BACKGROUND
protein acetylation is a very common modification with a significant impact on several cellular processes.
acetylation occurs both at lysine residues within proteins  and at the n-terminus of proteins .
in yeast, n-acetyltransferase  <dig>  complexes with arrest defective  <dig>  to generate a functional nata protein nα-acetyltransferase  <cit> , ard1p being the catalytic subunit.
proteins with ser-, thr-, gly-, or ala- n-termini are described to be substrates of nata after methionine cleavage  <cit> .
the yeast natb and natc complexes acetylates different subsets of methionine n-termini  <cit> .
almost all known n-terminally acetylated yeast proteins are products of one of these nat complexes <cit> .
protein n-terminal acetylation is generally believed to be a cotranslational process linked to the ribosome  <cit> .
hard <dig>  the human protein with highest sequence similarity to yeast ard <dig>  has been described on the genomic   <cit> , mrna  <cit> , protein, and enzyme activity levels  <cit> .
endogenous hard <dig> 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  <cit> .
in vitro translated mouse homologues, mnat <dig> and mard <dig>  have also been shown to interact and express n-acetyltransferase activity  <cit> .
in s. cerevisiae and d. melanogaster, a third subunit of the nata complex has been described and named nat5p and san, respectively  <cit> .
the function of this subunit is unknown, but sequence analysis suggests that nat5p/san is an acetyltransferase.
the human orthologue, hnat <dig>  was also recently demonstrated to be a part of the human nata complex  <cit> .
even though 80– <dig> % of all mammalian proteins and  <dig> % of yeast proteins are estimated to be cotranslationally nα-acetylated  <cit> , 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  <cit> .
using yeast null strains, nata activity has been demonstrated to be associated with go entry, cell growth, and the ability to sporulate  <cit> .
the importance of protein nα-acetylation has also been described in c. elegans, where knockdown of either the ard <dig> or nat <dig> homologues resulted in embryonal lethality  <cit> .
the human nata complex has also recently been demonstrated to be essential for normal cellular viability.
rna interference mediated knockdown of nath or hard <dig> induced apoptosis in hela cells  <cit> .
mouse ard <dig> was also reported to be implicated in the acetylation of lysine  <dig> of hif-1α, contributing to its degradation in normoxia  <cit> .
however, several independent investigations have reported that at least the wildtype hard <dig> protein does not mediate nε-acetylation of the lysine residue  <dig> of hif-1α  <cit> .
the hard <dig> gene is located on chromosome x .
database searches revealed the presence on chromosome  <dig>  of a putative human paralogue of the previously published hard <dig> gene .
we named this hypothetical human ard, hard <dig>
here we describe the cloning and expression of hard <dig>  the entire orf of hard <dig> is intronless, resembling a gene duplicate.
many gene duplicates are non-functional pseudogenes but some, including hard <dig>  are active genes producing mrnas and proteins  <cit> .
similar to hard <dig>  hard <dig> interacts with nath and expresses n-α-acetyltransferase activity.
RESULTS
hard <dig> cloning and expression
analysis of the genome sequence of hard <dig> suggests that the complete open reading frame is located within only one exon.
this is supported by the sequences of cdnas  .
one intron  and a non-coding second exon was predicted at the 3'-end of the gene.
the intron borders are defined by the common "gt-ag" consensus   <cit> .
using rt-pcr, human cdna and primers covering the predicted orf, we cloned hard <dig>  and demonstrated hard <dig> mrna expression in different cell lines .
to verify the presence of spliced hard <dig> mrna we used primers flanking the intron.
rt-pcr product of the expected size was detected  and dna sequencing verified its authenticity as a spliced hard <dig> mrna.
there has also been reported an hard <dig> cdna sequence covering another exon, 5' to the exon  <dig> indicated in figure 1a .
this cdna encodes a protein with extra amino acid residues in the n-terminal domain of the protein, while the reading frame is intact.
however, we were not able to verify the existence of such a transcript .
