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K63 polyubiquitination is a new modulator of the oxidative stress response.

Silva GM, Finley D, Vogel C - Nat. Struct. Mol. Biol. (2015)

Bottom Line: We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase.Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins.Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics and Systems Biology, New York University, New York, New York, USA.

ABSTRACT
Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.

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K63 ubiquitination is linked to active translation(a–c) Anti-K63 ubiquitin western blot of lysates from (a) WT, gcn2Δ and K63R mutant cells, (b) WT cells treated for 30 min with designated translation inhibitors prior to H2O2 treatment and (c) WT cells grown into Log phase OD600 = 0.4 or after 24 h in culture starting from OD600 = 0.2 (Stationary), in the presence (+) or absence (–) of the indicated compounds. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
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Figure 6: K63 ubiquitination is linked to active translation(a–c) Anti-K63 ubiquitin western blot of lysates from (a) WT, gcn2Δ and K63R mutant cells, (b) WT cells treated for 30 min with designated translation inhibitors prior to H2O2 treatment and (c) WT cells grown into Log phase OD600 = 0.4 or after 24 h in culture starting from OD600 = 0.2 (Stationary), in the presence (+) or absence (–) of the indicated compounds. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.

Mentions: Further, we found that K63 ubiquitination may be a new factor promoting translation during oxidative stress through polysome stabilization, and that K63 levels are affected by perturbation of translation regulatory mechanisms. For example, GCN2 encodes the kinase that inhibits translation in response to oxidative stress by phosphorylating the alpha subunit of the eukaryotic translation initiation factor 2(ref. 2324). We found that the Δgcn2 strain presents a high level of basal K63 ubiquitination, which is further enhanced in the presence of H2O2 (Fig. 6a). Moreover, cellular treatment with 5 μg/ml puromycin, a translation inhibitor which prematurely terminates protein synthesis by releasing ribosomes, increased the amount of K63 ubiquitin under stress (Fig. 6b). In comparison, the addition of 0.2 μg/ml rapamycin, which prevents translation initiation, and 200 μg/ml cycloheximide, which locks the ribosomes onto the mRNA and halts elongation, did not lead to increased K63 ubiquitin under stress (Fig. 6b). Our results also showed that K63 ubiquitin accumulates in cells grown to stationary phase even in the absence of exogenous H2O2, a physiological model of oxidative stress where translation is also inhibited (Fig. 6c). Interestingly, the levels of K63 ubiquitin under H2O2 treatment were much higher in cells grown to stationary phase than in cells grown to log phase.


K63 polyubiquitination is a new modulator of the oxidative stress response.

Silva GM, Finley D, Vogel C - Nat. Struct. Mol. Biol. (2015)

K63 ubiquitination is linked to active translation(a–c) Anti-K63 ubiquitin western blot of lysates from (a) WT, gcn2Δ and K63R mutant cells, (b) WT cells treated for 30 min with designated translation inhibitors prior to H2O2 treatment and (c) WT cells grown into Log phase OD600 = 0.4 or after 24 h in culture starting from OD600 = 0.2 (Stationary), in the presence (+) or absence (–) of the indicated compounds. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
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Figure 6: K63 ubiquitination is linked to active translation(a–c) Anti-K63 ubiquitin western blot of lysates from (a) WT, gcn2Δ and K63R mutant cells, (b) WT cells treated for 30 min with designated translation inhibitors prior to H2O2 treatment and (c) WT cells grown into Log phase OD600 = 0.4 or after 24 h in culture starting from OD600 = 0.2 (Stationary), in the presence (+) or absence (–) of the indicated compounds. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
Mentions: Further, we found that K63 ubiquitination may be a new factor promoting translation during oxidative stress through polysome stabilization, and that K63 levels are affected by perturbation of translation regulatory mechanisms. For example, GCN2 encodes the kinase that inhibits translation in response to oxidative stress by phosphorylating the alpha subunit of the eukaryotic translation initiation factor 2(ref. 2324). We found that the Δgcn2 strain presents a high level of basal K63 ubiquitination, which is further enhanced in the presence of H2O2 (Fig. 6a). Moreover, cellular treatment with 5 μg/ml puromycin, a translation inhibitor which prematurely terminates protein synthesis by releasing ribosomes, increased the amount of K63 ubiquitin under stress (Fig. 6b). In comparison, the addition of 0.2 μg/ml rapamycin, which prevents translation initiation, and 200 μg/ml cycloheximide, which locks the ribosomes onto the mRNA and halts elongation, did not lead to increased K63 ubiquitin under stress (Fig. 6b). Our results also showed that K63 ubiquitin accumulates in cells grown to stationary phase even in the absence of exogenous H2O2, a physiological model of oxidative stress where translation is also inhibited (Fig. 6c). Interestingly, the levels of K63 ubiquitin under H2O2 treatment were much higher in cells grown to stationary phase than in cells grown to log phase.

Bottom Line: We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase.Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins.Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics and Systems Biology, New York University, New York, New York, USA.

ABSTRACT
Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.

Show MeSH
Related in: MedlinePlus