Search

Background Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. Here, we tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. Results Using metabolomic and lipidomic approaches, we found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting our results, we find a strong association of NAA40 expression with insulin sensitivity in obese patients. Conclusions Overall, our findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders.

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitor is competitive to the peptide substrate and noncompetitive to the cofactor, suggesting NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific towards NatD, displaying about 1,000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z&rsquo, factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

N-alpha-acetyltransferase 40 (NAA40) catalyzes the transfer of an acetyl moiety to the alpha-amino group of serine 1 (S1) on histones H4 and H2A. Our previous studies linked NAA40 and its corresponding N-terminal acetylation of histone H4 (N-acH4) to colorectal cancer (CRC). However, the role of NAA40 in CRC development was not investigated. Here, we show that NAA40 protein and mRNA levels are commonly increased in CRC primary tissues compared to non-malignant specimens. Importantly, depletion of NAA40 inhibits cell proliferation and survival of CRC cell lines and increases their sensitivity to 5-Fluorouracil (5-FU) treatment. Moreover, the absence of NAA40 significantly delays the growth of human CRC xenograft tumors. Intriguingly, we found that NAA40 knockdown and loss of N-acH4 reduce the levels of symmetric dimethylation of histone H4 (H4R3me2s) through transcriptional downregulation of protein arginine methyltransferase 5 (PRMT5). NAA40 depletion and subsequent repression of PRMT5 results in altered expression of key oncogenes and tumor suppressor genes leading to inhibition of CRC cell growth. Consistent with this, NAA40 mRNA levels correlate with those of PRMT5 in CRC patient tissues. Taken together, our results establish the oncogenic function of the epigenetic enzyme NAA40 in colon cancer and support its potential as a therapeutic target.

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation (Nt-acetylation) of histone H4 known to be involved in cell growth. Here we report that NatD promotes the migratory and invasive capabilities of lung cancer cells in vitro and in vivo. Depletion of NatD suppresses the epithelial-to-mesenchymal transition (EMT) of lung cancer cells by directly repressing the expression of transcription factor Slug, a key regulator of EMT. We found that Nt-acetylation of histone H4 antagonizes histone H4 serine 1 phosphorylation (H4S1ph), and that downregulation of Nt-acetylation of histone H4 facilitates CK2α binding to histone H4 in lung cancer cells, resulting in increased H4S1ph and epigenetic reprogramming to suppress Slug transcription to inhibit EMT. Importantly, NatD is commonly upregulated in primary human lung cancer tissues where its expression level correlates with Slug expression, enhanced invasiveness, and poor clinical outcomes. These findings indicate that NatD is a crucial epigenetic modulator of cell invasion during lung cancer progression., NatD is an acetyltransferase responsible for N-α-terminal acetylation of the histone H4 and H2A and has been linked to cell growth. Here the authors show that NatD-mediated acetylation of histone H4 serine 1 competes with the phosphorylation by CK2α at the same residue thus leading to the upregulation of Slug and tumor progression.

Cotranslational N-terminal (Nt-) acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently, there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study, we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala, or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs.

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license 17 1829 1843 Cited By :2

Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers. Electronic supplementary material The online version of this article (doi:10.1007/s10495-015-1207-0) contains supplementary material, which is available to authorized users.

Mosquitoes transmit a number of diseases in animals and humans, including Dengue, Chikungunya and Zika viruses that affect millions of people each year. Controlling the disease-transmitting mosquitoes has proven to be a successful strategy to reduce the viruses transmission. Polyamines are required for the life cycle of the RNA viruses, Chikungunya virus and Zika virus, and a depletion of spermidine and spermine in the host via induction of spermine N-acetyltransferase restricts their replication. Spermine N-acetyltransferase is a key catabolic enzyme in the polyamine pathway, however there is no information of the enzyme identification in any insects. Aliphatic polyamines play a fundamental role in tissue growth and development in organisms. They are acetylated by spermidine/spermine N-1-acetyl-transferase (SAT). In this study we provided a molecular and biochemical identification of SAT from Aedes aegypti mosquitoes. Screening of purified recombinant proteins against polyamines established that aaNAT5b, named previously based on sequence similarity with identified aaNAT1 in insects, is active to spermine and spermidine. A crystal structure was determined and used in molecular docking in this study. Key residues were identified to be involved in spermine binding using molecular docking and simulation. In addition, SAT transcript was down regulated by blood feeding using a real time PCR test. Based on its substrate profile and transcriptional levels after blood feeding, together with previous reports for polyamines required in arboviruses replication, SAT might be potentially used as a target to control arboviruses with human interference. National Natural Science Foundation of China [31472186] This work was supported by National Natural Science Foundation of China [Grant Number 31472186] (QH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Summary N-terminal acetylation is among the most common protein modifications in eukaryotes and is mediated by evolutionarily conserved N-terminal acetyltransferases (NATs). NatD is among the most selective NATs; its only known substrates are histones H4 and H2A, containing the N-terminal sequence SGRGK in humans. Here we characterize the molecular basis for substrate-specific acetylation by NatD by reporting its crystal structure bound to cognate substrates and performing related biochemical studies. A novel N-terminal segment wraps around the catalytic core domain to make stabilizing interactions, and the α1-α2 and β6-β7 loops adopt novel conformations to properly orient the histone N termini in the binding site. Ser1 and Arg3 of the histone make extensive contacts to highly conserved NatD residues in the substrate binding pocket, and flanking glycine residues also appear to contribute to substrate-specific binding by NatD, together defining a Ser-Gly-Arg-Gly recognition sequence. These studies have implications for understanding substrate-specific acetylation by NAT enzymes.