we then demonstrated endogenous hard <dig> protein expression using an antibody specific for amino acids 192– <dig> within hard <dig>  a region specific to hard <dig> as compared to hard <dig>  to confirm the specificity of the antibody, xpress-hard <dig> and xpress-hard <dig> was expressed in hek <dig> cells.
the resulting cell lysates were analyzed by sds-page and western blotting.
anti-hard <dig> only detected x-press-hard <dig> and anti-hard <dig> only detected x-press-hard <dig>  while anti-xpress detected both proteins as expected .
then, hek <dig> cells were transfected with a plasmid encoding wildtype hard <dig> and the resulting western blot displayed a strong band at the expected size of hard <dig>  in the untransfected control  there was a weaker band at the same position strongly indicating the presence of an endogenous hard <dig> .
endogenous hard <dig> protein was detected in several human cell lines indicating that it is commonly expressed in human cell cultures .
particularly high expression was detected in the mcf- <dig> breast carcinoma cell line.
the expression of hard <dig> in the same cell lines indicates that hard <dig> and hard <dig> are simultaneously present in the cells .
evolution of the hard <dig> gene
the hard <dig> and hard <dig> genbank identifiers were used to identify the relevant protein family in taed  <cit> .
the protein sequences of all members of this family were blasted against all ensembl peptides , identifying them as members of ensembl family 'n terminal acetyltransferase complex ard <dig> subunit homolog ec_ <dig> .1'.
a non-redundant set of the union of the taed and ensembl families was produced,-a multiple alignment of the peptide sequences was calculated , and an initial phylogenetic tree was produced.
using this tree and the synteny information available through ensembl suggested that ard <dig> is a mammalian specific duplication.
to confirm this we searched for expression of the ensembl rat and mouse ard <dig> genes .
rt-pcr experiments of rat and mouse cdna using primers flanking each gene confirmed the expression of the mouse gene ensmusg <dig> , which is in a region of synteny with hard <dig>  and the rat gene ensrnog <dig>  which is not.
all the ard2s have single exon open reading frames, indicating that they might have arisen through a mammalian specific retrotransposition event.
furthermore, we used cdna and genomic dna from kangaroo  to check for the presence of ard gene and in an attempt to pinpoint the time at which the duplication occurred.
degenerate primers made from human hard <dig> and hard <dig> flanking the orf and one exon were used to amplify and topo-ta clone kangaroo ard from cdna and genomic dna, respectively.
blasting hard <dig> and hard <dig> against the opossum genome predicted peptides available from ensembl showed only one ard gene, with a gene structure the same as hard <dig>  the kangaroo and opossum ards and the ensembl putative dog ard <dig> and ard <dig> were aligned with the original set of protein sequences , a coding sequence alignment was produced from this , the best aligned section was extracted  , and from this a new tree was generated  all kangaroo sequences clustered together in one clade, with the opossum sequence as an outgroup.
the tree therefore suggests the presence of only one kangaroo ard gene .
this tree was rooted to give the final tree , which shows that the speciation of kangaroo and opossum precedes the gene duplication that results in ard <dig>  thus, ard <dig> seems not to be present in kangaroo and opossum and thus the emergence of ard <dig> probably represents a eutherian mammal specific retrotransposition event.
also, kangaroo muscle tissue was lysed and analysed by sds-page and western blotting using anti-hard <dig> and anti-hard <dig> .
a kangaroo protein of the expected size was observed when using anti-ard <dig> but not when using anti-hard <dig>  the opossum ard sequence is identical to hard <dig> in the region recognized by anti-hard <dig>  however, the posterior probability supporting the split separating metatherians from the eutherian ard1/ard <dig> divergence is only  <dig> .
further, a band of  <dig> nucleotides was observed by pcr of genomic kangaroo material, although this kangaroo ard band did cluster as a metatherian sequence, rather than with the ard <dig> clade .
