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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 polyubiquitin transiently accumulates in response to H2O2a, Anti-K63- and K48- specific ubiquitin western blot of lysates from WT and K63R cells upon treatment with, and subsequent recovery from, 0.6 mM H2O2. b, Anti-K63 ubiquitin western blot of lysate from WT cells treated with H2O2 for different amounts of time. c, Histogram showing dynamics of K63 and K48 ubiquitin linkages measured by quantitative targeted mass spectrometry. Plot shows mean of two biological replicates with two technical replicates each, and error bars indicate the range of values across the replicates. d, Anti-K63 and anti-K48 ubiquitin western blot of lysate from WT cells subjected to indicated compounds and heat-shock for designated times. e, Anti-K63 ubiquitin western blot of lysate from WT cells treated with the indicated oxidizing agents for 30 min. 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 1: K63 polyubiquitin transiently accumulates in response to H2O2a, Anti-K63- and K48- specific ubiquitin western blot of lysates from WT and K63R cells upon treatment with, and subsequent recovery from, 0.6 mM H2O2. b, Anti-K63 ubiquitin western blot of lysate from WT cells treated with H2O2 for different amounts of time. c, Histogram showing dynamics of K63 and K48 ubiquitin linkages measured by quantitative targeted mass spectrometry. Plot shows mean of two biological replicates with two technical replicates each, and error bars indicate the range of values across the replicates. d, Anti-K63 and anti-K48 ubiquitin western blot of lysate from WT cells subjected to indicated compounds and heat-shock for designated times. e, Anti-K63 ubiquitin western blot of lysate from WT cells treated with the indicated oxidizing agents for 30 min. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.

Mentions: We set out to characterize the role of polyubiquitination during the oxidative stress response, and monitored the dynamics of the three most abundant ubiquitin linkages (K11, K48 and K63) in a wild-type yeast strain expressing a single ubiquitin gene (WT - SUB280). While both K48 and K63 ubiquitin responded strongly and rapidly to H2O2 treatment (Fig. 1a and Supplementary Fig. 1a, b), K11 response was very weak and seemed limited to few targets (Supplementary Fig. 1c). K48 levels sustained over four hours in the recovery medium, but K63 polyubiquitination rose and declined rapidly, falling below detection levels immediately during the recovery phase in fresh medium (Fig. 1a) or after 90 min of prolonged incubation with H2O2 (Fig. 1b). This strong pulse of K63 ubiquitination during the oxidative stress response has not been reported before.


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

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

K63 polyubiquitin transiently accumulates in response to H2O2a, Anti-K63- and K48- specific ubiquitin western blot of lysates from WT and K63R cells upon treatment with, and subsequent recovery from, 0.6 mM H2O2. b, Anti-K63 ubiquitin western blot of lysate from WT cells treated with H2O2 for different amounts of time. c, Histogram showing dynamics of K63 and K48 ubiquitin linkages measured by quantitative targeted mass spectrometry. Plot shows mean of two biological replicates with two technical replicates each, and error bars indicate the range of values across the replicates. d, Anti-K63 and anti-K48 ubiquitin western blot of lysate from WT cells subjected to indicated compounds and heat-shock for designated times. e, Anti-K63 ubiquitin western blot of lysate from WT cells treated with the indicated oxidizing agents for 30 min. 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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Related In: Results  -  Collection

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Figure 1: K63 polyubiquitin transiently accumulates in response to H2O2a, Anti-K63- and K48- specific ubiquitin western blot of lysates from WT and K63R cells upon treatment with, and subsequent recovery from, 0.6 mM H2O2. b, Anti-K63 ubiquitin western blot of lysate from WT cells treated with H2O2 for different amounts of time. c, Histogram showing dynamics of K63 and K48 ubiquitin linkages measured by quantitative targeted mass spectrometry. Plot shows mean of two biological replicates with two technical replicates each, and error bars indicate the range of values across the replicates. d, Anti-K63 and anti-K48 ubiquitin western blot of lysate from WT cells subjected to indicated compounds and heat-shock for designated times. e, Anti-K63 ubiquitin western blot of lysate from WT cells treated with the indicated oxidizing agents for 30 min. Anti-GAPDH was used as loading control. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
Mentions: We set out to characterize the role of polyubiquitination during the oxidative stress response, and monitored the dynamics of the three most abundant ubiquitin linkages (K11, K48 and K63) in a wild-type yeast strain expressing a single ubiquitin gene (WT - SUB280). While both K48 and K63 ubiquitin responded strongly and rapidly to H2O2 treatment (Fig. 1a and Supplementary Fig. 1a, b), K11 response was very weak and seemed limited to few targets (Supplementary Fig. 1c). K48 levels sustained over four hours in the recovery medium, but K63 polyubiquitination rose and declined rapidly, falling below detection levels immediately during the recovery phase in fresh medium (Fig. 1a) or after 90 min of prolonged incubation with H2O2 (Fig. 1b). This strong pulse of K63 ubiquitination during the oxidative stress response has not been reported before.

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