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Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities., (© 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease., (© The Author 2014. Published by Oxford University Press.)

Maturation of protein N-termini occurs in all kingdoms of life, with major protein modifications being proteolytic processing (e.g., removal of initiator methionines) and N-terminal acetylation. The functional consequences of these modifications are only known for a few substrates, and techniques to study such modifications have begun to emerge only recently. We here report on a method enabling targeted, mass spectrometry based analysis of protein N-termini from polyacrylamide gel-separated proteins. In our method, stable isotope incorporation by in-gel N-acetylation of free primary amines permits calculating the extent of in vivo N-terminal acetylation, proven to reveal crucial information with reference to N-terminal protein biology.

Protein N-terminal acetylation is a major modification of eukaryotic proteins. Its functional implications include regulation of protein-protein interactions and targeting to membranes, as demonstrated by studies of a handful of proteins. Fifty years after its discovery, a potential general function of the N-terminal acetyl group carried by thousands of unique proteins remains enigmatic. However, recent functional data suggest roles for N-terminal acetylation as a degradation signal and as a determining factor for preventing protein targeting to the secretory pathway, thus highlighting N-terminal acetylation as a major determinant for the life and death of proteins. These contributions represent new and intriguing hypotheses that will guide the research in the years to come., Competing Interests: The author has declared that no competing interests exist.

Although locating at the protein ends, N- and C-termini are at the center of numerous cellular functions. This topic engages an increasing number of scientists, recently forming the International Society of Protein Termini (ISPT). Protein Termini 2022 gathered this interdisciplinary community to discuss how protein ends may steer protein functionality. [ABSTRACT FROM AUTHOR]

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK. The chaperone HYPK regulates the human N-terminal acetyltransferase E (NatE) complex, but the mechanism is not fully understood. Here, the authors study the interplay of NatE and HYPK by cryo-EM and biochemical approaches, providing insights into the molecular basis of N-terminal acetylation of human proteins. [ABSTRACT FROM AUTHOR]

N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast. [ABSTRACT FROM AUTHOR]

The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets. We report the x-ray crystal structure of Naa50p bound to a native substrate peptide fragment and CoA. We found that the peptide backbone of the substrate is anchored to the protein through a series of backbone hydrogen bonds with the first methionine residue specified through multiple van der Waals contacts, together creating an α-amino methionine-specific pocket. We also employed structure-based mutagenesis; the results support the importance of the α-amino methionine-specific pocket of Naa50p and are consistent with the proposal that conserved histidine and tyrosine residues play important catalytic roles. Superposition of the ternary Naa50p complex with the peptide-bound Gcn5 histone acetyltransferase revealed that the two enzymes share a Gcn5-related N-acetyltransferase fold but differ in their respective substrate-binding grooves such that Naa50p can accommodate only an α-amino substrate and not a side chain lysine substrate that is acetylated by lysine acetyltransferase enzymes such as Gcn5. The structure of the ternary Naa50p complex also provides the first molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: The hNaa10p (hArd1) protein is the catalytic subunit of the human NatA Nα- terminal acetyltransferase complex. The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed. In the flexible C-terminal tail of hNaa10p, there are several potential phosphorylation sites that might serve as points of regulation. Findings: Using 2D-gel electrophoresis and hNaa10p specific antibodies, we have investigated whether hNaa10p is phosphorylated in HEK293 cells. Several differently charged forms of hNaa10p are present in HEK293 cells and treatment with Calf Intestine Alkaline Phophatase (CIAP) strongly suggests that hNaa10p is phosphorylated at multiple sites under various cell culture conditions. A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively. Conclusion: We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nα-terminal acetyltransferases in human cells. [ABSTRACT FROM AUTHOR]

Abstract: The human hARD1–NATH complex, cotranslationally acetylating the α-amino groups of proteins, was recently described. In S. cerevisiae and D. melanogaster this NatA complex contains a third subunit, Nat5p or San, respectively. Based on phylogenetic analyses and database searches, we here describe the human homologue, hNAT5, of these proteins. RT-PCR experiments demonstrated that hNat5 mRNA was expressed in several human cell lines. The candidacy of hNAT5 as a third subunit of the hARD1–NATH complex was investigated using anti-NATH or anti-hARD1 in co-immunoprecipitation experiments followed by Mass Spectrometry analysis of tryptic peptides. Oligopeptides specific for hNAT5 were identified. This verified the expression of endogenous hNAT5 protein in human cells and also identified hNAT5 as a NATH and hARD1 interacting partner. hNAT5 localized to the cytoplasm in accordance with hNAT5–hARD1–NATH complexes playing a role in cotranslational N-α-acetylation. Sequence alignment revealed a high degree of similarity of the NAT5 protein between species supporting its conserved role as a part of the complex. The predicted acetyltransferase domain within hNAT5 suggested that this protein, like hARD1, is an enzymatically active component. In summary, we present the first description of the human homologue of Nat5p/San, hNAT5, the third component of the human NatA N-α-acetyltransferase complex. [Copyright &y& Elsevier]

Abstract: Hypoxia inducible factor-1α (HIF-1α) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1α and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1α and thereby enhances HIF-1α ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1α. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1α. However, we find that hARD1 specifically binds HIF-1α, suggesting a putative, still unclear, connection between these proteins. [Copyright &y& Elsevier]

Protein N-terminal acetylation is a very common modification, but has during the past decades received relatively little attention. In order to put this neglected field back on the scientific map, we have in May 2007 and September 2008 arranged two international NAT symposia in Bergen, Norway. This supplement contains selected proceedings from these symposia reflecting the current status of the field, including an overview of protein N-terminal acetylation in yeast and humans, a novel nomenclature system for the N-terminal acetyltransferases (NATs) and methods for studying protein N-terminal acetylation in vitro and in vivo. [ABSTRACT FROM AUTHOR]

N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast. [ABSTRACT FROM AUTHOR]

Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine Nϵ-acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. Nα-terminal acetylation occurs on the ribosome where the a amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ardip) and Naa15p (Natip). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an Nα-acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities. [ABSTRACT FROM AUTHOR]