altogether, this provides some level of support for a eutherian origin of ard <dig>  knowledge of the kangaroo gene structure and full gene sequence will improve the confidence level of this conclusion.
structural comparison between hard <dig> and hard2
alignment of the mammalian ard <dig> and ard2s and identification of the location of their n-acetyltransferase domains , shows only conservative substitutions from ard <dig> to ard <dig>  except for an alanine to proline substitution at aa <dig> in human ard <dig> .
as mentioned above, the hard <dig> sequence is highly similar to hard <dig> in the first ~ <dig> amino acids.
the alanine amino acid residue at position  <dig> within the predicted acetyltransferase domain of hard <dig> <cit>  is substituted by a proline in hard <dig>  however, structure prediction analysis indicates that this residue is a part of a loop in both hard <dig> and hard <dig> .
this prediction was made by selecting a protein structure that also matches to pfam domain pf <dig> , aligning it and hard <dig> and hard <dig> to the pfam hidden markov model using hmmer  <cit> , running swissmodel  <cit>  and making a molscript figure  <cit> .
importantly, this proline residue is not conserved within the mammalian ard2s .
the difference in the c-terminal region may also account for changes in the enzymatic activity.
several serine and threonine amino acid residues have changed between the two proteins and this could result in a change in the regulation of the enzymes by kinases.
prediction of the disorder and globularity  <cit>  of hard <dig> and hard <dig> indicates that the c-terminal part of these two proteins to some extent diverge .
overexpressed hard <dig> co-immunoprecipitate nath and hif-1α
the nath-hard <dig> complex constitutes a functional protein n-acetyltransferase in human cells  <cit> .
to study whether or not a complex of nath and hard <dig> could exist, we performed immunoprecipitation experiments.
extract of hek <dig> cells transfected with a plasmid encoding v5-tagged hard <dig> was made and used for immunoprecipitation with anti-v <dig>  western blotting analysis of the immunoprecipitates revealed that a minor fraction of nath interacts with hard2-v <dig> .
the interaction with nath is to be expected since the removal of the c-terminal  <dig> amino acids of hard <dig> did not abolish nath binding  <cit>  and the remaining n-terminal  <dig> amino acids of the hard <dig> sequence is as mentioned above highly similar to hard <dig> .
the interaction between hard <dig> and hif-1α has been described  <cit> .
to investigate whether also hard <dig> is capable of interacting with hif-1α, we cotransfected mcf- <dig> cells with plasmids encoding xpress-hard <dig> 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-hard <dig> .
subcellular localization of hard2
subcellular localization of hard <dig> was studied by expressing v5-tagged hard <dig> in hela cells followed by immunofluorescence staining .
similarly to hard <dig>  <cit> , hard2-v <dig> was present both in the cytoplasm and in the nucleus, but the majority of the protein appeared to be located in the cytoplasm.
thus, hard2-nath complexes may potentially have a function in the cytoplasm.
n-α-acetyltransferase activity of hard2
to investigate whether or not hard <dig> is a functionally active protein n-acetyltransferase, xpress-hard <dig> was immunoprecipitated from hek <dig> cells and the n-acetyltransferase activity of the xpress-hard <dig> was determined and compared to a negative control using xpress-lacz and a positive control using xpress-hard <dig> .
we found that hard <dig> expresses n-acetyltransferase activity, demonstrated by the acetylation of the n-terminus of corticotropin .
the activity data was normalized using the quantified protein amount in the agarose beads analyzed by western blotting .
the radioactivity of the xpress-lacz samples were defined as background and subtracted from the xpress-hard1/hard <dig> values.
the activity of the xpress-hard <dig> in three independent experiments gave a mean value of  <dig> % relative to xpress-hard <dig>  these results demonstrate that hard <dig> expresses n-acetyltransferase activity, but also suggest that its specific activity is lower than that of hard <dig> under the assay conditions used.
hard <dig> and hard <dig> are differently regulated during nb <dig> cell differentiation
it has been demonstrated that mard <dig> and mnat <dig> mrnas are downregulated during neuronal differentiation of p <dig> cells  <cit> .
