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Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex.

Ametzazurra A, Gázquez C, Lasa M, Larrea E, Prieto J, Aldabe R - BMC Proc (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. aametzazurra@proteomika.com

ABSTRACT

Background: Human Nalpha-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.

Methods: 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.

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

hNatB purification and enzymatic activity. A. Coimmunoprecipitation of GST-hNaa25p and hNaa20p-CTAP or GST-hNaa25p and hNaa20p. B. NAT activity of immunoprecipitated hNatB complexes from Hela cells overexpressing GST-hNaa25p and hNaa20p-CTAP with anti-hNaa20p or anti-GST antibodies. Human tropomyosin-1 or p53 aminoterminal peptides synthesized by Dr Francisco Borrás-Cuesta at CIMA University of Navarra as previously described [27] were used as substrates. The results are representative of three independent experiments.
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Figure 3: hNatB purification and enzymatic activity. A. Coimmunoprecipitation of GST-hNaa25p and hNaa20p-CTAP or GST-hNaa25p and hNaa20p. B. NAT activity of immunoprecipitated hNatB complexes from Hela cells overexpressing GST-hNaa25p and hNaa20p-CTAP with anti-hNaa20p or anti-GST antibodies. Human tropomyosin-1 or p53 aminoterminal peptides synthesized by Dr Francisco Borrás-Cuesta at CIMA University of Navarra as previously described [27] were used as substrates. The results are representative of three independent experiments.

Mentions: NAT enzymatic complexes are composed by a catalytic subunit and one or several accessory subunits. In the case of hNatA there are two possible catalytic subunits [24] and several accessory subunits [25] but hNatB complex is formed by the catalytic subunit hNaa20p that interacts with hNaa25p to generate an active hNatB complex [5]. We have coexpressed hNaa20p with a carboxi terminal protein tag (CTAP) and hNaa25p fused to GST in Hela cells, detecting an interaction between both fusion proteins (Figure 3A) as it has been described in yeast and human cells before [3-5]. In order to determine if the hNatB enzymatic complex immunopurified with the GST antibodies is enzymatically active we performed an in vitro acetyltransferase assay using as substrate a peptide corresponding to human tropomyosin-1 amino terminus, that has been demonstrated to be a hNatB substrate [12]. We observed that immunopurified hNatB complexes, using GST-Naa25p as bait, rescue active enzymatic complexes that acetylate efficiently the tropomyosin-1 aminoterminal peptide, as well as the hNatB complexes immunopurified with a hNaa20p specific antibody (Figure 3B). Thereafter we analyzed if the immunopurified hNatB complexes were able to acetylate p53 aminoterminal peptide, as this protein is a potential NatB substrate based on its first two amino acids (Met-Glu-). Surprisingly, none of the purified hNatB complexes acetylated this peptide.


Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex.

Ametzazurra A, Gázquez C, Lasa M, Larrea E, Prieto J, Aldabe R - BMC Proc (2009)

hNatB purification and enzymatic activity. A. Coimmunoprecipitation of GST-hNaa25p and hNaa20p-CTAP or GST-hNaa25p and hNaa20p. B. NAT activity of immunoprecipitated hNatB complexes from Hela cells overexpressing GST-hNaa25p and hNaa20p-CTAP with anti-hNaa20p or anti-GST antibodies. Human tropomyosin-1 or p53 aminoterminal peptides synthesized by Dr Francisco Borrás-Cuesta at CIMA University of Navarra as previously described [27] were used as substrates. The results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2722097&req=5

Figure 3: hNatB purification and enzymatic activity. A. Coimmunoprecipitation of GST-hNaa25p and hNaa20p-CTAP or GST-hNaa25p and hNaa20p. B. NAT activity of immunoprecipitated hNatB complexes from Hela cells overexpressing GST-hNaa25p and hNaa20p-CTAP with anti-hNaa20p or anti-GST antibodies. Human tropomyosin-1 or p53 aminoterminal peptides synthesized by Dr Francisco Borrás-Cuesta at CIMA University of Navarra as previously described [27] were used as substrates. The results are representative of three independent experiments.
Mentions: NAT enzymatic complexes are composed by a catalytic subunit and one or several accessory subunits. In the case of hNatA there are two possible catalytic subunits [24] and several accessory subunits [25] but hNatB complex is formed by the catalytic subunit hNaa20p that interacts with hNaa25p to generate an active hNatB complex [5]. We have coexpressed hNaa20p with a carboxi terminal protein tag (CTAP) and hNaa25p fused to GST in Hela cells, detecting an interaction between both fusion proteins (Figure 3A) as it has been described in yeast and human cells before [3-5]. In order to determine if the hNatB enzymatic complex immunopurified with the GST antibodies is enzymatically active we performed an in vitro acetyltransferase assay using as substrate a peptide corresponding to human tropomyosin-1 amino terminus, that has been demonstrated to be a hNatB substrate [12]. We observed that immunopurified hNatB complexes, using GST-Naa25p as bait, rescue active enzymatic complexes that acetylate efficiently the tropomyosin-1 aminoterminal peptide, as well as the hNatB complexes immunopurified with a hNaa20p specific antibody (Figure 3B). Thereafter we analyzed if the immunopurified hNatB complexes were able to acetylate p53 aminoterminal peptide, as this protein is a potential NatB substrate based on its first two amino acids (Met-Glu-). Surprisingly, none of the purified hNatB complexes acetylated this peptide.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. aametzazurra@proteomika.com

ABSTRACT

Background: Human Nalpha-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.

Methods: 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.

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus