Illuminating the impact of N-terminal acetylation: from protein to physiology

Abstract

N-terminal acetylation is a highly abundant protein modification in eukaryotic cells. This modification is catalysed by N-terminal acetyltransferases acting co- or post-translationally. Here, we review the eukaryotic N-terminal acetylation machinery: the enzymes involved and their substrate specificities. We also provide an overview of the impact of N-terminal acetylation, including its effects on protein folding, subcellular targeting, protein complex formation, and protein turnover. In particular, there may be competition between N-terminal acetyltransferases and other enzymes in defining protein fate. At the organismal level, N-terminal acetylation is highly influential, and its impairment was recently linked to cardiac dysfunction and neurodegenerative diseases.

Fig. 1. Protein N-terminal acetylation reaction.

N-terminal acetyltransferases (NATs) catalyse the transfer of an acetyl moiety (red) from acetyl-coenzyme A (Ac-CoA) to the α-amino group (blue) at the extreme N-terminus of a polypeptide or protein.

Fig. 2. NAT machinery, conservation and Nt-acetylome in eukaryotes.

A The eukaryotic family of N-terminal acetyltransferases (NATs) comprises eight enzyme types (NatA–NatH). NatA–NatE act co-translationally, modifying nascent polypeptides during their synthesis on the ribosome. In contrast, NatF–NatH operate post-translationally, targeting proteins after their synthesis. NatF is localised on the cytosolic side of the Golgi apparatus (and of the plasma membrane in plants), modifying transmembrane proteins^,. NatG is associated with plastids^,, and NatH specifically Nt-acetylates actins. The catalytic subunits of each NAT are designated NAA10–NAA90, with some NATs requiring auxiliary subunits (NAA15, HYPK, NAA25, NAA35, NAA38 and PFN2) for ribosome anchoring and modulation of enzymatic activity. Each NAT complex exhibits distinct substrate specificity, primarily determined by the first two to four amino acids. The indicated substrate specificity is defined by human and/or yeast NATs (and appears to be similar in plants), except for NatG, which is based on plant enzymes^,. B All co-translational NATs (NatA–NatE) are conserved from yeast to metazoans and plants, but yeast NatE is likely catalytically inactive. NatF is present in both plants and metazoans, whereas NatG is exclusive to plants and NatH is only found in metazoans. C Approximately 50–80% of the eukaryotic proteome is N-terminally acetylated (Nt-acetylome). In yeast, metazoans and plants, NatA accounts for the largest share of the Nt-acetylome, followed by NatB. NatC, NatE and NatF have overlapping substrate specificities in vitro, with varying coverage of the Nt-acetylome in eukaryotes^–.

Fig. 3. N-terminal acetylation and its impact on protein fate.

N-terminal acetylation has varying effects depending on the function of the substrate. Here, four main functions of Nt-acetylation are summarised. A Nt-acetylation may facilitate correct protein folding, maintaining protein solubility and function, while the non-Nt-acetylated variant is prone to aggregation^,,,. B Protein-protein interactions may depend on Nt-acetylation, increasing the ability to bind hydrophobic binding grooves and enhancing the formation of functional protein complexes^,,. C Some substrates depend on Nt-acetylation to achieve correct subcellular localisation, ensuring they reach their functional destinations within the cell^,–. D Nt-acetylation can protect proteins from degradation, contributing to their stability and longevity^,,. However, some Nt-acetylated proteins may be targeted for degradation through conditional mechanisms, such as correct stoichiometry, folding and protein-protein interactions^,.

Fig. 4. Nt-acetylation-dependent regulation of protein stability and degradation.

The major co-translational NATs (NatA, NatB and NatC) Nt-acetylate nascent polypeptides at the ribosome. NatA Nt-acetylates small amino acids (Ala, Ser, Thr, Val, Gly, Cys) after the initiator methionine (iMet) is excised by MetAPs^,,. NatB Nt-acetylates N-termini with a retained iMet followed by Asp, Glu, Asn, or Gln. Similarly, NatC acts on iMet-retained N-termini, but with a hydrophobic or basic amino acid in the second position (Leu, Ile, Phe, Val, Tyr, Met, His, or Lys). Nt-acetylation imprints protein stability by protecting proteins from proteasomal degradation mediated by ubiquitin (Ub) E3 ligases. However, the Ub E3 ligase MARCHF6 can recognise certain Nt-acetylated N-termini and confer conditional protein degradation through the Ac/N-degron pathway, potentially as a protein quality control mechanism. In cells with impaired NAT function, non-acetylated N-termini can be recognised by specific Ub E3 ligases and targeted for proteasomal degradation through the N-degron pathways. In human cells, several Ub E3 ligases recognise non-Nt-acetylated NatA substrates. In the GASTC/N-degron pathway, inhibitor of apoptosis proteins (IAPs), including XIAP, BIRC2, BIRC3 and BIRC6, act on N-termini starting with Ala or Ser, while the Cullin 2-RING E3 Ub ligase (CRL2) substrate receptors, ZYG11B and ZER1, recognise Gly, Ala, Ser, Thr, or Cys-starting N-termini^,. Additionally, UBRs can mediate the degradation of Cys-starting proteins through the Arg/N-degron pathway following oxidation by ADO and subsequent arginylation by ATE1. This degradation route is likely less common for non-Nt-acetylated Cys-starting substrates of NatA, as NatA and ADO have distinct substrate preferences. In A. thaliana, global proteome destabilisation was observed following NatA depletion, but the responsible Ub E3 ligases are not identified. The Ub E3 ligases UBR4–KCMF1, UBR1 and UBR2 mediate degradation of non-Nt-acetylated NatC substrates. NatB may shield a certain subset of proteins from degradation. Non-Nt-acetylated NatB substrates are potentially recognised by UBR1 in D. melanogaster and by UBR1 and UBR4 in mice. Human Ub ligases that recognise non-Nt-acetylated NatB substrates are not established, but Ube2w may be involved in degradation of non-acetylated αSyn in human cells.

Fig. 5. The cellular and organismal impact of Nt-acetylation.

A Summary of the physiological impact of NATs in A. thaliana. B NatC prevents age-dependent motility loss in D. melanogaster. NatC KO fruit flies exhibit muscle developmental defects and motility loss, and these phenotypes are rescued upon UbcE2M overexpression. C In yeast, NatD-mediated Nt-acetylation (Ac) of histone H4 normally antagonises the methylation (Me) of H4 Arg3, which regulates yeast lifespan. Under calorie restriction, yeast NatD and Nt-acetylation of histone H4 are downregulated, resulting in increased levels of H4 Arg3 methylation and induction of stress response genes such as PNC1, which promotes yeast longevity. D Post-translational Nt-acetylation of actin mediated by NAA80 in human cells affects cytoskeletal dynamics. Human NAA80 KO cells lacking Nt-acetylation of actin display more cell protrusions and increased cell motility, implicating NAA80 as a cell migration regulator. KD knockdown, KO knockout.

Fig. 6. Overview of pathogenic NAT variants.

Schematic representation of the pathogenic variants identified in various NAT subunits, their associated phenotypes and the number of affected individuals. Impairment of the major co-translational NATs (NatA-NatC) results in severe and general phenotypes^–,–. In contrast, impairment of the specific post-translational NatF (NAA60) and NatH (NAA80) is linked to brain calcification or abnormal hearing and muscles, respectively. The Gcn5-related N-acetyltransferase (GNAT) domains of the catalytic NAT subunits are shown in dark blue.