Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-α-acetyltransferase
© Arnesen et al; licensee BioMed Central Ltd. 2006
Received: 21 November 2005
Accepted: 25 April 2006
Published: 25 April 2006
Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing ε-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (h uman Ar rest D efective 1). hARD1 interacts with NATH (N-A cetyl T ransferase H uman) forming a complex expressing protein N-terminal α-acetylation activity.
We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-α-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable.
A human protein N-α-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-α-acetylation in human cells as compared to lower organisms which only have one ARD.
Protein acetylation is a very common modification with a significant impact on several cellular processes. Acetylation occurs both at lysine residues within proteins (Nε-acetylation) and at the N-terminus of proteins (Nα-acetylation). In yeast, N-acetyltransferase 1 (Nat1p) complexes with Arrest defective 1 (Ard1p) to generate a functional NatA protein Nα-acetyltransferase , Ard1p being the catalytic subunit. Proteins with Ser-, Thr-, Gly-, or Ala- N-termini are described to be substrates of NatA after methionine cleavage . The yeast NatB and NatC complexes acetylates different subsets of methionine N-termini [2–4]. Almost all known N-terminally acetylated yeast proteins are products of one of these Nat complexes. Protein N-terminal acetylation is generally believed to be a cotranslational process linked to the ribosome [6–10]. hARD1, the human protein with highest sequence similarity to yeast ARD1, has been described on the genomic (TE2, GenBank [NM_003491]) , mRNA , protein, and enzyme activity levels . Endogenous hARD1 was demonstrated to interact with NATH and express protein Nα-acetyltransferase activity. The complex was found to interact with ribosomal subunits supporting its function in cotranslational acetylation . In vitro translated mouse homologues, mNAT1 and mARD1, have also been shown to interact and express N-acetyltransferase activity . In S. cerevisiae and D. melanogaster, a third subunit of the NatA complex has been described and named Nat5p and San, respectively [8, 14]. The function of this subunit is unknown, but sequence analysis suggests that Nat5p/San is an acetyltransferase. The human orthologue, hNAT5, was also recently demonstrated to be a part of the human NatA complex .
Even though 80–90 % of all mammalian proteins and 50 % of yeast proteins are estimated to be cotranslationally Nα-acetylated [4, 16–20], only a few examples exist describing the functional importance of proper Nα-acetylation. For instance, the function of the yeast proteins Orc1p and Sir3p in telomeric silencing is dependent on proper NatA-mediated Nα-acetylation of these proteins [21, 22].
Using yeast null strains, NatA activity has been demonstrated to be associated with Go entry, cell growth, and the ability to sporulate [23–26]. The importance of protein Nα-acetylation has also been described in C. elegans, where knockdown of either the ard1 or nat1 homologues resulted in embryonal lethality . The human NatA complex has also recently been demonstrated to be essential for normal cellular viability. RNA interference mediated knockdown of NATH or hARD1 induced apoptosis in HeLa cells .
Mouse ARD1 was also reported to be implicated in the acetylation of lysine 532 of HIF-1α, contributing to its degradation in normoxia . However, several independent investigations have reported that at least the wildtype hARD1 protein does not mediate Nε-acetylation of the lysine residue 532 of HIF-1α [29–32].
The hARD1 gene is located on chromosome X (Xq28). Database searches revealed the presence on chromosome 4 (4q21.23) of a putative human paralogue of the previously published hARD1 gene (GeneID:84779, hypothetical protein [MGC10646]). We named this hypothetical human ARD, hARD2.
Here we describe the cloning and expression of hARD2. The entire ORF of hARD2 is intronless, resembling a gene duplicate. Many gene duplicates are non-functional pseudogenes but some, including hARD2, are active genes producing mRNAs and proteins [33–35]. Similar to hARD1, hARD2 interacts with NATH and expresses N-α-acetyltransferase activity.
hARD2 cloning and expression
Evolution of the hARD2 gene
Structural comparison between hARD1 and hARD2
Overexpressed hARD2 co-immunoprecipitate NATH and HIF-1α
The interaction between hARD1 and HIF-1α has been described . To investigate whether also hARD2 is capable of interacting with HIF-1α, we cotransfected MCF-7 cells with plasmids encoding Xpress-hARD2 and HA-HIF-1α. HA-HIF-1α will then accumulate under these normoxic conditions probably due to saturation of VHL-mediated degradation of HIF-1α. The resulting cell lysates were subjected to immunoprecipitation with the anti-Xpress antibody. Western blotting analysis using anti-HA demonstrated that a fraction of HA-HIF-1α co-immunoprecipitates with Xpress-hARD2 (Figure 5B).
