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Stablization of ACOs by NatB mediated N-terminal acetylation is required for ethylene homeostasis.

ABSTRACT: N-terminal acetylation (NTA) is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) in eukaryotes. However, the plant NATs and their biological functions have been poorly explored. Here we reveal that loss of function of CKRC3 and NBC-1, the auxiliary subunit (Naa25) and catalytic subunit (Naa20) of Arabidopsis NatB, respectively, led to defects in skotomorphogenesis and triple responses of ethylene. Proteome profiling and WB test revealed that the 1-amincyclopropane-1-carboxylate oxidase (ACO, catalyzing the last step of ethylene biosynthesis pathway) activity was significantly down-regulated in natb mutants, leading to reduced endogenous ethylene content. The defective phenotypes could be fully rescued by application of exogenous ethylene, but less by its precursor ACC. The present results reveal a previously unknown regulation mechanism at the co-translational protein level for ethylene homeostasis, in which the NatB-mediated NTA of ACOs render them an intracellular stability to maintain ethylene homeostasis for normal growth and responses.

SUBMITTER: Liu HQ

PROVIDER: S-EPMC8254318 | biostudies-literature |

REPOSITORIES: biostudies-literature

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NatB-mediated protein N-?-terminal acetylation is a potential therapeutic target in hepatocellular carcinoma.

Project description:The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-?-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-?-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

| S-EPMC5522283 | biostudies-literature

N-Terminal Acetylation by NatB Is Required for the Shutoff Activity of Influenza A Virus PA-X.

Project description:N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.

| S-EPMC6296758 | biostudies-literature

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

Project description: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 ?-synuclein (?Syn). Human NatB (hNatB) mediated N-terminal acetylation of ?Syn 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-?Syn 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 ?Syn 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.

| S-EPMC7494357 | biostudies-literature

Mutation of an Arabidopsis NatB N-alpha-terminal acetylation complex component causes pleiotropic developmental defects.

Project description:N-?-terminal acetylation is one of the most common, but least understood modifications of eukaryotic proteins. Although a high degree of conservation exists between the N-?-terminal acetylomes of plants and animals, very little information is available on this modification in plants. In yeast and humans, N-?-acetyltransferase complexes include a single catalytic subunit and one or two auxiliary subunits. Here, we report the positional cloning of TRANSCURVATA2 (TCU2), which encodes the auxiliary subunit of the NatB N-?-acetyltransferase complex in Arabidopsis. The phenotypes of loss-of-function tcu2 alleles indicate that NatB complex activity is required for flowering time regulation and for leaf, inflorescence, flower, fruit and embryonic development. In double mutants, tcu2 alleles synergistically interact with alleles of ARGONAUTE10, which encodes a component of the microRNA machinery. In summary, NatB-mediated N-?-terminal acetylation of proteins is pleiotropically required for Arabidopsis development and seems to be functionally related to the microRNA pathway.

| S-EPMC3828409 | biostudies-literature

pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli.

Project description:N-terminal acetylation is one of the most common co- and post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular ?-synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely N?-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely N?-acetylate our model substrate ?-synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.

| S-EPMC6040700 | biostudies-literature

N-terminal acetylation by NatB in Arabidopsis thaliana

Project description:N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes and is catalysed in humans by seven N-acetyltransferases. In this study, we investigated the Arabidopsis thaliana NatB catalytic (NAA20) and auxiliary subunit (NAA25) and their influence on protein N-terminal acetylation using the SILProNAQ approach

2019-11-21 | PXD016209 | Pride

NatB N-terminal acetyltransferase (hNAT5) inhibition

Project description:Search for genes differently expressed when we inhibit NatB N-terminal acetyltransferase. We compare gene expression after inhibiting hNAT5 expression in Hela cells with specific siRNAs expressed with adenoviruses. Keywords: gene expression comparison

2008-03-12 | GSE10779 | GEO

Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.

Project description:Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants.

| S-EPMC4973746 | biostudies-literature

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD<sup>+</sup> biosynthetic enzymes in <i>Saccharomyces cerevisiae</i>.

Project description: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 (t ½) 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.

| S-EPMC7247314 | biostudies-literature

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Project description:Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-? yeast strain partially complements the natB-? phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

| S-EPMC3412031 | biostudies-literature

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