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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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Related in: MedlinePlus

Conserved amino acid residues within the MAGE protein family.Alignment of the N-terminal (A) (aa 89 to 199 of S.p. Nse3) and C-terminal (B) (aa 211 to 301 of S.p. Nse3) part of MHD domain of Nse3/MAGEG1 subfamily. The Nse3/MAGEG1 orthologs are from S. pombe (S.p.), Aspergillus nidulans (A.n.), Neosartorya fischeri (N.f.), Aspergillus terreus (A.t.), Aspergillus clavatus (A.c.), Neurospora crassa (N.c.), Magnaporthe grisea (M.g.), Aspergillus oryzae (A.o.), S. cerevisiae (S.c.), Danio rerio (D.r.), Xenopus tropicalis (X.t.), Galus galus (G.g.), Ornithorhynchus anatinus (O.a.), Monodelphis domestica (M.d.), Dasypus novemcinctus (D.n.), Canis lupus familiaris (C.f.), Mus musculus (M.m.), Homo sapiens (H.s.). Secondary structure derived from the 3D-structure model of Nse3 is indicated above the alignment: cyan rectangle, helix; orange arrow, beta-sheet. Most of the conserved residues were mutated (mut) in the S. pombe Nse3 sequence to alanine; m, mutated residue; red rectangles indicate Nse1- and/or Nse4-specific mutants, respectively; Y264 and L265 residues are labelled with asterisk (B). NSE4b-specific residues of MAGEG1 protein are also indicated in red below the MAGEG1 sequence (B). (C) Alignment of C-terminal part of MHD domain of human MAGE proteins. Shading represents amino acid groups conserved across the family: dark green, hydrophobic and aromatic; light green, polar; blue, acidic; pink, basic; all glycine and proline residues are highlighted in yellow.
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pone-0017270-g002: Conserved amino acid residues within the MAGE protein family.Alignment of the N-terminal (A) (aa 89 to 199 of S.p. Nse3) and C-terminal (B) (aa 211 to 301 of S.p. Nse3) part of MHD domain of Nse3/MAGEG1 subfamily. The Nse3/MAGEG1 orthologs are from S. pombe (S.p.), Aspergillus nidulans (A.n.), Neosartorya fischeri (N.f.), Aspergillus terreus (A.t.), Aspergillus clavatus (A.c.), Neurospora crassa (N.c.), Magnaporthe grisea (M.g.), Aspergillus oryzae (A.o.), S. cerevisiae (S.c.), Danio rerio (D.r.), Xenopus tropicalis (X.t.), Galus galus (G.g.), Ornithorhynchus anatinus (O.a.), Monodelphis domestica (M.d.), Dasypus novemcinctus (D.n.), Canis lupus familiaris (C.f.), Mus musculus (M.m.), Homo sapiens (H.s.). Secondary structure derived from the 3D-structure model of Nse3 is indicated above the alignment: cyan rectangle, helix; orange arrow, beta-sheet. Most of the conserved residues were mutated (mut) in the S. pombe Nse3 sequence to alanine; m, mutated residue; red rectangles indicate Nse1- and/or Nse4-specific mutants, respectively; Y264 and L265 residues are labelled with asterisk (B). NSE4b-specific residues of MAGEG1 protein are also indicated in red below the MAGEG1 sequence (B). (C) Alignment of C-terminal part of MHD domain of human MAGE proteins. Shading represents amino acid groups conserved across the family: dark green, hydrophobic and aromatic; light green, polar; blue, acidic; pink, basic; all glycine and proline residues are highlighted in yellow.

Mentions: To gain further insight into the interaction surfaces, we have mutated most of the conserved residues in the S. pombe Nse3 MHD region (aa 93 to 301, Supplementary Table S1, Figures 2A and B). Each Nse3 mutant was tested for its ability to interact with both Nse1 and Nse4 using the yeast two-hybrid system. 37 out of a total of 82 mutants exhibited no defect in binding to either Nse1 or Nse4 (Table 1). In contrast, 13 mutants lost the ability to interact with both partners. The other 32 mutants disrupted interaction with either Nse1 or Nse4. In order to interpret these data we modelled the structure of Nse3 on the structures of the MHD of MAGEA4 (PDB entry 2WA0) and the recently published structure of MAGEG1 (PDB entry 3NW0 [14]). The structure (Figure 3) is comprised of two domains of approximately equal size, the N-terminal domain being made up of three alpha helices (H1 to H3) and two beta sheets (S1 and S2), whereas the C-terminal domain comprises five helices (H4-8) and two beta sheets (S3 and S4).