Subcellular localization of hARD2
N-α-acetyltransferase activity of hARD2
hARD1 and hARD2 are differently regulated during NB4 cell differentiation
We here report on the presence of a human gene encoding a protein acetyltransferase at the mRNA, protein and enzyme activity levels. The ARD2 acetyltransferase has originated through a retrotransposition event and represents an additional ARD gene that can evolve independently from ARD1. Our results suggest that the duplication event is eutherian mammal specific. Amino acid sequence differences between hARD1 and hARD2 demonstrate that changes already have taken place, particularly in the C-terminal region of hARD2. As proposed for hARD1, hARD2 could have several different roles in the cell. The interaction with NATH indicates a role in the cytoplasm, possibly cotranslational N-acetylation. It is likely that hARD1 and hARD2 bind the same region within NATH and therefore compete for NATH binding. The difference in activity between hARD1 and hARD2 could suggest that the ACTH peptide used in this assay is not an optimal substrate for hARD2. It is not unlikely that the substrate preference is different between these two enzymes. A putative HIF-1α associated function is supported by the immunoprecipitation results demonstrating the potential interaction between hARD2 and HIF-1α (Figure 5B). Whether or not HIF-1α is a direct substrate for hARD2 mediated acetylation has not been investigated in this study and recent studies question the role of hARD1 in destabilizing HIF-1α [29–32]. Thus, the potential link between HIF-1α and hARD1/hARD2 remains an unresolved issue.
Unfortunately, the multiple sequence alignment outside of the acetyl transferase domain used for phylogenetic studies is not good enough for evolutionary studies involving calculation of substitution rates at synonymous and nonsynonymous sites or other likelihood ratio tests. Future sequencing from additional mammals should improve alignment quality to enable such studies. The ultimate question regarding differences in function between hARD1 and hARD2 is one of selective pressures and neofunctionalization vs. subfunctionalization (see for example, [44, 45]), which requires knowledge of the expression patterns and functions of the ancestral state. The metatherian, frog, and fish sequences, which are currently available, can serve as proxies for the ancestral state as they appear not to have duplicate ARD copies. In an attempt to address the function of zebrafish ARD, an antibody to hARD1 was directed against zebrafish tissue, but no binding was detected, probably due to sequence divergence (data not shown). Future work in zebrafish or other non-eutherian mammal species closer to human can address the function of the ancestral state and the relative selective pressures on hARD1 and hARD2.
There are several indications that hARD1 and NATH may be linked to differentiation. As mentioned, mNAT1 and mARD1 mRNAs are downregulated during neuronal differentiation in mouse . In the present study, we show that hARD1 and NATH proteins were both downregulated after induction of differentiation in promyelocytic leukaemia cells. This is the first description of NAT-ARD1 downregulation induced by differentiation in human cells and also the first verification of downregulation at the protein level. Interestingly, the hARD1 gene was one of twelve genes identified to be elevated in dedifferentiated hepatocellular carcinomas . It should also be noted that the yeast ARD1 gene originally was implicated in controlling the switch between the mitotic cell cycle and developmental pathways . Whether or not hARD1 and NATH influences differentiation per se, awaits further studies, but our present results add support to a link between the NATH-hARD1 complex and differentiation. The lack of hARD2 downregulation during granulocytic differentiation could suggest that a specific subset of proteins then is acetylated at a stable level. In contrast, the hARD1 specific substrates would be less acetylated during differentiation. This balance between acetylation of different subsets of proteins could have an impact on the differentiation process itself or alternatively facilitate cellular adaptations associated with the process.
In summary, we have identified and characterized a human protein N-α-acetyltransferase which probably originates from a recent gene duplication event. The hARD2 protein displays similar properties as hARD1 in terms of subcellular localization and potential interactions with NATH and HIF-1α. Further expression and enzymatic studies are required to assess the overall functional contribution of hARD2 in cellular processes.
hARD2 cloning and expression
NCBI BLAST database was employed in the search of human homologues of yeast ARD1. The second best hit after hARD1 was the protein MGC10646, termed hARD2. Primers pr1 and pr3 were designed to clone the hARD2 gene and to detect the presence of hARD2 mRNA. Plasmid encoding V5- and Xpress-tagged hARD2 was constructed from a gene-specific PCR of cDNA made from total RNA isolated from human ARO cells. PCR product was inserted into the TOPO TA vectors pcDNA3.1/V5His and pcDNA 4/HisMax (Invitrogen). Simultaneously a vector encoding wildtype hARD2 was made using a reverse primer including the hARD2 stop codon. cDNA was made as previously described . The primers for amplifying the hARD2 gene (Figure 1) were as follows: pr1 (hARD2 forward), 5'-ATG AAC ATC CGC AAC GCT CAG-3'; pr3 (hARD2 rev), 5'- GGA GGT GGA ATC CGA GCT TTC-3'; pr2 (hARD2 545forward), 5'-CAG CAC ACT TTC TGA TTC TGA AG-3'; pr4 (hARD2 926reverse), 5'-GTA ATG GCA GGT CTC AAA GTTC-3'. β-Actin primers: Actin-F, 5'- GGC ACC ACA CCT TCT ACA 3'; Actin-R, 5'- AGG AAG GCT GGA AGA GTG 3'. Primers for amplifying genomic M. giganteus ARD: For: 5'-gtg aar cgy tcn cac cgg cgc cty ggy ctg-3'; Rev: 5'-ctc ttc ctg rcr tgy agr gas acr tay ttk gc-3'. Primers for amplifying M. giganteus ARD from cDNA: kARD1/2 ORFf, 5'-ATG AAC ATC CGC AAY GCK MRG CCA GAS GAC C; kARD1/2 ORFr, 5'-CTA GGA GGY KGA RTC SGA GSY YTC TGA GCT GTC C (r = a + g, y = c + t, n = a + c + t + g, s = c + g, k = g + t).
