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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

Cleavage of Wss1-AQA mutant.Partially purified HA-Wss1-AQA mutant (500 μg/ml of total protein) and wild-type protein (100 μg/ml) were incubated for the indicated time at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide) or 0.5 mM thiram (Th) was added at the beginning of the incubation. The reaction was analyzed by gel electrophoresis and Coomassie staining and Wss1 protein fragments were analyzed using Adobe Photoshop software. The positions of common (black) and unique (false-colored) fragments are shown for AQA mutant (blue) and WT protein (red).DOI:http://dx.doi.org/10.7554/eLife.06763.009
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fig2s3: Cleavage of Wss1-AQA mutant.Partially purified HA-Wss1-AQA mutant (500 μg/ml of total protein) and wild-type protein (100 μg/ml) were incubated for the indicated time at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide) or 0.5 mM thiram (Th) was added at the beginning of the incubation. The reaction was analyzed by gel electrophoresis and Coomassie staining and Wss1 protein fragments were analyzed using Adobe Photoshop software. The positions of common (black) and unique (false-colored) fragments are shown for AQA mutant (blue) and WT protein (red).DOI:http://dx.doi.org/10.7554/eLife.06763.009

Mentions: Together these data suggest that, like some other metalloproteases, Wss1 is regulated by a cysteine-switch mechanism. Displacement of the cysteine from the active site Zn induces autoproteolytic cleavage and may serve to release an inhibitory protein fragment. Wss1 has three cysteine residues, two of which, though located within the WLM domain, are far from the active site (Figure 2—figure supplement 2C). Moreover, their orientation suggests that they may form a disulfide bridge, though the estimated distance of 5.6 Å implies a conformational change would be necessary. The third cysteine, C226, is located in a negatively charged C-terminal part of the protein that was released by self-cleavage. This residue is conserved within the WLM family (Figure 1—figure supplement 1) and is a perfect candidate for the regulatory cysteine. Unfortunately, we were unable to produce soluble C226S protein, probably because of rapid auto-proteolysis of this construct. To examine the role of V189-cleavage site (Figure 2A) in C-terminal fragment release and activation of Wss1, we produced a Wss1 mutant where the GKG residues preceding V189 were replaced by AQA (Figure 2—figure supplement 3). Upon thiram treatment, the AQA mutant efficiently self-processed, though producing different proteolytic fragments (Figure 2—figure supplement 3). This result suggests that the primary sequence within this protein region is not essential for Wss1 activation.


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)

Cleavage of Wss1-AQA mutant.Partially purified HA-Wss1-AQA mutant (500 μg/ml of total protein) and wild-type protein (100 μg/ml) were incubated for the indicated time at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide) or 0.5 mM thiram (Th) was added at the beginning of the incubation. The reaction was analyzed by gel electrophoresis and Coomassie staining and Wss1 protein fragments were analyzed using Adobe Photoshop software. The positions of common (black) and unique (false-colored) fragments are shown for AQA mutant (blue) and WT protein (red).DOI:http://dx.doi.org/10.7554/eLife.06763.009
© Copyright Policy
Related In: Results  -  Collection

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

fig2s3: Cleavage of Wss1-AQA mutant.Partially purified HA-Wss1-AQA mutant (500 μg/ml of total protein) and wild-type protein (100 μg/ml) were incubated for the indicated time at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide) or 0.5 mM thiram (Th) was added at the beginning of the incubation. The reaction was analyzed by gel electrophoresis and Coomassie staining and Wss1 protein fragments were analyzed using Adobe Photoshop software. The positions of common (black) and unique (false-colored) fragments are shown for AQA mutant (blue) and WT protein (red).DOI:http://dx.doi.org/10.7554/eLife.06763.009
Mentions: Together these data suggest that, like some other metalloproteases, Wss1 is regulated by a cysteine-switch mechanism. Displacement of the cysteine from the active site Zn induces autoproteolytic cleavage and may serve to release an inhibitory protein fragment. Wss1 has three cysteine residues, two of which, though located within the WLM domain, are far from the active site (Figure 2—figure supplement 2C). Moreover, their orientation suggests that they may form a disulfide bridge, though the estimated distance of 5.6 Å implies a conformational change would be necessary. The third cysteine, C226, is located in a negatively charged C-terminal part of the protein that was released by self-cleavage. This residue is conserved within the WLM family (Figure 1—figure supplement 1) and is a perfect candidate for the regulatory cysteine. Unfortunately, we were unable to produce soluble C226S protein, probably because of rapid auto-proteolysis of this construct. To examine the role of V189-cleavage site (Figure 2A) in C-terminal fragment release and activation of Wss1, we produced a Wss1 mutant where the GKG residues preceding V189 were replaced by AQA (Figure 2—figure supplement 3). Upon thiram treatment, the AQA mutant efficiently self-processed, though producing different proteolytic fragments (Figure 2—figure supplement 3). This result suggests that the primary sequence within this protein region is not essential for Wss1 activation.

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