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)

Conserved amino acid residues within the MAGE protein family.Alignment of the N-terminal (A) (aa 89 to 199 of S.p. Nse3) and C-terminal (B) (aa 211 to 301 of S.p. Nse3) part of MHD domain of Nse3/MAGEG1 subfamily. The Nse3/MAGEG1 orthologs are from S. pombe (S.p.), Aspergillus nidulans (A.n.), Neosartorya fischeri (N.f.), Aspergillus terreus (A.t.), Aspergillus clavatus (A.c.), Neurospora crassa (N.c.), Magnaporthe grisea (M.g.), Aspergillus oryzae (A.o.), S. cerevisiae (S.c.), Danio rerio (D.r.), Xenopus tropicalis (X.t.), Galus galus (G.g.), Ornithorhynchus anatinus (O.a.), Monodelphis domestica (M.d.), Dasypus novemcinctus (D.n.), Canis lupus familiaris (C.f.), Mus musculus (M.m.), Homo sapiens (H.s.). Secondary structure derived from the 3D-structure model of Nse3 is indicated above the alignment: cyan rectangle, helix; orange arrow, beta-sheet. Most of the conserved residues were mutated (mut) in the S. pombe Nse3 sequence to alanine; m, mutated residue; red rectangles indicate Nse1- and/or Nse4-specific mutants, respectively; Y264 and L265 residues are labelled with asterisk (B). NSE4b-specific residues of MAGEG1 protein are also indicated in red below the MAGEG1 sequence (B). (C) Alignment of C-terminal part of MHD domain of human MAGE proteins. Shading represents amino acid groups conserved across the family: dark green, hydrophobic and aromatic; light green, polar; blue, acidic; pink, basic; all glycine and proline residues are highlighted in yellow.
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pone-0017270-g002: Conserved amino acid residues within the MAGE protein family.Alignment of the N-terminal (A) (aa 89 to 199 of S.p. Nse3) and C-terminal (B) (aa 211 to 301 of S.p. Nse3) part of MHD domain of Nse3/MAGEG1 subfamily. The Nse3/MAGEG1 orthologs are from S. pombe (S.p.), Aspergillus nidulans (A.n.), Neosartorya fischeri (N.f.), Aspergillus terreus (A.t.), Aspergillus clavatus (A.c.), Neurospora crassa (N.c.), Magnaporthe grisea (M.g.), Aspergillus oryzae (A.o.), S. cerevisiae (S.c.), Danio rerio (D.r.), Xenopus tropicalis (X.t.), Galus galus (G.g.), Ornithorhynchus anatinus (O.a.), Monodelphis domestica (M.d.), Dasypus novemcinctus (D.n.), Canis lupus familiaris (C.f.), Mus musculus (M.m.), Homo sapiens (H.s.). Secondary structure derived from the 3D-structure model of Nse3 is indicated above the alignment: cyan rectangle, helix; orange arrow, beta-sheet. Most of the conserved residues were mutated (mut) in the S. pombe Nse3 sequence to alanine; m, mutated residue; red rectangles indicate Nse1- and/or Nse4-specific mutants, respectively; Y264 and L265 residues are labelled with asterisk (B). NSE4b-specific residues of MAGEG1 protein are also indicated in red below the MAGEG1 sequence (B). (C) Alignment of C-terminal part of MHD domain of human MAGE proteins. Shading represents amino acid groups conserved across the family: dark green, hydrophobic and aromatic; light green, polar; blue, acidic; pink, basic; all glycine and proline residues are highlighted in yellow.
Mentions: To gain further insight into the interaction surfaces, we have mutated most of the conserved residues in the S. pombe Nse3 MHD region (aa 93 to 301, Supplementary Table S1, Figures 2A and B). Each Nse3 mutant was tested for its ability to interact with both Nse1 and Nse4 using the yeast two-hybrid system. 37 out of a total of 82 mutants exhibited no defect in binding to either Nse1 or Nse4 (Table 1). In contrast, 13 mutants lost the ability to interact with both partners. The other 32 mutants disrupted interaction with either Nse1 or Nse4. In order to interpret these data we modelled the structure of Nse3 on the structures of the MHD of MAGEA4 (PDB entry 2WA0) and the recently published structure of MAGEG1 (PDB entry 3NW0 [14]). The structure (Figure 3) is comprised of two domains of approximately equal size, the N-terminal domain being made up of three alpha helices (H1 to H3) and two beta sheets (S1 and S2), whereas the C-terminal domain comprises five helices (H4-8) and two beta sheets (S3 and S4).

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