Protein N(alpha)-terminal acetylation (Nt-acetylation) is considered one of the most common protein modification in eukaryotes, and 80-90% of all soluble human proteins are modified in this way, with functional implications ranging from altered protein function and stability to translocation potency amongst others. Nt-acetylation is catalyzed by N-terminal acetyltransferases (NATs), and in yeast five NAT types are identified and denoted NatA-NatE. Higher eukaryotes additionally express NatF. Except for NatD, human orthologues for all yeast NATs are identified. yNatD is defined as the catalytic unit Naa40p (Nat4) which co-translationally Nt-acetylates histones H2A and H4. In this study we identified and characterized hNaa40p/hNatD, the human orthologue of the yeast Naa40p. An in vitro proteome-derived peptide library Nt-acetylation assay indicated that recombinant hNaa40p acetylates N-termini starting with the consensus sequence Ser-Gly-Gly-Gly-Lys-, strongly resembling the N-termini of the human histones H2A and H4. This was confirmed as recombinant hNaa40p Nt-acetylated the oligopeptides derived from the N-termini of both histones. In contrast, a synthetically Nt-acetylated H4 N-terminal peptide with all lysines being non-acetylated, was not significantly acetylated by hNaa40p, indicating that hNaa40p catalyzed H4 N(alpha)-acetylation and not H4 lysine N(epsilon)-acetylation. Also, immunoprecipitated hNaa40p specifically Nt-acetylated H4 in vitro. Heterologous expression of hNaa40p in a yeast naa40-Delta strain restored Nt-acetylation of yeast histone H4, but not H2A in vivo, probably reflecting the fact that the N-terminal sequences of human H2A and H4 are highly similar to each other and to yeast H4 while the N-terminal sequence of yeast H2A differs. Thus, Naa40p seems to have co-evolved with the human H2A sequence. Finally, a partial co-sedimentation with ribosomes indicates that hNaa40p co-translationally acetylates H2A and H4. Combined, our results strongly suggest that human Naa40p/NatD is conserved from yeast. Thus, the NATs of all classes of N-terminally acetylated proteins in humans now appear to be accounted for.

Protein acetylation is increasingly recognized as an important post-translational modification. Although a lot of protein acetyltransferases have been identified, a few putative acetyltransferases are yet to be studied. In this study, we identified a novel protein acetyltransferase, Patt1, which belongs to GNAT family. Patt1 exhibited histone acetyltransferase activity and auto-acetylation activity. Deletion and mutation analysis of the predicted acetyltransferase domain in Patt1 showed that the conserved Glu139 was an important residue for its protein acetyltransferase activity. Furthermore, we found that Patt1 was highly expressed in liver and significantly downregulated in hepatocellular carcinoma tissues. In addition, we showed that overexpression of Patt1 enhanced the apoptosis of hepatoma cells dependent on its acetyltransferase activity, whereas knockdown of Patt1 significantly protected Chang liver cells from apoptosis. These data suggest that Patt1 might be involved in the development of hepatocellular carcinoma, and could be served as a potential therapy target for hepatocellular carcinoma.

