Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Charting the N-Terminal Acetylome: A Comprehensive Map of Human NatA Substrates.

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

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.

N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.

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.

Developmental roles of protein N-terminal acetylation.

Discovered more than 50 years ago, N-terminal acetylation (N-Ac) is one of the most common protein modifications. Catalyzed by different N-terminal acetyltransferases (NATs), N-Ac was originally believed to mostly promote protein stability. However, several functional consequences at substrate level were recently described that yielded important new insights about the distinct molecular functions for this modification. The ubiquitous and apparent irreversible nature of this protein modification leads to the assumption that N-Ac mostly executes constitutive functions. In spite of the large number of substrates for each NAT, recent studies in multicellular organisms have nevertheless indicated very specific phenotypes after NAT loss. This raises the hypothesis that in vivo N-Ac is only functionally rate limiting for a small subset of substrates. In this review, we will discuss the function of N-Ac in the context of a developing organism. We will propose that some rate limiting NAT substrates may be tissue-specific leading to differential functions of N-Ac during development of multicellular organisms. Moreover, we will also propose the existence of tissue and developmental-specific mechanisms that differentially regulate N-Ac.

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice.

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 µM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.

Introns in the Naa50 gene act as strong enhancers of tissue-specific expression in Arabidopsis.

Naa50 is the catalytic subunit of N-terminal acetyltransferase complex E, which plays an important role in regulating plant development, endoplasmic reticulum stress and immune responses in Arabidopsis. In this study, the complete genomic sequence (but not the coding sequence) of Naa50 rescued the phenotype of Naa50 deletion mutants. Naa50 expression was noted in whole roots except for central root cap cells. The deletion of intron 1 resulted in a loss of Naa50 expression in the root meristem zone and in the epidermis, cortex and endodermis of the elongation zone and mature zone, while the deletion of intron 2 decreased Naa50 expression in the epidermis, cortex and endodermis of the root elongation zone and mature zone. The native Naa50 promoter together with introns 1 and 2 promotes the expression of Naa50 in sepal vascular bundles, filaments, pollen and stigmas; however, neither intron has positive effect on Naa50 expression in mature rosette leaves. The results of this study show that introns 1 and 2 in the Naa50 gene function as enhancers to promote the tissue-specific expression of Naa50.

The N-terminal acetyltransferase Naa50 regulates tapetum degradation and pollen development in Arabidopsis.

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9-11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

Pan-cancer analysis reveals NAA50 as a cancer prognosis and immune infiltration-related biomarker.

Background: N-Alpha-Acetyltransferase 50 (NAA50) has acetyltransferase activity and is important for chromosome segregation. However, the function and mechanism of NAA50 expression in cancer development was still unclear. Here, we systematically researched the function and mechanism of NAA50 in pan-cancer, and further verified the results of NAA50 in lung adenocarcinoma (LUAD). Methods: In this study, using the online databases TIMER2.0, SangerBox3.0, HPA, UCSC, GEPIA, cBioPortal, UALCAN, TISIDB, CancerSEA and LinkedOmics, we focused on the relevance between NAA50 and oncogenesis, progression, methylation, immune infiltration, function and prognosis. In addition, the proliferation of cells was detected by CCK-8 and Edu assay. Finally, we analyzed the relationship between the expression of NAA50 and cell cycle related proteins. Results: Pan-cancer analysis indicated that NAA50 was overexpressed in most cancers. And there was a significant correlation between NAA50 expression and the prognosis of cancer patients. In the meantime, NAA50 gene changes occur in a variety of tumors. Compared with normal tissues, the methylation level of NAA50 promoter increased in most cancer tissues. In addition, the results exhibited that in most cancers, NAA50 was significantly positively correlated with bone myeloid-derived suppressor cell (MDSC) infiltration and negatively correlated with T cell NK infiltration. Moreover, functional enrichment indicated that NAA50 regulates cell cycle and proliferation in LUAD. In vitro experiments testified that knockout of NAA50 could significantly inhibit the proliferation of LUAD. Conclusion: NAA50 may be a potential biomarker and oncogene of pan-cancer, especially LUAD, which may promote the occurrence and development of tumors through different mechanisms. Furthermore, NAA50 was bound up with to immune cell infiltration in pan-cancer, meaning NAA50 may be an important therapeutic target for human cancers.

Structural and functional characterization of the N-terminal acetyltransferase Naa50.

The majority of eukaryotic proteins is modified by N-terminal acetylation, which plays a fundamental role in protein homeostasis, localization, and complex formation. N-terminal acetyltransferases (NATs) mainly act co-translationally on newly synthesized proteins at the ribosomal tunnel exit. NatA is the major NAT consisting of Naa10 catalytic and Naa15 auxiliary subunits, and with Naa50 forms the NatE complex. Naa50 has recently been identified in Arabidopsis thaliana and is important for plant development and stress response regulation. Here, we determined high-resolution X-ray crystal structures of AtNaa50 in complex with AcCoA and a bisubstrate analog. We characterized its substrate specificity, determined its enzymatic parameters, and identified functionally important residues. Even though Naa50 is conserved among species, we highlight differences between Arabidopsis and yeast, where Naa50 is catalytically inactive but binds CoA conjugates. Our study provides insights into Naa50 conservation, species-specific adaptations, and serves as a basis for further studies of NATs in plants.

Structure and Mechanism of Acetylation by the N-Terminal Dual Enzyme NatA/Naa50 Complex.

NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We find that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. These studies have implications for understanding the role of the NatA/Naa50 complex in modulating the majority of the N-terminal acetylome in diverse species.

The biological functions of Naa10 - From amino-terminal acetylation to human disease.

N-terminal acetylation (NTA) is one of the most abundant protein modifications known, and the N-terminal acetyltransferase (NAT) machinery is conserved throughout all Eukarya. Over the past 50 years, the function of NTA has begun to be slowly elucidated, and this includes the modulation of protein-protein interaction, protein-stability, protein function, and protein targeting to specific cellular compartments. Many of these functions have been studied in the context of Naa10/NatA; however, we are only starting to really understand the full complexity of this picture. Roughly, about 40% of all human proteins are substrates of Naa10 and the impact of this modification has only been studied for a few of them. Besides acting as a NAT in the NatA complex, recently other functions have been linked to Naa10, including post-translational NTA, lysine acetylation, and NAT/KAT-independent functions. Also, recent publications have linked mutations in Naa10 to various diseases, emphasizing the importance of Naa10 research in humans. The recent design and synthesis of the first bisubstrate inhibitors that potently and selectively inhibit the NatA/Naa10 complex, monomeric Naa10, and hNaa50 further increases the toolset to analyze Naa10 function.