N-terminal Acetylation Research Articles

NAA10 p.(D10G) and NAA10 p.(L11R) Variants Hamper Formation of the NatA N-Terminal Acetyltransferase Complex.

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.

Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30–Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure–function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins.

Naa20, the catalytic subunit of NatB complex, contributes to hepatocellular carcinoma by regulating the LKB1\u2013AMPK\u2013mTOR axis

N-α-acetyltransferase 20 (Naa20), which is a catalytic subunit of the N-terminal acetyltransferase B (NatB) complex, has recently been reported to be implicated in hepatocellular carcinoma (HCC) progression and autophagy, but the underlying mechanism remains unclear. Here, we report that based on bioinformatic analysis of Gene Expression Omnibus and The Cancer Genome Atlas data sets, Naa20 expression is much higher in HCC tumors than in normal tissues, promoting oncogenic properties in HCC cells. Mechanistically, Naa20 inhibits the activity of AMP-activated protein kinase (AMPK) to promote the mammalian target of rapamycin signaling pathway, which contributes to cell proliferation, as well as autophagy, through its N-terminal acetyltransferase (NAT) activity. We further show that liver kinase B1 (LKB1), a major regulator of AMPK activity, can be N-terminally acetylated by NatB in vitro, but also probably by NatB and/or other members of the NAT family in vivo, which may have a negative effect on AMPK activity through downregulation of LKB1 phosphorylation at S428. Indeed, p-LKB1 (S428) and p-AMPK levels are enhanced in Naa20-deficient cells, as well as in cells expressing the nonacetylated LKB1-MPE mutant; moreover, importantly, LKB1 deficiency reverses the molecular and cellular events driven by Naa20 knockdown. Taken together, our findings suggest that N-terminal acetylation of LKB1 by Naa20 may inhibit the LKB1–AMPK signaling pathway, which contributes to tumorigenesis and autophagy in HCC.

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes.

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

Maturation of NAA20 Aminoterminal End Is Essential to Assemble NatB N-Terminal Acetyltransferase Complex

Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation.

NAA10 p.(N101K) disrupts N-terminal acetyltransferase complex NatA and is associated with developmental delay and hemihypertrophy

Nearly half of all human proteins are acetylated at their N-termini by the NatA N-terminal acetyltransferase complex. NAA10 is evolutionarily conserved as the catalytic subunit of NatA in complex with NAA15, but may also have NatA-independent functions. Several NAA10 variants are associated with genetic disorders. The phenotypic spectrum includes developmental delay, intellectual disability, and cardiac abnormalities. Here, we have identified the previously undescribed NAA10 c.303C>A and c.303C>G p.(N101K) variants in two unrelated girls. These girls have developmental delay, but they both also display hemihypertrophy a feature normally not observed or registered among these cases. Functional studies revealed that NAA10 p.(N101K) is completely impaired in its ability to bind NAA15 and to form an enzymatically active NatA complex. In contrast, the integrity of NAA10 p.(N101K) as a monomeric acetyltransferase is intact. Thus, this NAA10 variant may represent the best example of the impact of NatA mediated N-terminal acetylation, isolated from other potential NAA10-mediated cellular functions and may provide important insights into the phenotypes observed in individuals expressing pathogenic NAA10 variants.

N-terminal acetylation of a mastoparan-like peptide enhances PE/PG segregation in model membranes

The synthetic peptides L1A and its acetylated analog (acL1A) display potent Gram-negative bactericidal activities without being hemolytic. We have gathered evidence that the N-terminal acetylation of L1A enhances the lytic activity in anionic vesicles with high capability to insert into and disturb lipid packing of model membranes. Here, the impact of L1A and acL1A was evaluated on a model membrane that mimics the cytoplasmic membrane of Gram-negative bacteria, which is rich in phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), using 3:1 mixture of POPE/DOPG and a variety of techniques. We followed peptide adsorption and penetration by zeta potential determination of large unilamellar vesicles, accessibility of tryptophan residue to acrylamide by quenching assays, and Gibbs isotherms. The secondary structure of the peptide on the membranes was assessed using circular dichroism. Peptide mixing ability with the lipids and phase segregation was assessed by the observation of Langmuir monolayers with fluorescence microscopy, as well as with differential scanning calorimetry thermograms of multilamellar vesicles. All in all, the results indicate that both peptides adsorb and penetrate POPE/DOPG membranes with similar affinities, decreasing the surface charge, and adopting alpha structures. Both peptides mix with DOPG and demix from POPE, and consequently, persist at the interface to larger surface pressures in the presence of PG than in pure PE monolayers. This selective degree of mixing of the peptides with PE and PG leads to peptide-induced segregation of PG from PE, being the less charged peptide, acL1A, able to segregate the lipids more efficiently.