N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring on approximately 50% of yeast soluble proteins and about 80 to 90% of mammalian proteins (10, 13, 18). Eukaryotic cytosolic proteins initiate with methionine, which is cleaved from nascent chains of most proteins. Subsequently, N-terminal acetylation occurs on certain proteins either containing or lacking the methionine residue (10, 13, 18, 25, 27). Proteins susceptible to N-terminal acetylation have a variety of different N-terminal sequences, with no simple consensus motifs and with no dependence on a single type of residue (25, 27). Earlier workers attempted to determine the N-acetylation rules, and some distinguishing features were found in the sequences, mainly between residues 1 and 10 and fewer features between residues 16 and 24 and residues 30 and 40, but the precise nature of these features was not determined (2). Proteins with serine and alanine termini are the most frequently acetylated, and these residues, along with methionine, glycine, and threonine, account for over 95% of the N-terminal acetylated residues. However, proteins having these amino acids at their N termini are not always acetylated and none of these N-terminal residues guarantees acetylation, indicating that the enzymes recognize some structural characteristics of the N-terminal portion in addition to a particular amino acid at the N terminus (25). N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs) that transfer acetyl groups from acetyl coenzyme A to termini of α-amino groups. We have established that Saccharomyces cerevisiae contains the three major NATs, NatA, NatB, and NatC, with each having different catalytic subunits, Ard1, Nat3, and Mak3, respectively, and with each acting on a different group of proteins (Table ​(Table1)1) (28, 29). As previously summarized (21, 24, 25), subclasses of proteins with Ser, Ala, Gly, or Thr termini are acetylated by NatA; proteins with Met-Glu or Met-Asp termini and subclasses of proteins with Met-Asn and Met-Met termini are acetylated by NatB; subclasses of proteins with Met-Ile, Met-Leu, Met-Trp, or Met-Phe termini are acetylated by NatC. TABLE 1. The five types of yeast N-terminal acetyltransferases NatA activity requires two subunits, the catalytic subunit Ard1p and the auxiliary subunit Nat1 (22). nat1 and ard1 mutants were unable to N-terminally acetylate in vivo the same subset of 24 normally acetylated proteins, including those with Ala and Ser termini (1). In addition to lacking NAT activity, both nat1-Δ and ard1-Δ mutants exhibited slower growth, derepression of the silent mating-type gene HMLα, and failure to enter Go, due in part to the lack of acetylation of Sir3 and Orc1 (15, 45). Overexpression of both Ard1 and Nat1 subunits are required for increased NAT activity in vivo (22), and both interact with each other to form an active complex in vitro (23). A putative N-terminal acetyltransferase, Nat5, homologous to catalytic subunits Ard1, Nat3, and Mak3, was shown to copurify with NatA at about a 1:1:1 ratio (14). Although the substrates of Nat5 have not been identified, the nat5-Δ strain produces a phenotype that is distinct from those found in NatA, NatB, NatC, and NatD mutants (see below), and Nat5 is not required for NatA activity, suggesting that Nat5 forms a distinct NAT, which we have denoted NatE (Table ​(Table11). NatB acetyltransferase is composed of at least two subunits, the catalytic subunit Nat3 (29) and the auxiliary subunit Mdm20 (16), which also is required for acetylation of the NatB substrates (30, 38). The corresponding deletion mutants have similar phenotypes, including slow growth, temperature and osmotic sensitivity, calcium and caffeine sensitivity, deficiency in utilization of nonfermentable carbon sources, reduced mating efficiency, sensitivity to antimitotic drugs, and susceptibility to DNA-damaging agents (30). NatC contains three subunits, Mak3, Mak10, and Mak31 (34). Tercero et al. (41) described the MAK3 gene, which encodes the catalytic subunit of NatC and is required for the N-terminal acetylation of the viral major coat protein, gag, with an Ac-Met-Leu-Arg-Phe terminus (42). We demonstrated that each of the Mak3, Mak10, and Mak31 subunits are required for acetylation of the NatC-type sequences in vivo (26). In addition, all three deletion strains had similar phenotypes, including slower growth on nonfermentable carbon sources at elevated temperature. Recently, an additional NAT, Nat4 (NatD), was shown to acetylate the N termini of highly conserved histones H2A and H4, which have Ser-Gly-Gly-Lys-Gly and Ser-Gly-Arg-Gly-Lys N termini, respectively (39). Song et al. (39) also demonstrated that Nat4 acetylates in vitro a 23-amino-acid-long synthetic peptide corresponding to the N terminus of histone H4, but not an H3 peptide, which is not normally acetylated, nor a adrenocorticotropin peptide, which is an NatA substrate. However no obvious nat4-Δ phenotype was reported (39). So far, there is no evidence that Nat4 acetylates any other yeast protein, as judged by two-dimensional (2D) gel analysis of the soluble proteins prepared from normal and nat4-Δ mutant strains (39). We have designated this NAT activity as NatD, to be consistent with our established nomenclature. In this work we investigated the NatD requirements for N-terminal acetylation and uncovered nat4-Δ phenotypes. Surprisingly, NatD differs from the other NATs in its requirement for an extended N-terminal region for efficient acetylation. Mutant forms of iso-1-cytochrome c with extensions with as many as 23 amino acid residues corresponding to the N terminus of H4 histone were only partially acetylated by Nat4. In contrast, only five or fewer N-terminal amino acid replacements were necessary to acetylate mutant forms of iso-1-cytochrome c (iso-1) by NatA, NatB, or NatC. For example, iso-1 with eight N-terminal amino acids corresponding to the N-terminal region of H2B histone was sufficient for acetylation by NatA (29). In addition, we have investigated the site of action of NatD in the cell and demonstrated that Nat4 is a cytoplasmic protein that colocalizes with mono- and polyribosomes in a sucrose density gradient. Also, we have uncovered nat4-Δ phenotypes by using a variety of special media, and these phenotypes include sensitivity to 3-aminotriazol (3-AT), benomyl, salt, and thiabendazole (TBZ). Interestingly, nat4-Δ showed a synthetic growth defect in the strain with an altered histone H4 protein having K5R K8R K12R replacements and therefore lacking acetylated lysine residues in the N-terminal region.

A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.