Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb;22(2):116-23.
doi: 10.1038/nsmb.2955. Epub 2015 Jan 26.

K63 polyubiquitination is a new modulator of the oxidative stress response

Gustavo M Silva  1 , Daniel Finley  2 , Christine Vogel  1
Affiliations

K63 polyubiquitination is a new modulator of the oxidative stress response

Gustavo M Silva et al. Nat Struct Mol Biol. 2015 Feb.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. K63 polyubiquitin transiently accumulates in response to H2O2
a, 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.
Figure 2
Figure 2. Rad6-Bre1 conjugate K63 polyubiquitination in response to H2O2
(a,b) Anti-K63 ubiquitin western blot of lysates from (a) E2 and (b) Rad6-interacting-E3 deleted cells in the presence and absence of 0.6 mM H2O2. Anti-GAPDH was used as loading control. WTcol, wild-type cells S288c used with the deletion collection. MW, molecular weight.
Figure 3
Figure 3. Ubp2 is reversibly inhibited by H2O2
a, Histogram with activity of intracellular DUBs from WT cells treated with the indicated concentrations of H2O2. Activity was measured after incubation of cellular lysate with the Ub-AMC fluorogenic substrate. Error bar, s.d. (P values were calculated using paired, one-tailed Student's t-test, n = 3 independent cell growth). (b,c) Anti-K63 ubiquitin western blot of lysates from (b) DUB deleted cells in the presence or absence of H2O2 and (c) WT and ubp2Δ after H2O2 treatment for different amount of times. d, Scatter plot with activity of native purified TAP-tagged Ubp2 (black) and 0.5 mM H2O2-treated Ubp2 (grey), measured after incubation with Ub-AMC fluorogenic substrate for the indicated times. Arrow indicates addition of 10 mM DTT. e, SDS-PAGE gel of 1μg K63 tetra-ubiquitin chains after incubation for the indicated times with purified Ubp2 treated with 10 mM DTT or 0.5 mM H2O2. f, Anti-K63 ubiquitin blot of lysate from cells treated with H2O2 after incubation with native or H2O2-treated TAP-tagged Ubp2. Samples were incubated without DTT to prevent activation of intracellular DUBs. Anti-GAPDH was used as loading control. WTDUBs, wild-type yeast strain SUB62. a.u., fluorescence arbitrary units. MW, molecular weight.
Figure 4
Figure 4. Ribosomal proteins are a main target of K63 polyubiquitination under H2O2 stress
a, 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) is represented in light grey and the 40S small unit (PDB ID 3O2Z) 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.
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7. Lack of K63 ubiquitin impacts cellular resistance to oxidative stress
a, 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.
See this image and copyright information in PMC

References

    1. Herrero E, Ros J, Belli G, Cabiscol E. Redox control and oxidative stress in yeast cells. Biochim Biophys Acta. 2008;1780:1217–1235. - PubMed
    1. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual review of plant biology. 2004;55:373–399. - PubMed
    1. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annual review of pharmacology and toxicology. 2004;44:239–267. - PubMed
    1. Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82:47–95. - PubMed
    1. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239–247. - PubMed

Publication types

MeSH terms