Cell culture and transfection
Cells were cultured at 37°C, 5% CO2 in DMEM (HEK293, embryonal kidney, ATCC CRL-1573 and HeLa, epithelial cervix adenocarcinoma, ATCC CCL-2), RPMI 1640 (NB4, acute promyelocytic leukemia, DSMZ ACC 207 and MCF-7, epithelial mammary gland, breast adenocarcinoma, ATCC HTB-22) or EMEM (SK-MEL2, malignant melanoma, ATCC HTB-68) supplemented with 10% FBS and 3% l-glutamine. Transfections were performed using Fugene6 (Roche) according to the instruction manual. The plasmid pHA-HIF-1α has been described . A hARD2 specific rabbit antibody was generated by Biogenes GmbH using a peptide corresponding to amino acids 192–206 of hARD2. Western blotting was performed as described . Dilutions: anti-hARD2 1:500; anti-hARD1  1:500; anti-NATH  1:500; anti-V5 (Invitrogen) 1:1000; anti-β-tubulin (Sigma) 1:1000; anti-HA (Sigma) 1:1000.
Immunofluorescence and immunoprecipitation
HeLa cells were transfected using Fugene6 and grown 24 h on coverslips. Then cells were prepared for immunofluorescence as described . HEK293 or MCF-7 cells (~2 × 106) were transfected using Fugene6 and incubated 48 hours before harvesting and lysis in 300 μl lysis buffer. Immunoprecipitation was performed as previously described .
Immunoprecipitation of X-press-lacZ, X-press-hARD1 or X-press-hARD2 was performed as described above. Pellets of Protein A/G-Agarose bound X-press-hARD2 was added 10 μl ACTH (0.5 mM, human corticotropin fragment 1–24, Calbiochem), 4 μl [3H]Acetyl-CoA (1 μCi, 107 GBq/mmol, Amersham Biosciences) and 136 μl 0.2 M K2HPO4 (pH 8.1). The mixture was incubated for 2 hours at 37°C. After centrifugation the supernatant was added to 150 μl SP Sepharose (50% slurry in 0.5 M Acetic acid, Sigma) and incubated on a rotor for 5 min. The mixture was centrifuged and the pellet was washed three times with 0.5 M acetic acid and finally with methanol. Radioactivity in the ACTH containing pellet was determined by scintillation counting.
Alignment and tree building
Peptide sequence alignments were made using T-Coffee  with the default settings. Coding sequence alignments were produced by aligning the coding sequences with reference to the alignment of the corresponding peptide sequences. The phylogenetic tree from the initial peptide sequence alignment was built using MrBayes  with the Jones matrix of substitution run on four MCMC chains for 750000 generations, to generate a majority rule consensus tree of all compatible partitions of the final 500000 generations (sampled rate 100). MrBayes was also used to produce the coding sequence alignment based tree, with the same settings as before except for a nucleotide model of substitution allowing different rates for transitions and transversions and a gamma distribution of rates across sites. Gene trees were rooted by mapping them on to the NCBI tree of life whilst minimizing gene duplication and loss events and allowing poorly supported branches (those with posterior probabilities less than 0.7) to be rearranged according to the NCBI taxonomy as a reference species tree (Berglund, Steffansson, Betts and Liberles, Manuscript submitted).
hypoxia inducible factor-1α
polymerase chain reaction.
We thank C. Baldersheim, K. Jacobsen and C. Hoff for technical assistance and S. Knappskog for advice. R. McKinnon, A. El-Merhibi, G.H. Robstad, Kristiansand Zoo and C. Krakstad are thanked for providing kangaroo and rodent specimens. This work was supported by The Norwegian Cancer Society (Grants to TA, JEV, JRL), The Locus of Experimental Cancer Research (University of Bergen), The Meltzer Foundation (Grant to TA), FUGE and the International Agency for Research on Cancer (grant to FP).
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