N-Terminal Acetyltransferases Are Cancer-Essential Genes Prevalently Upregulated in Tumours.

Simple SummaryCancer remains one of the leading causes of mortality globally. Ultimately, cancers are driven by the disruption of normal mechanisms that control the growth and behaviour of our cells. Improvements in our knowledge of how these normal cell control mechanisms are disrupted in cancers can potentially lead to better diagnosis and new treatments. In this study, we examined the involvement of a specific gene family, encoding protein N-terminal acetyltransferases (NATs), in various types of tumours by analysing available large-scale cancer-associated datasets. We report several novel findings relating to how NATs are disrupted in cancers highlighting specific tumours where NATs can be of biological importance and may serve as therapeutic targets.N-terminal acetylation (Nt-Ac) is an abundant eukaryotic protein modification, deposited in humans by one of seven N-terminal acetyltransferase (NAT) complexes composed of a catalytic and potentially auxiliary subunits. The involvement of NATs in cancers is being increasingly recognised, but a systematic cross-tumour assessment is currently lacking. To address this limitation, we conducted here a multi-omic data interrogation for NATs. We found that tumour genomic alterations of NATs or of their protein substrates are generally rare events, with some tumour-specific exceptions. In contrast, altered gene expression of NATs in cancers and their association with patient survival constitute a widespread cancer phenomenon. Examination of dependency screens revealed that (i), besides NAA60 and NAA80 and the NatA paralogues NAA11 and NAA16, the other ten NAT genes were within the top 80th percentile of the most dependent genes (ii); NATs act through distinct biological processes. NAA40 (NatD) emerged as a NAT with particularly interesting cancer biology and therapeutic potential, especially in liver cancer where a novel oncogenic role was supported by its increased expression in multiple studies and its association with patient survival. In conclusion, this study generated insights and data that will be of great assistance in guiding further research into the function and therapeutic potential of NATs in cancer.

Molecular basis for N-terminal alpha-synuclein acetylation by human NatB.

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (αSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in the pathogenesis of Parkinson's disease and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-αSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for αSyn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.

NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity.

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

Abstract 4690: ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation

Abstract The transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2) is regarded as one of the main orchestrators of the cellular antioxidant and carcinogen-detoxifying response. NRF2 plays a pivotal role in protecting normal cells from carcinogenic insults, but it may also accelerate the proliferation and progression of cancer cells. Abnormally elevated expression or activation of NRF2 has been attributed to mutation, especially in the sequences involved in interaction with its negative regulator KEAP1. However, other mechanisms responsible for NRF2 overactivation/overexpression are less well understood. Arrest defective (ARD1), an N-terminal acetyltransferase that catalyzes N-terminal acetylation of target proteins, has lysine acetyltransferase activity and is involved in mediating various (patho)physiological processes, such as proliferation, apoptosis, autophagy, and differentiation. Aberrant overexpression of ARD1 is correlated with metastasis and poor prognosis in several types of cancer, suggesting that it may act as a tumor promoter. In the present study, we found that NRF2, acteyl-NRF2, and ARD1 were highly expressed in some human colon cancer cell lines (HCT-116, HCT-15, DLD1) and tissues obtained from colorectal cancer patients. Furthermore, overexpression of NRF2 and ARD1 was also verified by quantitative immunofluorescence analysis of colorectal tissue microarray. We noticed that silencing of NRF2 and ARD1 individually in human colon cancer cells (HCT-116) led to the decreases in viability, anchorage-independent growth, and migration capacity of HCT-116 cells. While there was no change in the protein level of ARD1 when NRF2 expression was knock down by siRNA, protein expression of Nrf2 and acteyl-NRF2 was markedly reduced by silencing of the ARD1 gene. This prompted us to explore the association between ARD1 and NRF2 in the human colon cancer cell proliferation and progression. Co-immunoprecipitation and immunocytochemical analyses showed that two proteins colocalized in the cytosol, and ARD1 directly bound to NRF2. Taken together, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer cells by acetylating and stabilizing this transcription factor. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea. Citation Format: Xizhu Fang, Yeon-Hwa Lee, Jie Zheng, Seong Hoon Kim, Jeong-Hoon Jang, Do-Hee Kim, Young-Joon Surh. ARD1-mediated NRF2 acetylation promotes human colon cancer cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4690.

