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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 ubiquitin modifies proteins in monosome and polysome fractions(a,b), Sucrose sedimentation profiles of polysomes from the (a) WT and (b) K63R mutant cells extracted using a physiological MgCl2 concentration (3 mM). (c–e) Anti-K63 ubiquitin western blot from stabilized polysomes extracted using 30 mM MgCl2 from (c) WT and (d) K63R mutant cells or (e) WTcol, rad6Δ and bre1Δ cells. Ponceau-S loading control is shown in Supplementary Fig. 5. (*) Half of the sample volume was loaded for better visualization. WT, wild-type SUB280 yeast strain, K63R, ubiquitin K63R mutant SUB413 yeast strain. WTcol, wild-type cells S288c used with the deletion collection. MW, molecular weight.
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Figure 5: K63 ubiquitin modifies proteins in monosome and polysome fractions(a,b), Sucrose sedimentation profiles of polysomes from the (a) WT and (b) K63R mutant cells extracted using a physiological MgCl2 concentration (3 mM). (c–e) Anti-K63 ubiquitin western blot from stabilized polysomes extracted using 30 mM MgCl2 from (c) WT and (d) K63R mutant cells or (e) WTcol, rad6Δ and bre1Δ cells. Ponceau-S loading control is shown in Supplementary Fig. 5. (*) Half of the sample volume was loaded for better visualization. WT, wild-type SUB280 yeast strain, K63R, ubiquitin K63R mutant SUB413 yeast strain. WTcol, wild-type cells S288c used with the deletion collection. MW, molecular weight.

Mentions: Next, we showed that ribosomal K63 ubiquitination under oxidative stress appears to help stabilizing the complete 80S complex and the formation of polysomes. When we monitored polysome levels by sucrose sedimentation profiling at physiological ionic strength (3 mM MgCl2), the K63R mutant displayed strongly increased peaks of the unassembled 40S and 60S subunits (Fig. 5). H2O2 is known to reduce the levels of polysomes43, however the K63R mutant subjected to H2O2 almost completely lacked polysomes, compared to the wild-type strain with largely intact polysomes (Fig. 5a,b). This phenotype was partially rescued at high-ionic strength which inhibits ribonucleases and stabilizes polysomes (30 mM MgCl2, Supplementary Fig. 5a). Using this setup, we further validated ribosomal K63 ubiquitination in two different background strains (Figure 5c-e). The majority of K63 labels was distributed across both the monosomal and polysomal fractions in a WT yeast strain exposed to H2O2 (Fig. 5c), and, expectedly, no meaningful K63 ubiquitin labeling was detected in the K63R strain treated with H2O2 (Fig. 5d). However, the K63R mutant strain still displayed K48 ubiquitin in the polysomal fraction (Supplementary Fig. 5c). Importantly, rad6Δ and bre1Δ strains did not show detectable levels of K63 ubiquitination in polysomes after H2O2 treatment (Fig. 5e), corroborating the role of Rad6-Bre1 in mediating K63 polyubiquitination of ribosomal proteins in response to oxidative stress.


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

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

K63 ubiquitin modifies proteins in monosome and polysome fractions(a,b), Sucrose sedimentation profiles of polysomes from the (a) WT and (b) K63R mutant cells extracted using a physiological MgCl2 concentration (3 mM). (c–e) Anti-K63 ubiquitin western blot from stabilized polysomes extracted using 30 mM MgCl2 from (c) WT and (d) K63R mutant cells or (e) WTcol, rad6Δ and bre1Δ cells. Ponceau-S loading control is shown in Supplementary Fig. 5. (*) Half of the sample volume was loaded for better visualization. WT, wild-type SUB280 yeast strain, K63R, ubiquitin K63R mutant SUB413 yeast strain. WTcol, wild-type cells S288c used with the deletion collection. MW, molecular weight.
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Related In: Results  -  Collection

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Figure 5: K63 ubiquitin modifies proteins in monosome and polysome fractions(a,b), Sucrose sedimentation profiles of polysomes from the (a) WT and (b) K63R mutant cells extracted using a physiological MgCl2 concentration (3 mM). (c–e) Anti-K63 ubiquitin western blot from stabilized polysomes extracted using 30 mM MgCl2 from (c) WT and (d) K63R mutant cells or (e) WTcol, rad6Δ and bre1Δ cells. Ponceau-S loading control is shown in Supplementary Fig. 5. (*) Half of the sample volume was loaded for better visualization. WT, wild-type SUB280 yeast strain, K63R, ubiquitin K63R mutant SUB413 yeast strain. WTcol, wild-type cells S288c used with the deletion collection. MW, molecular weight.
Mentions: Next, we showed that ribosomal K63 ubiquitination under oxidative stress appears to help stabilizing the complete 80S complex and the formation of polysomes. When we monitored polysome levels by sucrose sedimentation profiling at physiological ionic strength (3 mM MgCl2), the K63R mutant displayed strongly increased peaks of the unassembled 40S and 60S subunits (Fig. 5). H2O2 is known to reduce the levels of polysomes43, however the K63R mutant subjected to H2O2 almost completely lacked polysomes, compared to the wild-type strain with largely intact polysomes (Fig. 5a,b). This phenotype was partially rescued at high-ionic strength which inhibits ribonucleases and stabilizes polysomes (30 mM MgCl2, Supplementary Fig. 5a). Using this setup, we further validated ribosomal K63 ubiquitination in two different background strains (Figure 5c-e). The majority of K63 labels was distributed across both the monosomal and polysomal fractions in a WT yeast strain exposed to H2O2 (Fig. 5c), and, expectedly, no meaningful K63 ubiquitin labeling was detected in the K63R strain treated with H2O2 (Fig. 5d). However, the K63R mutant strain still displayed K48 ubiquitin in the polysomal fraction (Supplementary Fig. 5c). Importantly, rad6Δ and bre1Δ strains did not show detectable levels of K63 ubiquitination in polysomes after H2O2 treatment (Fig. 5e), corroborating the role of Rad6-Bre1 in mediating K63 polyubiquitination of ribosomal proteins in response to oxidative stress.

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