Characterization of the human Nα-terminal acetyltransferase B enzymatic complex
© Ametzazurra et al; licensee BioMed Central Ltd. 2009
Published: 4 August 2009
Skip to main content
© Ametzazurra et al; licensee BioMed Central Ltd. 2009
Published: 4 August 2009
Human Nα-acetyltransferase complex B (hNatB) is integrated by hNaa20p (hNAT5/hNAT3) and hNaa25p (hMDM20) proteins. Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells.
We have silenced hNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB.
hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast.
hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins.
Protein modification is a mechanism for regulate and improve proteins function and activity, being Nα-terminal acetylation one of the most abundant activities. Although the two cotranslational events, cleavage of the initiator methionine and Nα-acetylation, are common and highly conserved from bacteria to higher eukaryotes, their function it is not well understood [1, 2]. Obviously, the removal of the initiator residue allows diversity of aminoterminal sequences and therefore enhances the functional repertoire of the polypeptide.
Nα-acetyltransferase complexes are composed by one catalytic subunit and one or several auxiliary subunits. NatB enzymatic complex is integrated by the catalytic subunit Naa20p and the auxiliary subunit Naa25p, as has been observed in yeast and human cells [3–5].
In yeast, proteins with Met-Asp-, Met-Glu-, Met-Asn- and Met-Met- amino termini constitute potential NatB substrates. However, whereas Met-Asp- and Met-Glu- termini appear to be acetylated in 100% of the cases, only some of the experimentally investigated Met-Asn- and Met-Met- termini have been found to constitute true NatB targets . In the meantime all mammals proteins with Met-Asn- and Met-Met- termini analyzed present the methionine acetylated and consequently they are considered as NatB substrates .
Despite the widespread occurrence of Nα-terminal acetylation in eukaryotes, the biological relevance of this protein modification has only been deduced for a few substrates, as the tropomyosin and actin. It has been documented that yeast tropomyosin-1 and actin are NatB-mediated acetylated being this modification important for the formation of stable and functional actin cables [3, 4]. Tropomyosin activity is also regulated by N-terminal acetylation in fission yeast  and N-terminal acetylation of Dictiostelum actin strengthens interaction of actin and myosin . In addition, actin aminoterminal processing is ligated to the acetylation of the initial methionine as the removal of the methionine in class I and class II actins and of the cysteine in class II actins that occurs in an acetylation-dependent manner [9, 10]. Hence, a proper N-terminal processing is very important for regulating actin function .
Human NatB complex has been recently characterized showing that it is composed at least by the catalytic subunit hNaa20p and the auxiliary subunit hNaa25p . The two main aminoterminal acetyltransferase activities, NatA and NatB, are important for human cell cycle progression [5, 12], but unlike NatA, there is not an induction of apoptosis when NatB is inhibited [5, 12, 13].
We have extended the analysis of NatB function to two human hepatocarcinoma cell lines concluding its implication in cellular proliferation. We have also studied in vitro hNatB activity identifying some new in vitro substrates and establishing that not all the peptides with Met-Asp- and Met-Glu- amino termini are good in vitro hNatB substrates.
hNAA20-CTAP expression plasmid was generated excising hNAA20 from the pcDNA3-TOPO-TA-hNAA20 plasmid and inserting it in the plasmid pCTAP (Stratagene, CA, USA). The plasmid pDEST27-NAA25 was obtained from Imagenes (Berlin, Germany).
hNAA20 expression in HepG2 and Hep3B cell lines has been inhibited using recombinant adenoviruses that express specific siRNAs. These sequences and adenovirus production have been described previously .
Human Hela, HepG2, Hep3B and 293 cell lines were purchased from the ATCC and 293 Cre4 cell line was provided by Dr Hardy. All the cell lines were cultured in DMEM supplemented with 10% foetal calf serum. Cre4 cells were also supplemented with 500 μg/ml G418. All reagents were from Gibco-BRL (Paisley, UK).
