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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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Ribosomal proteins are a main target of K63 polyubiquitination under H2O2 stressa, Scheme describing the SILAC LC-MS/MS approach used to identify and quantify K63 conjugates by high-resolution mass spectrometry. b, Chart showing gene ontology (GO) annotation for K63 ubiquitinated targets using DAVID functional annotation tool. (*) GO enrichment significant at FDR < 0.005 %. c, Surface 3D structure shows mapping of ribosomal proteins modified by K63 ubiquitination (blue) onto the 80S ribosome particle. The 60S large unit (PDB ID 3O58)59 is represented in light grey and the 40S small unit (PDB ID 3O2Z)59 is in dark grey. In pink, we highlight proteins in the mRNA-tRNA interaction sites (top panel) and in the ribosome exit tunnel (bottom panel). WT, wild-type SILAC GMS280 yeast strain. K63R, ubiquitin K63R mutant SILAC GMS413 yeast strain.
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Figure 4: Ribosomal proteins are a main target of K63 polyubiquitination under H2O2 stressa, Scheme describing the SILAC LC-MS/MS approach used to identify and quantify K63 conjugates by high-resolution mass spectrometry. b, Chart showing gene ontology (GO) annotation for K63 ubiquitinated targets using DAVID functional annotation tool. (*) GO enrichment significant at FDR < 0.005 %. c, Surface 3D structure shows mapping of ribosomal proteins modified by K63 ubiquitination (blue) onto the 80S ribosome particle. The 60S large unit (PDB ID 3O58)59 is represented in light grey and the 40S small unit (PDB ID 3O2Z)59 is in dark grey. In pink, we highlight proteins in the mRNA-tRNA interaction sites (top panel) and in the ribosome exit tunnel (bottom panel). WT, wild-type SILAC GMS280 yeast strain. K63R, ubiquitin K63R mutant SILAC GMS413 yeast strain.

Mentions: To further characterize the cellular role of K63 polyubiquitin during the oxidative stress response, we identified the K63-ubiquitinated targets by quantitative proteomics. To-date, no proteomics method was available to analyze targets of a specific ubiquitin linkage type. We developed a new approach in which we used the K63-TUBE isolation system (LifeSensors) to enrich for K63 linkages (Supplementary Fig. 4a), and subjected the proteins to LC-MS/MS analysis. To eliminate contaminants, validate true K63 conjugates, and reduce experimental biases, we performed a stable isotope labeling (SILAC)-based mass spectrometry experiment which paired the WT with the ubiquitin K63R strain (SUB413) after H2O2 treatment (Fig. 4a - see Methods and Supplementary Notes for details). The K63R strain expresses a ubiquitin mutant with lysine 63 substituted by arginine, which specifically prevents K63-chain formation (Fig. 1a). In this setup, we identified 115 potential K63 targets in two replicate experiments (Supplementary Table 1), as defined by an at least 50 % increase in abundance relative to the K63R negative control at a 5 % FDR (Supplementary Fig. 4b). In addition, in Supplementary Table 2 we described a core dataset of even higher confidence, reporting 52 K63-ubiquitinated targets identified by two independent search engines (MaxQuant and Proteome Discoverer).


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

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

Ribosomal proteins are a main target of K63 polyubiquitination under H2O2 stressa, Scheme describing the SILAC LC-MS/MS approach used to identify and quantify K63 conjugates by high-resolution mass spectrometry. b, Chart showing gene ontology (GO) annotation for K63 ubiquitinated targets using DAVID functional annotation tool. (*) GO enrichment significant at FDR < 0.005 %. c, Surface 3D structure shows mapping of ribosomal proteins modified by K63 ubiquitination (blue) onto the 80S ribosome particle. The 60S large unit (PDB ID 3O58)59 is represented in light grey and the 40S small unit (PDB ID 3O2Z)59 is in dark grey. In pink, we highlight proteins in the mRNA-tRNA interaction sites (top panel) and in the ribosome exit tunnel (bottom panel). WT, wild-type SILAC GMS280 yeast strain. K63R, ubiquitin K63R mutant SILAC GMS413 yeast strain.
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Figure 4: Ribosomal proteins are a main target of K63 polyubiquitination under H2O2 stressa, Scheme describing the SILAC LC-MS/MS approach used to identify and quantify K63 conjugates by high-resolution mass spectrometry. b, Chart showing gene ontology (GO) annotation for K63 ubiquitinated targets using DAVID functional annotation tool. (*) GO enrichment significant at FDR < 0.005 %. c, Surface 3D structure shows mapping of ribosomal proteins modified by K63 ubiquitination (blue) onto the 80S ribosome particle. The 60S large unit (PDB ID 3O58)59 is represented in light grey and the 40S small unit (PDB ID 3O2Z)59 is in dark grey. In pink, we highlight proteins in the mRNA-tRNA interaction sites (top panel) and in the ribosome exit tunnel (bottom panel). WT, wild-type SILAC GMS280 yeast strain. K63R, ubiquitin K63R mutant SILAC GMS413 yeast strain.
Mentions: To further characterize the cellular role of K63 polyubiquitin during the oxidative stress response, we identified the K63-ubiquitinated targets by quantitative proteomics. To-date, no proteomics method was available to analyze targets of a specific ubiquitin linkage type. We developed a new approach in which we used the K63-TUBE isolation system (LifeSensors) to enrich for K63 linkages (Supplementary Fig. 4a), and subjected the proteins to LC-MS/MS analysis. To eliminate contaminants, validate true K63 conjugates, and reduce experimental biases, we performed a stable isotope labeling (SILAC)-based mass spectrometry experiment which paired the WT with the ubiquitin K63R strain (SUB413) after H2O2 treatment (Fig. 4a - see Methods and Supplementary Notes for details). The K63R strain expresses a ubiquitin mutant with lysine 63 substituted by arginine, which specifically prevents K63-chain formation (Fig. 1a). In this setup, we identified 115 potential K63 targets in two replicate experiments (Supplementary Table 1), as defined by an at least 50 % increase in abundance relative to the K63R negative control at a 5 % FDR (Supplementary Fig. 4b). In addition, in Supplementary Table 2 we described a core dataset of even higher confidence, reporting 52 K63-ubiquitinated targets identified by two independent search engines (MaxQuant and Proteome Discoverer).

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