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Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Balakirev MY, Mullally JE, Favier A, Assard N, Sulpice E, Lindsey DF, Rulina AV, Gidrol X, Wilkinson KD - Elife (2015)

Bottom Line: Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins.In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent.Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches en technologies et sciences pour le vivant-Biologie à Grande Echelle, Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France.

ABSTRACT
Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

No MeSH data available.


Related in: MedlinePlus

Structural basis for Wss1-Cdc48 interaction.(A) RMN spectrum of Wss1 VIM (209–219) peptide used for structure determination. Fingerprint region of 800 MHz TOCSY (black) and NOESY (red) spectra recorded at T = 278 K. (B) Different views showing positioning of Wss1 VIM (green) and Cdc48 SIM (blue) within the central groove separating Nc and Nn-Cdc48 subdomains. (C) Identification of Wss1 VIM residues involved in Cdc48 binding. The RMN structure of Wss1 VIM peptide (in red) was docked into a model of N-Cdc48 domain based on the gp78 VIM-p97N complex (see Supplementary file 8). Both Wss1 VIM (red) and gp78 VIM show (blue) similar positioning of the side chains of the key amino acids implicated in interaction. (D) Conserved Wss1 residues from VIM and SHP domain are involved in Cdc48 binding. The MBP-Wss1/GST-Cdc48 complexes was pre-formed from purified proteins and isolated on amylose beads. Left part of the gel: Wss1 VIM peptide (209–219) does not inhibit the formation of stoichiometric 1:1 MBP-Wss1/GST-Cdc48 complex at concentrations up to 5 mg/ml, probably because it is too short to compete out Wss1, and/or because another part of Wss1 (SHP domain) is also involved in Cdc48 interaction. Right part of the gel: mutation of the conserved residues from VIM and SHP domains inhibits Cdc48 binding. F2R construct has apart from R218,219S VIM mutation, the mutation of conserved phenylalanine residue in SHP domain (F152S) known to be important for Cdc48 binding (see Figure 1—figure supplement 1B). Histograms shows GST-Cdc48: MBP-Wss1 ratio within the complexes quantified with ImageJ. See also corresponding .pse file: Supplementary file 8.DOI:http://dx.doi.org/10.7554/eLife.06763.019
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fig5s3: Structural basis for Wss1-Cdc48 interaction.(A) RMN spectrum of Wss1 VIM (209–219) peptide used for structure determination. Fingerprint region of 800 MHz TOCSY (black) and NOESY (red) spectra recorded at T = 278 K. (B) Different views showing positioning of Wss1 VIM (green) and Cdc48 SIM (blue) within the central groove separating Nc and Nn-Cdc48 subdomains. (C) Identification of Wss1 VIM residues involved in Cdc48 binding. The RMN structure of Wss1 VIM peptide (in red) was docked into a model of N-Cdc48 domain based on the gp78 VIM-p97N complex (see Supplementary file 8). Both Wss1 VIM (red) and gp78 VIM show (blue) similar positioning of the side chains of the key amino acids implicated in interaction. (D) Conserved Wss1 residues from VIM and SHP domain are involved in Cdc48 binding. The MBP-Wss1/GST-Cdc48 complexes was pre-formed from purified proteins and isolated on amylose beads. Left part of the gel: Wss1 VIM peptide (209–219) does not inhibit the formation of stoichiometric 1:1 MBP-Wss1/GST-Cdc48 complex at concentrations up to 5 mg/ml, probably because it is too short to compete out Wss1, and/or because another part of Wss1 (SHP domain) is also involved in Cdc48 interaction. Right part of the gel: mutation of the conserved residues from VIM and SHP domains inhibits Cdc48 binding. F2R construct has apart from R218,219S VIM mutation, the mutation of conserved phenylalanine residue in SHP domain (F152S) known to be important for Cdc48 binding (see Figure 1—figure supplement 1B). Histograms shows GST-Cdc48: MBP-Wss1 ratio within the complexes quantified with ImageJ. See also corresponding .pse file: Supplementary file 8.DOI:http://dx.doi.org/10.7554/eLife.06763.019

Mentions: To map the interactions within the Wss1/Doa1/Cdc48 complex, we performed a series of pull-down experiments with purified protein domains (Figure 5D). The results show that the N-terminus of Wss1 binds the PFU domain of Doa1, while the SHP and VIM motifs of Wss1 interact with the N-terminus of Cdc48 (Figure 5E). Finally, we determined the NMR structure of the Wss1 VIM motif and used molecular docking and structure-based mutagenesis to identify R218, R219, F152 as key residues involved in Cdc48 binding (Figure 5—figure supplement 3). Mutation of all three of these residues (F2R) ablates Cdc48 binding (Figure 5—figure supplement 3D).


Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Balakirev MY, Mullally JE, Favier A, Assard N, Sulpice E, Lindsey DF, Rulina AV, Gidrol X, Wilkinson KD - Elife (2015)

Structural basis for Wss1-Cdc48 interaction.(A) RMN spectrum of Wss1 VIM (209–219) peptide used for structure determination. Fingerprint region of 800 MHz TOCSY (black) and NOESY (red) spectra recorded at T = 278 K. (B) Different views showing positioning of Wss1 VIM (green) and Cdc48 SIM (blue) within the central groove separating Nc and Nn-Cdc48 subdomains. (C) Identification of Wss1 VIM residues involved in Cdc48 binding. The RMN structure of Wss1 VIM peptide (in red) was docked into a model of N-Cdc48 domain based on the gp78 VIM-p97N complex (see Supplementary file 8). Both Wss1 VIM (red) and gp78 VIM show (blue) similar positioning of the side chains of the key amino acids implicated in interaction. (D) Conserved Wss1 residues from VIM and SHP domain are involved in Cdc48 binding. The MBP-Wss1/GST-Cdc48 complexes was pre-formed from purified proteins and isolated on amylose beads. Left part of the gel: Wss1 VIM peptide (209–219) does not inhibit the formation of stoichiometric 1:1 MBP-Wss1/GST-Cdc48 complex at concentrations up to 5 mg/ml, probably because it is too short to compete out Wss1, and/or because another part of Wss1 (SHP domain) is also involved in Cdc48 interaction. Right part of the gel: mutation of the conserved residues from VIM and SHP domains inhibits Cdc48 binding. F2R construct has apart from R218,219S VIM mutation, the mutation of conserved phenylalanine residue in SHP domain (F152S) known to be important for Cdc48 binding (see Figure 1—figure supplement 1B). Histograms shows GST-Cdc48: MBP-Wss1 ratio within the complexes quantified with ImageJ. See also corresponding .pse file: Supplementary file 8.DOI:http://dx.doi.org/10.7554/eLife.06763.019
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Related In: Results  -  Collection

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fig5s3: Structural basis for Wss1-Cdc48 interaction.(A) RMN spectrum of Wss1 VIM (209–219) peptide used for structure determination. Fingerprint region of 800 MHz TOCSY (black) and NOESY (red) spectra recorded at T = 278 K. (B) Different views showing positioning of Wss1 VIM (green) and Cdc48 SIM (blue) within the central groove separating Nc and Nn-Cdc48 subdomains. (C) Identification of Wss1 VIM residues involved in Cdc48 binding. The RMN structure of Wss1 VIM peptide (in red) was docked into a model of N-Cdc48 domain based on the gp78 VIM-p97N complex (see Supplementary file 8). Both Wss1 VIM (red) and gp78 VIM show (blue) similar positioning of the side chains of the key amino acids implicated in interaction. (D) Conserved Wss1 residues from VIM and SHP domain are involved in Cdc48 binding. The MBP-Wss1/GST-Cdc48 complexes was pre-formed from purified proteins and isolated on amylose beads. Left part of the gel: Wss1 VIM peptide (209–219) does not inhibit the formation of stoichiometric 1:1 MBP-Wss1/GST-Cdc48 complex at concentrations up to 5 mg/ml, probably because it is too short to compete out Wss1, and/or because another part of Wss1 (SHP domain) is also involved in Cdc48 interaction. Right part of the gel: mutation of the conserved residues from VIM and SHP domains inhibits Cdc48 binding. F2R construct has apart from R218,219S VIM mutation, the mutation of conserved phenylalanine residue in SHP domain (F152S) known to be important for Cdc48 binding (see Figure 1—figure supplement 1B). Histograms shows GST-Cdc48: MBP-Wss1 ratio within the complexes quantified with ImageJ. See also corresponding .pse file: Supplementary file 8.DOI:http://dx.doi.org/10.7554/eLife.06763.019
Mentions: To map the interactions within the Wss1/Doa1/Cdc48 complex, we performed a series of pull-down experiments with purified protein domains (Figure 5D). The results show that the N-terminus of Wss1 binds the PFU domain of Doa1, while the SHP and VIM motifs of Wss1 interact with the N-terminus of Cdc48 (Figure 5E). Finally, we determined the NMR structure of the Wss1 VIM motif and used molecular docking and structure-based mutagenesis to identify R218, R219, F152 as key residues involved in Cdc48 binding (Figure 5—figure supplement 3). Mutation of all three of these residues (F2R) ablates Cdc48 binding (Figure 5—figure supplement 3D).

Bottom Line: Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins.In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent.Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches en technologies et sciences pour le vivant-Biologie à Grande Echelle, Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France.

ABSTRACT
Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

No MeSH data available.


Related in: MedlinePlus