Infections with hNAA20 siRNA expressing adenoviruses were performed in HepG2 and Hep3B cells. The day prior to infection 250,000 HepG2 and 150,000 Hep3B cells were plated in a 6 well plate and infection was performed in DMEM-2% foetal calf serum at a multiplicity of infection (MOI) of 10.
To extract protein from cultured cells, the cells were collected in 100 μl of RIPA solution (150 mM NaCl, 50 mM Tris pH 7.5, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM Na3VO4 and a protease inhibitor cocktail from Roche). Protein extracts were collected after sonication and centrifugation at 16,000 g for 15 minutes, and supernatants were stored at -80°C.
Cultured cells protein extracts (20 μg) were loaded in SDS polyacrylamide gels and after electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) and detected by incubation with specific antibodies. Protein bands were visualised using the enhanced chemiluminescence detection system (Perkin Elmer, Boston, Massachusetts, USA) and membranes were autoradiographed.
Antibodies used were: chicken anti-hNAA20 from Genway Biotech (California, USA), goat anti-GST from Abcam (Cambridge, UK), mouse anti-GAPDH from Biogenesis (Bournemouth, UK), rabbit anti-Bcl-2, mouse anti-Mdm2, mouse anti-p53 (DO-1), goat anti-p21(WAF/CIP1)(WAF1/CIP1) from Santa Cruz Biotechnology (Santa Cruz, California, USA), anti-rabbit IgG HRPO from Cell Signalling (Beverly, Massachusetts, USA), anti-goat IgG HRPO and anti-mouse IgG HRPO from Sigma, rabbit anti-IgY from Upstate and rabbit anti-IgY HRPO from Pierce (Illinois, USA).
GST-hNAA25 and hNAA20-CTAP expressing plasmids were transfected in Hela cells using linear polyethylenimine 25 kDa (Polysciences, Warrington, PA, USA) as described previously . 48 hours after transfection the samples were harvested in RIPA buffer and incubated with anti-Naa20p or anti-GST antibodies to immunoprecipitate the protein complexes.
The NAT assay was performed basically as described previously [15, 16] but using hNaa20p or GST-hNaa25p immunoprecipitated complexes that were incubated with 138 μl of 0.2 M K2HPO4 (pH 8.1), 10 μl of the substrate peptide (0.5 mM) and 1 μCi of [3H]acetyl-CoA (99.9 GBq/mmol, GE Healthcare) for 2 hours at 37°C. The samples were centrifuged and the supernatant was incubated with SP-Sepharose (50% in 0.5 M acetic acid) for 10 minutes on a rotor before washing the SP-Sepharose three times with acetic acid 0.5 M and once with methanol. The radioactivity incorporated in the peptides was determined by scintillation counting.
HepG2 and Hep3B cells were seeded in 15 mm cover glasses and infected with siRNA expressing adenovirus pAdsiRNA2 or pAdsiRNA350 for 24, 48 and 72 hours, BrdU labeled for 1 hour and processed using the 5-bromo-2'-deoxyuridine Labeling and Detection Kit I (Roche Applied Science, Penzberg, Germany) following the manufacturer's instructions.
HepG2 and Hep3B cells were harvested after 72 hours of infection with the adenovirus pAdsiRNA2 or pAdsiRNA350 and processed using the CycleTEST PLUS DNA reagent kit (Becton Dickinson) following the manufacturer's instructions. Flow cytometric analysis of the different samples was performed using BD FACScalibur flow cytometer and DNA content was analyzed using the MODFIT software.
HepG2 and Hep3B cells were incubated in 15 mm cover glasses and infected with adenovirus pAdsiRNA2 or pAdsiRNA350 for 48 hours prior adding the proteasome inhibitor MG132 (5 μM) (Calbiochem, Darmstadt, Germany) for an additional 8 hours. Cells were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4). TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer's instructions. Cell apoptosis percentage was obtained as indicated in cell proliferation assay.
