10.5) of a lysine residue deprotonates to become a nucleophile and attacks the thioester carbonyl of an acyl-CoA to generate a stable amide-linked protein modification (Fig. 1A)[13]. We show that lysine N-acylation by acyl-CoAs is enhanced by interactions between acyl-CoAs and nucleotide-binding sites on the protein surface, and that this mechanism may contribute to elevated protein N-acylation in vivo
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