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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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Lack of K63 ubiquitin impacts cellular resistance to oxidative stressa, Anti-DNP (oxidation), anti-K63 and anti-K48 ubiquitin western blots of cell lysate from WT and K63R mutant cells upon treatment with, and subsequent recovery from, 4 mM H2O2. Anti-GAPDH was used as loading control. b, Serial dilution assays from WT and K63R mutant cells treated with different amounts of H2O2. After stress induction cells were spotted onto YPD plates without H2O2. c, Correlation plot showing log base 2 SILAC ratio K63R/WT for the mass spectrometry data from cell lysate replicates. We highlighted individual examples of stress-related proteins with decreased expression to less than 25 % in the K63R mutant compared to the levels found in the WT. Pearson correlation coefficient is 0.60. d, Model of the role of K63 polyubiquitination during the translation cycle in response to H2O2. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
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Figure 7: Lack of K63 ubiquitin impacts cellular resistance to oxidative stressa, Anti-DNP (oxidation), anti-K63 and anti-K48 ubiquitin western blots of cell lysate from WT and K63R mutant cells upon treatment with, and subsequent recovery from, 4 mM H2O2. Anti-GAPDH was used as loading control. b, Serial dilution assays from WT and K63R mutant cells treated with different amounts of H2O2. After stress induction cells were spotted onto YPD plates without H2O2. c, Correlation plot showing log base 2 SILAC ratio K63R/WT for the mass spectrometry data from cell lysate replicates. We highlighted individual examples of stress-related proteins with decreased expression to less than 25 % in the K63R mutant compared to the levels found in the WT. Pearson correlation coefficient is 0.60. d, Model of the role of K63 polyubiquitination during the translation cycle in response to H2O2. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.

Mentions: Next, we investigated whether K63 polyubiquitination is important for cellular resistance to oxidative stress. In cells deficient of K63 ubiquitination, high levels of H2O2 (4 mM) increased the amount of oxidized, and therefore, damaged proteins compared to the wild-type (Fig. 7a). This condition also triggered high levels of K48 ubiquitinated proteins which presumably were en route to proteasomal degradation. At 0.6 mM H2O2, which is the concentration used throughout our study, the K63R and WT strains accumulated similar amounts of oxidized proteins (Supplementary Fig. 6a). These results suggest that, consistent with impaired translation as indicated by dissociated polysomes and higher levels of oxidative damage, K63R cells are more sensitive to stress than wild-type cells (Fig. 7b).


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

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

Lack of K63 ubiquitin impacts cellular resistance to oxidative stressa, Anti-DNP (oxidation), anti-K63 and anti-K48 ubiquitin western blots of cell lysate from WT and K63R mutant cells upon treatment with, and subsequent recovery from, 4 mM H2O2. Anti-GAPDH was used as loading control. b, Serial dilution assays from WT and K63R mutant cells treated with different amounts of H2O2. After stress induction cells were spotted onto YPD plates without H2O2. c, Correlation plot showing log base 2 SILAC ratio K63R/WT for the mass spectrometry data from cell lysate replicates. We highlighted individual examples of stress-related proteins with decreased expression to less than 25 % in the K63R mutant compared to the levels found in the WT. Pearson correlation coefficient is 0.60. d, Model of the role of K63 polyubiquitination during the translation cycle in response to H2O2. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
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Figure 7: Lack of K63 ubiquitin impacts cellular resistance to oxidative stressa, Anti-DNP (oxidation), anti-K63 and anti-K48 ubiquitin western blots of cell lysate from WT and K63R mutant cells upon treatment with, and subsequent recovery from, 4 mM H2O2. Anti-GAPDH was used as loading control. b, Serial dilution assays from WT and K63R mutant cells treated with different amounts of H2O2. After stress induction cells were spotted onto YPD plates without H2O2. c, Correlation plot showing log base 2 SILAC ratio K63R/WT for the mass spectrometry data from cell lysate replicates. We highlighted individual examples of stress-related proteins with decreased expression to less than 25 % in the K63R mutant compared to the levels found in the WT. Pearson correlation coefficient is 0.60. d, Model of the role of K63 polyubiquitination during the translation cycle in response to H2O2. WT, wild-type SUB280 yeast strain. K63R, ubiquitin K63R mutant SUB413 yeast strain. MW, molecular weight.
Mentions: Next, we investigated whether K63 polyubiquitination is important for cellular resistance to oxidative stress. In cells deficient of K63 ubiquitination, high levels of H2O2 (4 mM) increased the amount of oxidized, and therefore, damaged proteins compared to the wild-type (Fig. 7a). This condition also triggered high levels of K48 ubiquitinated proteins which presumably were en route to proteasomal degradation. At 0.6 mM H2O2, which is the concentration used throughout our study, the K63R and WT strains accumulated similar amounts of oxidized proteins (Supplementary Fig. 6a). These results suggest that, consistent with impaired translation as indicated by dissociated polysomes and higher levels of oxidative damage, K63R cells are more sensitive to stress than wild-type cells (Fig. 7b).

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