Thus hNatB inhibition promotes a reduction in cellular proliferation in all cell lines tested, according to the effects of hNatA inhibition [13, 17]. It seems therefore that N-terminal acetylation of proteins is an important function for a proper cell growth and division as has been described for protein lysine acetylation/deacetylation .
In many instances inhibition of cellular proliferation is associated with an increase of apoptosis . Inhibition of NatB activity in Hela cells sensitizes the cells to a proapoptotic stimulus, like the proteasome inhibitor MG132 , but it is not associated with an increase of cellular apoptosis. We observed that hNAA20 knockdown in HepG2 and Hep3B is not associated with apoptosis induction (data not shown ) but it sensitizes the cells to the proapoptotic treatment with MG132 as it is presented in Figure 2B where there is a clear induction of apoptosis in HepG2 and Hep3B cells that express hNAA20 siRNA, siRNA2. This effect correlates in HepG2 with a reduction of BCL2 (Figure 2A), which is an important antiapoptotic molecule .
We have observed also that Hep3B cell line is more sensitive to MG132 treatment as Hep3B cells that express the unrelated siRNA present some sensitivity to MG132. This is Hep3B specific because a prolonged exposure of the cells to this proapoptotic stimulus induces an apoptotic cell death that it is not observed in Hela and HepG2 cells (data not shown). These findings are also reminiscent of the increased sensitivity to environmental stress when yeast strains were deleted of yNatB enzymatic complex (naa20-Δ, naa25-Δ) [3, 4, 6].
Identification of hNatB in vitro substrates.
Characterization of hNatB in vitroacetylating sequences.
Actin N-terminal processing is very important for a proper actin function  being N-terminal acetylation a significant step in this procedure [9, 10]. Therefore we extended the analysis to the human β-actin aminoterminal peptide with the same substitutions as the tropomyosin peptide. Surprisingly, all the mutated β-actin peptides were as good substrates as the original sequence (Table 2). Consequently, in some cases there are other amino acids besides the first two aminoterminal that dictate the competence of a peptide as hNatB substrate.
In conclusion, we have determined that hNatB enzymatic complex is necessary for a proper cell cycle progression and resistance to proapoptotic stimuli in hepatic cell lines as has been observed before in other cell types. hNaa20p downregulation induces cell growth arrest in a p53 dependent and independent manner. hNatB acetylates in vitro most of the peptides with Met-Asp- or Met-Glu- amino termini, being more important the aspartic or glutamic acid than the initial methionine for a proper acetylation.
14-3-3 protein epsilon
Apoptosis regulator BAX, membrane isoform alpha
Breast cancer type 1 susceptibility protein
Cell division protein kinase 3
Cell division protein kinase 8
Alpha crystallin B chain
FADD protein (FAS-associating death domain-containing protein)
40S ribosomal protein S28
Troponin C, slow skeletal and cardiac muscles (TN-C)
N-acetyltransferase 2 (arylamine N-acetyltransferase)
Adenylate kinase isoenzyme 1
Band 3 anion transport protein
Tyrosine-protein phosphatase non-receptor type 1
We thank Dr Francisco Borrás-Cuesta and Virginia Belsué for peptides synthesis and Sandra Jusue and Beatriz Carte for excellent technical assistance. AA was supported by a FPU training fellowship (AP2001-1567) and RA was supported by a FIS research contract.
CIBEREHD is funded by Instituto de Salud Carlos III and this research was partially supported by grants from Ministerio de Sanidad y Consumo (02/3054), Departamento de Educación, Gobierno de Navarra (GNE-Enzima N) and UTE project CIMA.
This article has been published as part of BMC Proceedings Volume 3 Supplement 6, 2009: Proceedings of the 2007 and 2008 Symposia on Protein N-terminal Acetylation. The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/3?issue=S6
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.