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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.


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Wss1 cleavage of SUMO1-FP and His6-Ub-SUMO-HA substrates.(A) Proteolysis of SUMO1-FP substrate. Fluorescence anisotropy measurements with 100 nM SUMO1-FP substrate were performed on a MOS-450 spectrometer (BioLogic, Inc.) in a 150-μl quartz cuvette at 25°C essentially as described (Geurink et al., 2012). The proteolysis was initiated at 200 s by addition of 1 μg/ml (final concentration) of SUMO isopeptidase Ulp1, or 20 μg/ml of HA-Wss1 with or without 0.5 mM thiram (Th). (B) Proteolysis of His6-Ub-SUMO-HA substrate. Purified His6-Ub-SUMO-HA substrate (100 μg/ml) was incubated with 100 μg/ml of recombinant HA-Wss1 (Wss1), 50 μg/ml of SUMO isopeptidase Ulp1, or 50 μg/ml of Ub isopeptidase Usp2 (catalytic domain) for 3 hr at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide), 0.5 mM thiram (Th), or 3 mM OPA was added. The reaction was analyzed by western blotting with α-SUMO antibody.DOI:http://dx.doi.org/10.7554/eLife.06763.015
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fig3s1: Wss1 cleavage of SUMO1-FP and His6-Ub-SUMO-HA substrates.(A) Proteolysis of SUMO1-FP substrate. Fluorescence anisotropy measurements with 100 nM SUMO1-FP substrate were performed on a MOS-450 spectrometer (BioLogic, Inc.) in a 150-μl quartz cuvette at 25°C essentially as described (Geurink et al., 2012). The proteolysis was initiated at 200 s by addition of 1 μg/ml (final concentration) of SUMO isopeptidase Ulp1, or 20 μg/ml of HA-Wss1 with or without 0.5 mM thiram (Th). (B) Proteolysis of His6-Ub-SUMO-HA substrate. Purified His6-Ub-SUMO-HA substrate (100 μg/ml) was incubated with 100 μg/ml of recombinant HA-Wss1 (Wss1), 50 μg/ml of SUMO isopeptidase Ulp1, or 50 μg/ml of Ub isopeptidase Usp2 (catalytic domain) for 3 hr at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide), 0.5 mM thiram (Th), or 3 mM OPA was added. The reaction was analyzed by western blotting with α-SUMO antibody.DOI:http://dx.doi.org/10.7554/eLife.06763.015

Mentions: (A) Activation of Wss1 cleavage by 4-aminophenylmercuric acetate (APMA). Recombinant HA-Wss1 protein (200 μg/ml) was incubated with or without 1 mM APMA for the indicated time at 25°C. Where indicated 3 mM OPA was added at the beginning of the incubation. The reaction was analyzed by non-reducing gel electrophoresis and Coomassie staining and quantified by ImageJ. The positions of full-length HA-Wss1 (FL), large N-terminal fragment (N-Wss1), small cleavage products (asterisk), and HA-Wss1 oligomers (Wss1)n are shown. (B) Kinetics of Wss1 cleavage. Diagram shows the time course of the level of full-length HA-Wss1 at various conditions. The data are obtained by ImageJ quantification of the experiments shown on Figure 3B and Figure 3—figure supplement 1A. (C) Localization of the cysteines C96 and C108 within the WLM domain. Their mutual orientation suggest possible disulfide bond, though the estimated distance of 5.6 A is too long. The cysteines are shown in yellow. The active site Zn ligands are shown in red. The conserved structural hydrophobic residue of the active site is shown in green. The same WLM structure as in Figure 1—figure supplement 2 is used.


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)

Wss1 cleavage of SUMO1-FP and His6-Ub-SUMO-HA substrates.(A) Proteolysis of SUMO1-FP substrate. Fluorescence anisotropy measurements with 100 nM SUMO1-FP substrate were performed on a MOS-450 spectrometer (BioLogic, Inc.) in a 150-μl quartz cuvette at 25°C essentially as described (Geurink et al., 2012). The proteolysis was initiated at 200 s by addition of 1 μg/ml (final concentration) of SUMO isopeptidase Ulp1, or 20 μg/ml of HA-Wss1 with or without 0.5 mM thiram (Th). (B) Proteolysis of His6-Ub-SUMO-HA substrate. Purified His6-Ub-SUMO-HA substrate (100 μg/ml) was incubated with 100 μg/ml of recombinant HA-Wss1 (Wss1), 50 μg/ml of SUMO isopeptidase Ulp1, or 50 μg/ml of Ub isopeptidase Usp2 (catalytic domain) for 3 hr at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide), 0.5 mM thiram (Th), or 3 mM OPA was added. The reaction was analyzed by western blotting with α-SUMO antibody.DOI:http://dx.doi.org/10.7554/eLife.06763.015
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559962&req=5

fig3s1: Wss1 cleavage of SUMO1-FP and His6-Ub-SUMO-HA substrates.(A) Proteolysis of SUMO1-FP substrate. Fluorescence anisotropy measurements with 100 nM SUMO1-FP substrate were performed on a MOS-450 spectrometer (BioLogic, Inc.) in a 150-μl quartz cuvette at 25°C essentially as described (Geurink et al., 2012). The proteolysis was initiated at 200 s by addition of 1 μg/ml (final concentration) of SUMO isopeptidase Ulp1, or 20 μg/ml of HA-Wss1 with or without 0.5 mM thiram (Th). (B) Proteolysis of His6-Ub-SUMO-HA substrate. Purified His6-Ub-SUMO-HA substrate (100 μg/ml) was incubated with 100 μg/ml of recombinant HA-Wss1 (Wss1), 50 μg/ml of SUMO isopeptidase Ulp1, or 50 μg/ml of Ub isopeptidase Usp2 (catalytic domain) for 3 hr at 25°C. Where indicated 2.5 μM DNA (DNA, 70b mbpTop1d oligonucleotide), 0.5 mM thiram (Th), or 3 mM OPA was added. The reaction was analyzed by western blotting with α-SUMO antibody.DOI:http://dx.doi.org/10.7554/eLife.06763.015
Mentions: (A) Activation of Wss1 cleavage by 4-aminophenylmercuric acetate (APMA). Recombinant HA-Wss1 protein (200 μg/ml) was incubated with or without 1 mM APMA for the indicated time at 25°C. Where indicated 3 mM OPA was added at the beginning of the incubation. The reaction was analyzed by non-reducing gel electrophoresis and Coomassie staining and quantified by ImageJ. The positions of full-length HA-Wss1 (FL), large N-terminal fragment (N-Wss1), small cleavage products (asterisk), and HA-Wss1 oligomers (Wss1)n are shown. (B) Kinetics of Wss1 cleavage. Diagram shows the time course of the level of full-length HA-Wss1 at various conditions. The data are obtained by ImageJ quantification of the experiments shown on Figure 3B and Figure 3—figure supplement 1A. (C) Localization of the cysteines C96 and C108 within the WLM domain. Their mutual orientation suggest possible disulfide bond, though the estimated distance of 5.6 A is too long. The cysteines are shown in yellow. The active site Zn ligands are shown in red. The conserved structural hydrophobic residue of the active site is shown in green. The same WLM structure as in Figure 1—figure supplement 2 is used.

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