N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein ...N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.
Humans express six highly conserved actin isoforms, which differ the most at their N‐termini. Actin's N‐terminus undergoes co‐ and post‐translational processing unique among eukaryotic proteins. ...During translation, the initiator methionine of the two cytoplasmic isoforms is N‐terminally acetylated (Nt‐acetylated) and that of the four muscle isoforms is removed and the exposed cysteine is Nt‐acetylated. Then, an unidentified acetylaminopeptidase post‐translationally removes the Ac‐Met (or Ac‐Cys), and all six isoforms are re‐acetylated at the N‐terminus. Despite the vital importance of actin for cellular processes ranging from cell motility to organelle trafficking and cell division, the mechanism and functional consequences of Nt‐acetylation remained unresolved. Two recent studies significantly advance our understanding of actin Nt‐acetylation. Drazic et al. (2018, Proc Natl Acad Sci U S A, 115, 4399–4404) identify actin's dedicated N‐terminal acetyltransferase (NAA80/NatH), and demonstrate that Nt‐acetylation critically impacts actin assembly in vitro and in cells. NAA80 knockout cells display increased filopodia and lamellipodia formation and accelerated cell motility. In vitro, the absence of Nt‐acetylation leads to a decrease in the rates of filament depolymerization and elongation, including formin‐induced elongation. Goris et al. (2018, Proc Natl Acad Sci U S A, 115, 4405–4410 describe the structure of Drosophila NAA80 in complex with a peptide‐CoA bi‐substrate analog mimicking the N‐terminus of β‐actin. The structure reveals the source of NAA80's specificity for actin's negatively‐charged N‐terminus. Nt‐acetylation neutralizes a positive charge, thus enhancing the overall negative charge of actin's unique N‐terminus. Actin's N‐terminus is exposed in the filament and influences the interactions of many actin‐binding proteins. These advances open the way to understanding the many likely consequences and functional roles of actin Nt‐acetylation.
The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although ...profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.
Evasion of apoptosis and active cell proliferation are among the characteristics of cancer cells. Triggering the induction of apoptosis or reducing the proliferative rate will potentially be helpful ...for cancer treatment. Recently, several reports demonstrated that knockdown of the protein acetyltransferase hARD1 significantly reduced the growth rate of human cancer cell lines. Furthermore, hARD1 knockdown induced apoptosis or sensitized cells to drug induced apoptosis. hARD1 acts in complex with the NATH protein and catalyzes cotranslational acetylation of protein N-termini. Thus, it was suggested that the effects on cell proliferation and apoptosis induction are due to a reduced level of N-terminal acetylation of certain substrate proteins. NATH was originally identified as upregulated in thyroid papillary carcinomas and has lately also been found to correlate with aggressiveness and differentiation status of neuroblastic tumours. On the other hand, researchers recently reported that hARD1 acetylates Beta-catenin. Knockdown of hARD1 reduced the transcriptional activity of the Beta-Catenin/TCF4 complex, downregulating cyclin D1 and thereby promoting G1-arrest and inhibition of cell proliferation of lung cancer cells. Although the underlying molecular mechanisms need further clarification, several reports suggest that reduction of hARD1 negatively affects cell growth. Thus, hARD1 or the hARD1-NATH complex stands out as attractive drug targets in cancer treatment. One challenge will be to develop specific inhibitors that discriminate between hARD1 and the many other enzymes, including the histone acetyltransferases, using acetyl-coenzyme A as acetyl donor. This review focuses on the enzymatic and biological activities of hARD1, and potential mechanisms of functional inhibition.
N-terminal acetylation is being recognized as a factor affecting protein lifetime and proteostasis. It is a modification where an acetyl group is added to the N-terminus of proteins, and this occurs ...in 80 % of the human proteome. N-terminal acetylation is catalyzed by enzymes called N-terminal acetyltransferases (NATs). The various NATs acetylate different N-terminal amino acids, and methionine is a known target for some of the NATs. Currently, the acetylation status of most proteins can only be assessed with a limited number of methods, including mass spectrometry, which although powerful and robust, remains laborious and can only survey a fraction of the proteome. We here present testing of an antibody that was developed to specifically recognize Nt-acetylated methionine-starting proteins. We have used dot blots with synthetic acetylated and non-acetylated peptides in addition to protein analysis of lysates from NAT knockout cell lines to assess the specificity and application of this anti-Nt-acetylated methionine antibody (anti-NtAc-Met). Our results demonstrate that this antibody is indeed NtAc-specific and further show that it has selectivity for some subtypes of methionine-starting N-termini, specifically potential substrates of the NatC, NatE and NatF enzymes. We propose that this antibody may be a powerful tool to identify NAT substrates or to analyse changes in N-terminal acetylation for specific cellular proteins of interest.
