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.