Protein N-terminal acetylation is a widespread modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs). The various NATs and their specific substrate specificities and catalytic mechanisms are far from fully understood. We here describe an in vitro method based on reverse-phase HPLC to quantitatively measure in vitro acetylation of NAT oligopeptide substrates, enabling the determination of NAT specificity as well as kinetic parameters.

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators.

Nα-Acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. The NAT hNaa50p preferentially modifies peptides starting with methionine followed by a hydrophobic amino acid. hNaa50p also possesses Nϵ-autoacetylation activity. So far, no eukaryotic NAT has been mechanistically investigated. In this study, we used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. The NMR results, both the substrate binding study and the dynamic data, further indicate that the binding of acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein. This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant.

The impact of N(α)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(α)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(α)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(α)-acetyltransferase.

Deep exome resequencing is a powerful approach for delineating patterns of protein-coding variation among genes, pathways, individuals and populations.We analyzed exome data from 2,440 individuals of European and African ancestry as part of the National Heart, Lung, and Blood Institute's Exome Project, the aim of which is to discover novel genes and mechanisms that contribute to heart, lung and blood disorders.Each exome was sequenced to a mean coverage of 116×, allowing detailed inferences about the population genomic patterns of both common variation and rare coding variation.We identifi ed more than 500,000 single nucleotide variations, the majority of which were novel and rare (76% of variants had a minor allele frequency of less than 0.1%), refl ecting the recent dramatic increase in the size of the human population.The unprecedented magnitude of this dataset allowed us to rigorously characterize the large variation in nucleotide diversity among genes (ranging from 0 to 1.32%), as well as the role of positive and purifying selection in shaping patterns of proteincoding variation and the diff erential signatures of population structure from rare and common variation.This dataset provides a framework for personal genomics and is an important resource that will allow inferences of broad importance to human evolution and health.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

The human NatA protein N(alpha)-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G(0)/G(1) phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.

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 α 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 (Ard1p) and Naa15p (Nat1p). 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.

Protein N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells.

Protein N‐alpha‐terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, three major complexes, NatA, NatB and NatC catalyse near all N‐alpha‐terminal acetylation, acetylating specific subsets of proteins. In humans, the NatA complex is the most extensively studied. We here present the human NatB and NatC complexes (hNatB and hNatC). They are conserved from yeast with respect to subunit composition, substrate specificity and ribosome binding. Knockdown of hNatB subunits disrupts normal cell cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL‐62. Knockdown of hNatC subunits results in p53‐dependent cell death and reduced growth in human cell lines. Taken together, these studies emphasize the biological importance of N‐alpha‐terminal acetylation.

Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases.

Protein acetylation is among the most common protein modifications in eukaryotic cells. There are two types of acetylation; N‐epsilon and N‐alpha which are performed by distinct types of enzymes. N‐epsilon acetylation is performed by histone acetyltransferases (HAT) targeting epsilon‐amino group of lysines within histone proteins, a central modification in regulating gene transcription.Protein N‐terminal acetylation is performed by different N‐terminal acetyltransferase complexes (NAT). In yeast, three different NAT complexes are identified. One of them; NatA, is composed of two main subunits; Ard1p (catalytic subunit) and Nat1p (auxiliary subunit). Nat5p is a third subunit physically associated with NatA, but with unknown functions.We wanted to investigate the substrates of hNat5. Purified recombinant hNat5 and oligopeptide substrates were mixed and separated with reverse phase HPLC to study hNat5 acetylation activity. We found that hNat5 prefers substrates with N‐termini Met‐Leu‐Xxx‐Pro. In addition, mass spectrometry analysis of hNat5 incubated with acetyl CoA, demonstrated that hNat5 has autoacetylation activity. Autoacetylation and acetylation of other subset of substrates indicate activity similar to HAT.In conclusion, we here present a novel human acetyltransferase, hNat5 with a dual activity targeting both N‐terminal (NAT activity) and lysine ‐ amino groups (HAT activity).

N α -terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by y ARD1 and y NAT1 in yeast and h ARD1 and h NAT1 in humans. A yeast ard1 -Δ nat1 -Δ strain was phenotypically complemented by h ARD1 h NAT1 , suggesting that yNatA and hNatA are similar. However, heterologous combinations, h ARD1 y NAT1 and y ARD1 h NAT1 , were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1 -Δ nat1 -Δ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

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