The Meaning of an End: N-Terminal Acetyltransferase NAA50 Controls Plant Growth and Stress Responses.

At least 80% of eukaryotic proteins are estimated to undergo N-terminal acetylation (NTA), making it likely that your favorite protein is also regulated by NTA ([Linster and Wirtz, 2018][1]). This transfer of an acetyl group to the N terminus can modulate a protein’s interaction partners, i.e.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

Modulation of the bacterial CobB sirtuin deacylase activity by N-terminal acetylation

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.

The N-Terminal Acetyltransferase Naa50 Regulates Arabidopsis Growth and Osmotic Stress Response

N-terminal acetylation (Nt-acetylation) is one of the most common protein modifications in eukaryotes. The function of Naa50, the catalytic subunit of the evolutionarily conserved N-terminal acetyltransferase (Nat) E complex, has not been reported in Arabidopsis. In this study, we found that a loss of Naa50 resulted in a pleiotropic phenotype that included dwarfism and sterility, premature leaf senescence and a shortened primary root. Further analysis revealed that root cell patterning and various root cell properties were severely impaired in naa50 mutant plants. Moreover, defects in auxin distribution were observed due to the mislocalization of PIN auxin transporters. In contrast to its homologs in yeast and animals, Naa50 showed no co-immunoprecipitation with any subunit of the Nat A complex. Moreover, plants lacking Naa50 displayed hypersensitivity to abscisic acid and osmotic stress. Therefore, our results suggest that protein N-terminal acetylation catalyzed by Naa50 plays an essential role in Arabidopsis growth and osmotic stress responses.

Rapid and Easy Enrichment Strategy for Naturally Acetylated N Termini Based on LysN Digestion and Amine-Reactive Resin Capture.

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.

In Vitro N-Terminal Acetylation of Bacterially Expressed Parvalbumins by N-Terminal Acetyltransferases from Escherichia coli.

Most eukaryotic proteins are N-terminally acetylated (Nt-acetylated) by specific N-terminal acetyltransferases (NATs). Although this co-/post-translational protein modification may affect different aspects of protein functioning, it is typically neglected in studies of bacterially expressed eukaryotic proteins, lacking this modification. To overcome this limitation of bacterial expression, we have probed the efficiency of recombinant Escherichia coli NATs (RimI, RimJ, and RimL) with regard to in vitro Nt-acetylation of several parvalbumins (PAs) expressed in E. coli. PA is a calcium-binding protein of vertebrates, which is sensitive to Nt-acetylation. Our analyses revealed that only metal-free PAs were prone to Nt-acetylation (up to 100%), whereas Ca2+ binding abolished this modification, thereby indicating that Ca2+-induced structural stabilization of PAs impedes their Nt-acetylation. RimJ and RimL were active towards all PAs with N-terminal serine. Their activity towards PAs beginning with alanine was PA-specific, suggesting the importance of the subsequent residues. RimI showed the least activity regardless of the PA studied. Overall, NATs from E. coli are suited for post-translational Nt-acetylation of bacterially expressed eukaryotic proteins with decreased structural stability.

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae

NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.

Mechanism of actin N-terminal acetylation.

About 80% of human proteins are amino-terminally acetylated (Nt-acetylated) by one of seven Nt-acetyltransferases (NATs). Actin, the most abundant protein in the cytoplasm, has its own dedicated NAT, NAA80, which acts posttranslationally and affects cytoskeleton assembly and cell motility. Here, we show that NAA80 does not associate with filamentous actin in cells, and its natural substrate is the monomeric actin-profilin complex, consistent with Nt-acetylation preceding polymerization. NAA80 Nt-acetylates actin-profilin much more efficiently than actin alone, suggesting that profilin acts as a chaperone for actin Nt-acetylation. We determined crystal structures of the NAA80-actin-profilin ternary complex, representing different actin isoforms and different states of the catalytic reaction and revealing the first structure of NAT-substrate complex at atomic resolution. The structural, biochemical, and cellular analysis of mutants shows how NAA80 has evolved to specifically recognize actin among all cellular proteins while targeting all six actin isoforms, which differ the most at the amino terminus.