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Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.

Hudson JJ, Bednarova K, Kozakova L, Liao C, Guerineau M, Colnaghi R, Vidot S, Marek J, Bathula SR, Lehmann AR, Palecek J - PLoS ONE (2011)

Bottom Line: MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1).We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

ABSTRACT

Background: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.

Methodology/principal findings: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.

Conclusions/significance: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

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Interplay between MAGE and EID proteins in transcription activation system.(A) Effect of transfected FLAG-NSE4b and S-tagged MAGEG1 on transcriptional activation by SF-1 in HEK293 cells. “2×” indicates twice the concentration of MAGEG1 plasmid used in transfections. (B) Effects of FLAG-tagged EID1 or NSE4b on transcriptional activation by different MAGE proteins. The reporter activity in each column is normalised to the activity with nuclear receptor but with neither MAGE nor EID (column 2). Results show mean ± SEM of 3–5 independent transfections.
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pone-0017270-g006: Interplay between MAGE and EID proteins in transcription activation system.(A) Effect of transfected FLAG-NSE4b and S-tagged MAGEG1 on transcriptional activation by SF-1 in HEK293 cells. “2×” indicates twice the concentration of MAGEG1 plasmid used in transfections. (B) Effects of FLAG-tagged EID1 or NSE4b on transcriptional activation by different MAGE proteins. The reporter activity in each column is normalised to the activity with nuclear receptor but with neither MAGE nor EID (column 2). Results show mean ± SEM of 3–5 independent transfections.

Mentions: NSE4b/EID3 was first identified as a member of the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors and was shown to inhibit transcriptional activation from several promoters in HuH7 human hepatoma cells [20]. We were interested to see if the interaction between NSE4b and MAGEG1 might affect transcriptional activation, and to examine this, we used the Gal4-SF1 promoter system to study SF-1 mediated transcription activation [20]. Figure 6A confirms that, in HEK293 cells, nuclear receptor stimulates reporter activity some 5–10-fold (columns 1 and 2). Neither NSE4b nor MAGEG1 had much effect (columns 3 and 4), but there was a dramatic concentration-dependent stimulation of transcription activation when MAGEG1 and NSE4b were expressed together at two different concentrations of MAGEG1 (columns 8 and 16). To confirm that this transcriptional co-activation was the result of a functional interaction between MAGEG1 and NSE4b, we co-transfected the cells with NSE4b and the series of mutants of MAGEG1 that reduced or abolished its interaction with NSE4b (see Figure 5). As seen in Figure 6A, lanes 9–11 and lanes 17–19, transcriptional activation by the mutant MAGEG1 proteins and NSE4b was much less than with the corresponding concentration of wild-type MAGEG1 (Lanes 8 and 16).


Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.

Hudson JJ, Bednarova K, Kozakova L, Liao C, Guerineau M, Colnaghi R, Vidot S, Marek J, Bathula SR, Lehmann AR, Palecek J - PLoS ONE (2011)

Interplay between MAGE and EID proteins in transcription activation system.(A) Effect of transfected FLAG-NSE4b and S-tagged MAGEG1 on transcriptional activation by SF-1 in HEK293 cells. “2×” indicates twice the concentration of MAGEG1 plasmid used in transfections. (B) Effects of FLAG-tagged EID1 or NSE4b on transcriptional activation by different MAGE proteins. The reporter activity in each column is normalised to the activity with nuclear receptor but with neither MAGE nor EID (column 2). Results show mean ± SEM of 3–5 independent transfections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045436&req=5

pone-0017270-g006: Interplay between MAGE and EID proteins in transcription activation system.(A) Effect of transfected FLAG-NSE4b and S-tagged MAGEG1 on transcriptional activation by SF-1 in HEK293 cells. “2×” indicates twice the concentration of MAGEG1 plasmid used in transfections. (B) Effects of FLAG-tagged EID1 or NSE4b on transcriptional activation by different MAGE proteins. The reporter activity in each column is normalised to the activity with nuclear receptor but with neither MAGE nor EID (column 2). Results show mean ± SEM of 3–5 independent transfections.
Mentions: NSE4b/EID3 was first identified as a member of the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors and was shown to inhibit transcriptional activation from several promoters in HuH7 human hepatoma cells [20]. We were interested to see if the interaction between NSE4b and MAGEG1 might affect transcriptional activation, and to examine this, we used the Gal4-SF1 promoter system to study SF-1 mediated transcription activation [20]. Figure 6A confirms that, in HEK293 cells, nuclear receptor stimulates reporter activity some 5–10-fold (columns 1 and 2). Neither NSE4b nor MAGEG1 had much effect (columns 3 and 4), but there was a dramatic concentration-dependent stimulation of transcription activation when MAGEG1 and NSE4b were expressed together at two different concentrations of MAGEG1 (columns 8 and 16). To confirm that this transcriptional co-activation was the result of a functional interaction between MAGEG1 and NSE4b, we co-transfected the cells with NSE4b and the series of mutants of MAGEG1 that reduced or abolished its interaction with NSE4b (see Figure 5). As seen in Figure 6A, lanes 9–11 and lanes 17–19, transcriptional activation by the mutant MAGEG1 proteins and NSE4b was much less than with the corresponding concentration of wild-type MAGEG1 (Lanes 8 and 16).

Bottom Line: MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1).We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

ABSTRACT

Background: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.

Methodology/principal findings: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.

Conclusions/significance: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

Show MeSH
Related in: MedlinePlus