since this suggests a role of these proteins in differentiation, we wanted to investigate the endogenous protein expression of human homologues hard <dig>  hard <dig> and nath during differentiation.
for this purpose we used retinoic acid induced differentiation of the promyelocytic nb <dig> cell line.
protein levels of hard <dig> and nath significantly decreased during differentiation in nb <dig> cells .
this correlates well with the findings in mouse neuronal cells.
however, the levels hard <dig> protein is not significantly altered under these conditions .
discussion
we here report on the presence of a human gene encoding a protein acetyltransferase at the mrna, protein and enzyme activity levels.
the ard <dig> acetyltransferase has originated through a retrotransposition event and represents an additional ard gene that can evolve independently from ard <dig>  our results suggest that the duplication event is eutherian mammal specific.
amino acid sequence differences between hard <dig> and hard <dig> demonstrate that changes already have taken place, particularly in the c-terminal region of hard <dig>  as proposed for hard <dig>  hard <dig> 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 hard <dig> and hard <dig> bind the same region within nath and therefore compete for nath binding.
the difference in activity between hard <dig> and hard <dig> could suggest that the acth peptide used in this assay is not an optimal substrate for hard <dig>  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 hard <dig> and hif-1α .
whether or not hif-1α is a direct substrate for hard <dig> mediated acetylation has not been investigated in this study and recent studies question the role of hard <dig> in destabilizing hif-1α  <cit> .
thus, the potential link between hif-1α and hard1/hard <dig> 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 hard <dig> and hard <dig> is one of selective pressures and neofunctionalization vs. subfunctionalization , 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 hard <dig> was directed against zebrafish tissue, but no binding was detected, probably due to sequence divergence .
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 hard <dig> and hard <dig>
there are several indications that hard <dig> and nath may be linked to differentiation.
as mentioned, mnat <dig> and mard <dig> mrnas are downregulated during neuronal differentiation in mouse  <cit> .
in the present study, we show that hard <dig> and nath proteins were both downregulated after induction of differentiation in promyelocytic leukaemia cells.
this is the first description of nat-ard <dig> downregulation induced by differentiation in human cells and also the first verification of downregulation at the protein level.
interestingly, the hard <dig> gene was one of twelve genes identified to be elevated in dedifferentiated hepatocellular carcinomas  <cit> .
it should also be noted that the yeast ard <dig> gene originally was implicated in controlling the switch between the mitotic cell cycle and developmental pathways  <cit> .
whether or not hard <dig> and nath influences differentiation per se, awaits further studies, but our present results add support to a link between the nath-hard <dig> complex and differentiation.
the lack of hard <dig> downregulation during granulocytic differentiation could suggest that a specific subset of proteins then is acetylated at a stable level.
in contrast, the hard <dig> 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.
CONCLUSIONS
in summary, we have identified and characterized a human protein n-α-acetyltransferase which probably originates from a recent gene duplication event.
the hard <dig> protein displays similar properties as hard <dig> 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 hard <dig> in cellular processes.

