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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

Modification of protein sulfhydryl groups by thiuram disulfides.(i) Thiol-disulfide exchange reaction between protein sulfhydryl groups and thiram (Th) results in formation of protein–thiram mixed disulfide. When thiram is present in excess, this is the primary reaction product. (ii) With sub-stoichiometric amounts of thiram, the protein-thiram disulfide may further react with thiols producing inter- and intra-molecular disulfides. In general, protein modification by thiram can be fully reversed by an excess of thiols.DOI:http://dx.doi.org/10.7554/eLife.06763.007
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fig2s1: Modification of protein sulfhydryl groups by thiuram disulfides.(i) Thiol-disulfide exchange reaction between protein sulfhydryl groups and thiram (Th) results in formation of protein–thiram mixed disulfide. When thiram is present in excess, this is the primary reaction product. (ii) With sub-stoichiometric amounts of thiram, the protein-thiram disulfide may further react with thiols producing inter- and intra-molecular disulfides. In general, protein modification by thiram can be fully reversed by an excess of thiols.DOI:http://dx.doi.org/10.7554/eLife.06763.007

Mentions: During these experiments, we observed that the treatment of Wss1 with OPA induced protein oligomerization (Figure 2B). The oligomers are thiol-sensitive suggesting that they are disulfide linked (see Figure 4D below). We hypothesize that in Wss1 protein, the active site Zn is linked to a cysteine residue, which is released by OPA and promotes the formation of intermolecular crosslinks. This observation raises an interesting possibility that Wss1 is regulated by a cysteine-switch mechanism (Chakraborti et al., 2003). The metalloproteases regulated by a cysteine-switch mechanism can be activated by thiol-reacting reagents. By screening a panel of different electrophiles, we found that thiram (Balakirev and Zimmer, 1998), a compound that reversibly modifies cysteine residues, efficiently induces Wss1 self-cleavage (Figure 2B). Importantly, the cleavage pattern was the same as in refolding experiments (Figure 2A). Thiram belongs to the thiuram disulfide class of reactive compounds that modify protein sulfhydryl groups via thiol-disulfide exchange reaction (Neims et al., 1966; Vallari and Pietruszko, 1982) (Figure 2—figure supplement 1). Because thiram also inhibited OPA-induced Wss1 oligomerization (Figure 2B), it seems that it targets the same cysteine residue that is involved in Zn binding. In parallel, we found that toxic organomercury compound APMA, classically used for matrix metalloproteases activation, also produces Wss1 self-cleavage and inhibits OPA-dependent protein oligomerization (Figure 2—figure supplement 2A,B).10.7554/eLife.06763.011Figure 4.SUMO ligase activity of Wss1 protein.


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)

Modification of protein sulfhydryl groups by thiuram disulfides.(i) Thiol-disulfide exchange reaction between protein sulfhydryl groups and thiram (Th) results in formation of protein–thiram mixed disulfide. When thiram is present in excess, this is the primary reaction product. (ii) With sub-stoichiometric amounts of thiram, the protein-thiram disulfide may further react with thiols producing inter- and intra-molecular disulfides. In general, protein modification by thiram can be fully reversed by an excess of thiols.DOI:http://dx.doi.org/10.7554/eLife.06763.007
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559962&req=5

fig2s1: Modification of protein sulfhydryl groups by thiuram disulfides.(i) Thiol-disulfide exchange reaction between protein sulfhydryl groups and thiram (Th) results in formation of protein–thiram mixed disulfide. When thiram is present in excess, this is the primary reaction product. (ii) With sub-stoichiometric amounts of thiram, the protein-thiram disulfide may further react with thiols producing inter- and intra-molecular disulfides. In general, protein modification by thiram can be fully reversed by an excess of thiols.DOI:http://dx.doi.org/10.7554/eLife.06763.007
Mentions: During these experiments, we observed that the treatment of Wss1 with OPA induced protein oligomerization (Figure 2B). The oligomers are thiol-sensitive suggesting that they are disulfide linked (see Figure 4D below). We hypothesize that in Wss1 protein, the active site Zn is linked to a cysteine residue, which is released by OPA and promotes the formation of intermolecular crosslinks. This observation raises an interesting possibility that Wss1 is regulated by a cysteine-switch mechanism (Chakraborti et al., 2003). The metalloproteases regulated by a cysteine-switch mechanism can be activated by thiol-reacting reagents. By screening a panel of different electrophiles, we found that thiram (Balakirev and Zimmer, 1998), a compound that reversibly modifies cysteine residues, efficiently induces Wss1 self-cleavage (Figure 2B). Importantly, the cleavage pattern was the same as in refolding experiments (Figure 2A). Thiram belongs to the thiuram disulfide class of reactive compounds that modify protein sulfhydryl groups via thiol-disulfide exchange reaction (Neims et al., 1966; Vallari and Pietruszko, 1982) (Figure 2—figure supplement 1). Because thiram also inhibited OPA-induced Wss1 oligomerization (Figure 2B), it seems that it targets the same cysteine residue that is involved in Zn binding. In parallel, we found that toxic organomercury compound APMA, classically used for matrix metalloproteases activation, also produces Wss1 self-cleavage and inhibits OPA-dependent protein oligomerization (Figure 2—figure supplement 2A,B).10.7554/eLife.06763.011Figure 4.SUMO ligase activity of Wss1 protein.

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