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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

Proposed mechanism for Wss1 SUMO-ligase activity.(A) Wss1 protein has a positively charged N-terminal WLM domain and negatively charged C-terminal extension that contains two SIMs and conserved cysteine C226. This cysteine may be linked to the active site Zn counteracts, the interaction facilitated by charge interaction of the two protein parts. Wss1 E3-like ligase activity (Figure 2A,B) results from the juxtaposition of an E2∼SUMO thiol ester and an acceptor SUMO due to SUMO binding by two carboxy-terminal SIMs. The tendency of Wss1 to form oligomers further promotes Wss1-dependent sumoylation by multiplying SUMO binding sites. (B) Extraction of the active site Zn by o-Phenanthroline (OPA) release the cysteine 226, which is prone to produce intermolecular disulfide crosslinks with its homolog (forming Wss1 dimer) or one of the two cysteines localized in the WLM domain (forming Wss1 oligomers). Multiplication of the SIMs in Wss1 oligomers further promotes SUMO ligase activity.DOI:http://dx.doi.org/10.7554/eLife.06763.013
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fig4s2: Proposed mechanism for Wss1 SUMO-ligase activity.(A) Wss1 protein has a positively charged N-terminal WLM domain and negatively charged C-terminal extension that contains two SIMs and conserved cysteine C226. This cysteine may be linked to the active site Zn counteracts, the interaction facilitated by charge interaction of the two protein parts. Wss1 E3-like ligase activity (Figure 2A,B) results from the juxtaposition of an E2∼SUMO thiol ester and an acceptor SUMO due to SUMO binding by two carboxy-terminal SIMs. The tendency of Wss1 to form oligomers further promotes Wss1-dependent sumoylation by multiplying SUMO binding sites. (B) Extraction of the active site Zn by o-Phenanthroline (OPA) release the cysteine 226, which is prone to produce intermolecular disulfide crosslinks with its homolog (forming Wss1 dimer) or one of the two cysteines localized in the WLM domain (forming Wss1 oligomers). Multiplication of the SIMs in Wss1 oligomers further promotes SUMO ligase activity.DOI:http://dx.doi.org/10.7554/eLife.06763.013

Mentions: The WLM* mutant that is unable to bind Zn repeatedly showed higher SUMO ligase activity compared to WT protein. To see whether the release of Zn increases sumoylation, we produced Wss1 apoprotein by OPA titration and analyzed its auto-sumoylation (Figure 4C). Increasing OPA concentration promotes sumoylation of WT-Wss1. This effect was not due to stimulation of SUMO E1–E2 enzymes because OPA did not increase the sumoylation of the WLM* mutant. At 3 mM OPA, WT, and WLM* formed a similar level of SUMO conjugates (Figure 4C,D). It seems therefore that extraction of Zn from metalloprotease active site somehow promotes Wss1-dependent sumoylation. Although the mechanism of this effect is not completely clear, it could be explained by our finding that OPA induces Wss1 oligomerization (Figure 2B). The oligomers can be seen by non-reducing gel electrophoresis and disappear in the presence of dithiothreitol (DTT) suggesting that they are disulfide linked (Figure 4D). These data suggest that in the absence of Zn the regulatory cysteine 226 (Figure 2—figure supplement 4) induces intermolecular disulfide crosslinks, multiplication of SUMO-binding sites and increase in SUMO ligase activity (Figure 4—figure supplement 2).


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)

Proposed mechanism for Wss1 SUMO-ligase activity.(A) Wss1 protein has a positively charged N-terminal WLM domain and negatively charged C-terminal extension that contains two SIMs and conserved cysteine C226. This cysteine may be linked to the active site Zn counteracts, the interaction facilitated by charge interaction of the two protein parts. Wss1 E3-like ligase activity (Figure 2A,B) results from the juxtaposition of an E2∼SUMO thiol ester and an acceptor SUMO due to SUMO binding by two carboxy-terminal SIMs. The tendency of Wss1 to form oligomers further promotes Wss1-dependent sumoylation by multiplying SUMO binding sites. (B) Extraction of the active site Zn by o-Phenanthroline (OPA) release the cysteine 226, which is prone to produce intermolecular disulfide crosslinks with its homolog (forming Wss1 dimer) or one of the two cysteines localized in the WLM domain (forming Wss1 oligomers). Multiplication of the SIMs in Wss1 oligomers further promotes SUMO ligase activity.DOI:http://dx.doi.org/10.7554/eLife.06763.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559962&req=5

fig4s2: Proposed mechanism for Wss1 SUMO-ligase activity.(A) Wss1 protein has a positively charged N-terminal WLM domain and negatively charged C-terminal extension that contains two SIMs and conserved cysteine C226. This cysteine may be linked to the active site Zn counteracts, the interaction facilitated by charge interaction of the two protein parts. Wss1 E3-like ligase activity (Figure 2A,B) results from the juxtaposition of an E2∼SUMO thiol ester and an acceptor SUMO due to SUMO binding by two carboxy-terminal SIMs. The tendency of Wss1 to form oligomers further promotes Wss1-dependent sumoylation by multiplying SUMO binding sites. (B) Extraction of the active site Zn by o-Phenanthroline (OPA) release the cysteine 226, which is prone to produce intermolecular disulfide crosslinks with its homolog (forming Wss1 dimer) or one of the two cysteines localized in the WLM domain (forming Wss1 oligomers). Multiplication of the SIMs in Wss1 oligomers further promotes SUMO ligase activity.DOI:http://dx.doi.org/10.7554/eLife.06763.013
Mentions: The WLM* mutant that is unable to bind Zn repeatedly showed higher SUMO ligase activity compared to WT protein. To see whether the release of Zn increases sumoylation, we produced Wss1 apoprotein by OPA titration and analyzed its auto-sumoylation (Figure 4C). Increasing OPA concentration promotes sumoylation of WT-Wss1. This effect was not due to stimulation of SUMO E1–E2 enzymes because OPA did not increase the sumoylation of the WLM* mutant. At 3 mM OPA, WT, and WLM* formed a similar level of SUMO conjugates (Figure 4C,D). It seems therefore that extraction of Zn from metalloprotease active site somehow promotes Wss1-dependent sumoylation. Although the mechanism of this effect is not completely clear, it could be explained by our finding that OPA induces Wss1 oligomerization (Figure 2B). The oligomers can be seen by non-reducing gel electrophoresis and disappear in the presence of dithiothreitol (DTT) suggesting that they are disulfide linked (Figure 4D). These data suggest that in the absence of Zn the regulatory cysteine 226 (Figure 2—figure supplement 4) induces intermolecular disulfide crosslinks, multiplication of SUMO-binding sites and increase in SUMO ligase activity (Figure 4—figure supplement 2).

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