 protein acetylation is a very common modification with a significant impact on several cellular processes. acetylation occurs both at lysine residues within proteins and at the n-terminus of proteins. in yeast, n-acetyltransferase complexes with arrest defective 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. 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. hard the human protein with highest sequence similarity to yeast ard has been described on the genomic, mrna, protein, and enzyme activity levels. endogenous hard 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, mnat and mard 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. the function of this subunit is unknown, but sequence analysis suggests that nat5p/san is an acetyltransferase. the human orthologue, hnat was also recently demonstrated to be a part of the human nata complex. even though 80– % of all mammalian proteins and % of yeast proteins are estimated to be cotranslationally nα-acetylated, 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. using yeast null strains, nata activity has been demonstrated to be associated with go entry, cell growth, and the ability to sporulate. the importance of protein nα-acetylation has also been described in c. elegans, where knockdown of either the ard or nat 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 hard induced apoptosis in hela cells. mouse ard was also reported to be implicated in the acetylation of lysine of hif-1α, contributing to its degradation in normoxia. however, several independent investigations have reported that at least the wildtype hard protein does not mediate nε-acetylation of the lysine residue of hif-1α. the hard gene is located on chromosome x. database searches revealed the presence on chromosome of a putative human paralogue of the previously published hard gene. we named this hypothetical human ard, hard here we describe the cloning and expression of hard the entire orf of hard is intronless, resembling a gene duplicate. many gene duplicates are non-functional pseudogenes but some, including hard are active genes producing mrnas and proteins. similar to hard hard interacts with nath and expresses n-α-acetyltransferase activity.  hard cloning and expression analysis of the genome sequence of hard suggests that the complete open reading frame is located within only one exon. this is supported by the sequences of cdnas. one intron and a non-coding second exon was predicted at the 3'-end of the gene. the intron borders are defined by the common "gt-ag" consensus. using rt-pcr, human cdna and primers covering the predicted orf, we cloned hard and demonstrated hard mrna expression in different cell lines. to verify the presence of spliced hard mrna we used primers flanking the intron. rt-pcr product of the expected size was detected and dna sequencing verified its authenticity as a spliced hard mrna. there has also been reported an hard cdna sequence covering another exon, 5' to the exon indicated in figure 1a. this cdna encodes a protein with extra amino acid residues in the n-terminal domain of the protein, while the reading frame is intact. however, we were not able to verify the existence of such a transcript. we then demonstrated endogenous hard protein expression using an antibody specific for amino acids 192– within hard a region specific to hard as compared to hard to confirm the specificity of the antibody, xpress-hard and xpress-hard was expressed in hek cells. the resulting cell lysates were analyzed by sds-page and western blotting. anti-hard only detected x-press-hard and anti-hard only detected x-press-hard while anti-xpress detected both proteins as expected. then, hek cells were transfected with a plasmid encoding wildtype hard and the resulting western blot displayed a strong band at the expected size of hard in the untransfected control there was a weaker band at the same position strongly indicating the presence of an endogenous hard. endogenous hard protein was detected in several human cell lines indicating that it is commonly expressed in human cell cultures. particularly high expression was detected in the mcf- breast carcinoma cell line. the expression of hard in the same cell lines indicates that hard and hard are simultaneously present in the cells. evolution of the hard gene the hard and hard genbank identifiers were used to identify the relevant protein family in taed. the protein sequences of all members of this family were blasted against all ensembl peptides, identifying them as members of ensembl family'n terminal acetyltransferase complex ard subunit homolog ec_.1'. a non-redundant set of the union of the taed and ensembl families was produced,-a multiple alignment of the peptide sequences was calculated, and an initial phylogenetic tree was produced. using this tree and the synteny information available through ensembl suggested that ard is a mammalian specific duplication. to confirm this we searched for expression of the ensembl rat and mouse ard genes. rt-pcr experiments of rat and mouse cdna using primers flanking each gene confirmed the expression of the mouse gene ensmusg, which is in a region of synteny with hard and the rat gene ensrnog which is not. all the ard2s have single exon open reading frames, indicating that they might have arisen through a mammalian specific retrotransposition event. furthermore, we used cdna and genomic dna from kangaroo to check for the presence of ard gene and in an attempt to pinpoint the time at which the