•The function and regulation of Nt-acetylation is unknown for most proteins.•We evaluated a pan Nt-acetylation antibody as a method to monitor Nt-acetylation.•We used peptide dot blot, WB of NAT KO cell lysates, and MS of mouse tissue.•Met-hydrophobic starting N-termini are most suitable for NtAc-dependent detection with this antibody.•The antibody may be used for various Met-starting proteins, to be validated with peptide.
(Hyper)tension release by N-terminal acetylation Aksnes, Henriette; Drazic, Adrian; Arnesen, Thomas
Trends in biochemical sciences (Amsterdam. Regular ed.),
08/2015, Letnik:
40, Številka:
8
Journal Article
Recenzirano
A recent study links N-terminal acetylation and N-end rule degradation to blood pressure regulation. N-terminal mutants of Rgs2, a key G-protein regulator, are differentially processed by N-terminal ...acetyltransferases and the two branches of the N-end rule pathway. This leads to an imbalance in the signaling governing blood pressure.
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•N-terminal (Nt) acetylation and Nt-arginylation are mutually exclusive PTMs.•State-of-the-art proteomics defines β-actin Nt-modifications in mammalian cells.•Acetylation is the ...dominant Nt-modification of β-actin.•β-actin is not Nt-arginylated in wildtype cells.•Actin Nt-maturation is a complex and still not fully understood process.
Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic β-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, β-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of β-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated β-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved β-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified β-actin (DDI-) as well as cleaved and Nt-acetylated β-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of β-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.
Most human proteins are N‐terminally acetylated by N‐terminal acetyltransferases (NATs), which play crucial roles in many cellular functions. The NatC complex, comprising the catalytic subunit NAA30 ...and the auxiliary subunits NAA35 and NAA38, is estimated to acetylate up to 20% of the human proteome in a co‐translational manner. Several NAT enzymes have been linked to rare genetic diseases, causing developmental delay, intellectual disability, and heart disease. Here, we report a de novo heterozygous NAA30 nonsense variant c.244C>T (p.Q82*) (NM_001011713.2), which was identified by whole exome sequencing in a 5‐year‐old boy presenting with global development delay, autism spectrum disorder, hypotonia, tracheal cleft, and recurrent respiratory infections. Biochemical studies were performed to assess the functional impact of the premature stop codon on NAA30's catalytic activity. We find that NAA30‐Q82* completely disrupts the N‐terminal acetyltransferase activity toward a classical NatC substrate using an in vitro acetylation assay. This finding corresponds with structural modeling showing that the truncated NAA30 variant lacks the entire GNAT domain, which is required for catalytic activity. This study suggests that defective NatC‐mediated N‐terminal acetylation can cause disease, thus expanding the spectrum of NAT variants linked to genetic disease.
The vast majority of eukaryotic proteins are subjected to N-terminal (Nt) acetylation. This reaction is catalyzed by a group of N-terminal acetyltransferases (NATs), which co- or post-translationally ...transfer an acetyl group from Acetyl coenzyme A to the protein N-terminus. Nt-acetylation plays an important role in many cellular processes, but the functional consequences of this widespread protein modification are still undefined for most proteins. Several in vitro acetylation assays have been developed to study the catalytic activity and substrate specificity of NATs or other acetyltransferases. These assays are valuable tools that can be used to define substrate specificities of yet uncharacterized NAT candidates, assess catalytic impairment of pathogenic NAT variants, and determine the potency of chemical inhibitors. The enzyme input in acetylation assays is typically acetyltransferases that have been recombinantly expressed and purified or immunoprecipitated proteins. In this chapter, we highlight how cell lysates can also be used to assess NAT catalytic activity and impairment when used as input in a previously described isotope-based in vitro Nt-acetylation assay. This is a fast and highly sensitive method that utilizes isotope labeled
C-Ac-CoA and scintillation to detect the formation of Nt-acetylated peptide products.
N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased ...formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization.
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