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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 the regulation of Wss1 protease activity by cysteine switch mechanism.(I) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. (II) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione (Figure 3D). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. (III) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c).DOI:http://dx.doi.org/10.7554/eLife.06763.010
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fig2s4: Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism.(I) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. (II) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione (Figure 3D). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. (III) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c).DOI:http://dx.doi.org/10.7554/eLife.06763.010

Mentions: DNA may activate Wss1 in two ways (Figure 2—figure supplement 4). First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement from the active site of the negatively charged C-terminal peptide bearing the inhibitory cysteine. Additionally, DNA may facilitate Wss1 oligomerization and greatly promote proteolysis ‘in-trans’. Both processes may be linked in a ‘cooperative mechanism’ (Figure 2—figure supplement 4). The initial activation events seem to be rate-limiting in the DNA-dependent pathway and may account for an observed lag phase in Wss1 cleavage (Figure 2C). Indeed thiram greatly accelerates DNA-dependent Wss1 proteolysis suggesting a cooperative interaction between ‘in-cis’ and ‘in-trans’ cleavage (Figure 2B,C).


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 the regulation of Wss1 protease activity by cysteine switch mechanism.(I) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. (II) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione (Figure 3D). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. (III) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c).DOI:http://dx.doi.org/10.7554/eLife.06763.010
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559962&req=5

fig2s4: Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism.(I) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. (II) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione (Figure 3D). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. (III) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c).DOI:http://dx.doi.org/10.7554/eLife.06763.010
Mentions: DNA may activate Wss1 in two ways (Figure 2—figure supplement 4). First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement from the active site of the negatively charged C-terminal peptide bearing the inhibitory cysteine. Additionally, DNA may facilitate Wss1 oligomerization and greatly promote proteolysis ‘in-trans’. Both processes may be linked in a ‘cooperative mechanism’ (Figure 2—figure supplement 4). The initial activation events seem to be rate-limiting in the DNA-dependent pathway and may account for an observed lag phase in Wss1 cleavage (Figure 2C). Indeed thiram greatly accelerates DNA-dependent Wss1 proteolysis suggesting a cooperative interaction between ‘in-cis’ and ‘in-trans’ cleavage (Figure 2B,C).

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