duplication occurred. degenerate primers made from human hard and hard flanking the orf and one exon were used to amplify and topo-ta clone kangaroo ard from cdna and genomic dna, respectively. blasting hard and hard against the opossum genome predicted peptides available from ensembl showed only one ard gene, with a gene structure the same as hard the kangaroo and opossum ards and the ensembl putative dog ard and ard were aligned with the original set of protein sequences, a coding sequence alignment was produced from this, the best aligned section was extracted, and from this a new tree was generated all kangaroo sequences clustered together in one clade, with the opossum sequence as an outgroup. the tree therefore suggests the presence of only one kangaroo ard gene. this tree was rooted to give the final tree, which shows that the speciation of kangaroo and opossum precedes the gene duplication that results in ard thus, ard seems not to be present in kangaroo and opossum and thus the emergence of ard probably represents a eutherian mammal specific retrotransposition event. also, kangaroo muscle tissue was lysed and analysed by sds-page and western blotting using anti-hard and anti-hard. a kangaroo protein of the expected size was observed when using anti-ard but not when using anti-hard the opossum ard sequence is identical to hard in the region recognized by anti-hard however, the posterior probability supporting the split separating metatherians from the eutherian ard1/ard divergence is only. further, a band of nucleotides was observed by pcr of genomic kangaroo material, although this kangaroo ard band did cluster as a metatherian sequence, rather than with the ard clade. altogether, this provides some level of support for a eutherian origin of ard knowledge of the kangaroo gene structure and full gene sequence will improve the confidence level of this conclusion. structural comparison between hard and hard2 alignment of the mammalian ard and ard2s and identification of the location of their n-acetyltransferase domains, shows only conservative substitutions from ard to ard except for an alanine to proline substitution at aa in human ard. as mentioned above, the hard sequence is highly similar to hard in the first ~ amino acids. the alanine amino acid residue at position within the predicted acetyltransferase domain of hard is substituted by a proline in hard however, structure prediction analysis indicates that this residue is a part of a loop in both hard and hard. this prediction was made by selecting a protein structure that also matches to pfam domain pf, aligning it and hard and hard to the pfam hidden markov model using hmmer, running swissmodel and making a molscript figure. importantly, this proline residue is not conserved within the mammalian ard2s. the difference in the c-terminal region may also account for changes in the enzymatic activity. several serine and threonine amino acid residues have changed between the two proteins and this could result in a change in the regulation of the enzymes by kinases. prediction of the disorder and globularity of hard and hard indicates that the c-terminal part of these two proteins to some extent diverge. overexpressed hard co-immunoprecipitate nath and hif-1α the nath-hard complex constitutes a functional protein n-acetyltransferase in human cells. to study whether or not a complex of nath and hard could exist, we performed immunoprecipitation experiments. extract of hek cells transfected with a plasmid encoding v5-tagged hard was made and used for immunoprecipitation with anti-v western blotting analysis of the immunoprecipitates revealed that a minor fraction of nath interacts with hard2-v. the interaction with nath is to be expected since the removal of the c-terminal amino acids of hard did not abolish nath binding and the remaining n-terminal amino acids of the hard sequence is as mentioned above highly similar to hard. the interaction between hard and hif-1α has been described. to investigate whether also hard is capable of interacting with hif-1α, we cotransfected mcf- cells with plasmids encoding xpress-hard 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-hard. subcellular localization of hard2 subcellular localization of hard was studied by expressing v5-tagged hard in hela cells followed by immunofluorescence staining. similarly to hard, hard2-v was present both in the cytoplasm and in the nucleus, but the majority of the protein appeared to be located in the cytoplasm. thus, hard2-nath complexes may potentially have a function in the cytoplasm. n-α-acetyltransferase activity of hard2 to investigate whether or not hard is a functionally active protein n-acetyltransferase, xpress-hard was immunoprecipitated from hek cells and the n-acetyltransferase activity of the xpress-hard was determined and compared to a negative control using xpress-lacz and a positive control using xpress-hard. we found that hard expresses n-acetyltransferase activity, demonstrated by the acetylation of the n-terminus of corticotropin. the activity data was normalized using the quantified protein amount in the agarose beads analyzed by western blotting. the radioactivity of the xpress-lacz samples were defined as background and subtracted from the xpress-hard1/hard values. the activity of the xpress-hard in three independent experiments gave a mean value of % relative to xpress-hard these results demonstrate that hard expresses n-acetyltransferase activity, but also suggest that its specific activity is lower than that of hard under the assay conditions used. hard and hard are differently regulated during nb cell differentiation it has been demonstrated that mard and mnat mrnas are downregulated during neuronal differentiation of p cells. since this suggests a role of these proteins in differentiation, we wanted to investigate the endogenous protein expression of human homologues hard hard and nath during differentiation. for this purpose we used retinoic acid induced differentiation of the promyelocytic nb cell line. protein levels of hard and nath significantly decreased during differentiation in nb cells. this correlates well with the findings in mouse neuronal cells. however, the levels hard protein is not significantly altered under these conditions. discussion we here report on the presence of a human gene encoding a protein acetyltransferase at the mrna, protein and enzyme activity levels. the ard acetyltransferase has originated through a retrotransposition event and represents an additional ard gene that can evolve independently from ard our results suggest that the duplication event is eutherian mammal specific. amino acid sequence differences between hard and hard demonstrate that changes already have taken place, particularly in the c-terminal region of hard as proposed for hard hard 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 hard and hard bind the same region within nath and therefore compete for nath binding. the difference in activity between hard and hard could suggest that the acth peptide used in this assay is not an optimal substrate for hard 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 hard and hif-1α. whether or not hif-1α is a direct substrate for hard mediated acetylation has not been investigated in this study and recent studies question the role of hard in destabilizing hif-1α. thus, the potential link between hif-1α and hard1/hard 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 hard and hard is one of selective pressures and neofunctionalization vs. subfunctionalization, 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 hard was directed against zebrafish tissue, but no binding was detected, probably due to sequence divergence. 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 hard and hard there are several indications that hard and nath may be linked to differentiation. as mentioned, mnat and mard mrnas are downregulated during neuronal differentiation in mouse. in the present study, we show that hard and nath proteins were both downregulated after induction of differentiation in promyelocytic leukaemia cells. this is the first description of nat-ard downregulation induced by differentiation in human cells and also the first verification of downregulation at the protein level. interestingly, the hard gene was one of twelve genes identified to be elevated in dedifferentiated hepatocellular carcinomas. it should also be noted that the yeast ard gene originally was implicated in controlling the switch between the mitotic cell cycle and developmental pathways. whether or not hard and nath influences differentiation per se, awaits further studies, but our present results add support to a link between the nath-hard complex and differentiation. the lack of hard downregulation during granulocytic differentiation could suggest that a specific subset of proteins then is acetylated at a stable level. in contrast, the hard 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 hard protein displays similar properties as hard 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 hard in cellular processes. 
in nb cells undergoing retinoic acid mediated differentiation, the level of endogenous hard and nath protein decreases while the level of hard protein is stable. hard mrna and protein are expressed in several human cell lines. we here describe a human protein, hard with % sequence identity to hard the gene encoding hard most likely originates from a eutherian mammal specific retrotransposition event. 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 hard. a human protein n-α-acetyltransferase is herein described. immunoprecipitation experiments show that hard protein potentially interacts with nath, suggesting that hard2-nath complexes may be responsible for protein n-α-acetylation in human cells. ard potentially complements the functions of ard 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 increasingly recognized as an important mechanism regulating a variety of cellular functions.
 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 hard. hard interacts with nath forming a complex expressing protein n-terminal α-acetylation activity.  we here describe a human protein, hard with % sequence identity to hard the gene encoding hard most likely originates from a eutherian mammal specific retrotransposition event. hard mrna and protein are expressed in several human cell lines. immunoprecipitation experiments show that hard protein potentially interacts with nath, suggesting that hard2-nath complexes may be responsible for protein n-α-acetylation in human cells. in nb cells undergoing retinoic acid mediated differentiation, the level of endogenous hard and nath protein decreases while the level of hard protein is stable.  a human protein n-α-acetyltransferase is herein described. ard potentially complements the functions of ard adding more flexibility and complexity to protein n-α-acetylation in human cells as compared to lower organisms which only have one ard. 