Polyubiquitin Chains Linked by Lysine Residue 48 (K48) Selectively Target Oxidized Proteins In Vivo

Abstract

Aims: Ubiquitin is a highly conserved protein modifier that heavily accumulates during the oxidative stress response. Here, we investigated the role of the ubiquitination system, particularly at the linkage level, in the degradation of oxidized proteins. The function of ubiquitin in the removal of oxidized proteins remains elusive because of the wide range of potential targets and different roles that polyubiquitin chains play. Therefore, we describe in detail the dynamics of the K48 ubiquitin response as the canonical signal for protein degradation. We identified ubiquitin targets and defined the relationship between protein ubiquitination and oxidation during the stress response. Results: Combining oxidized protein isolation, linkage-specific ubiquitination screens, and quantitative proteomics, we found that K48 ubiquitin accumulated at both the early and late phases of the stress response. We further showed that a fraction of oxidized proteins are conjugated with K48 ubiquitin. We identified ∼750 ubiquitinated proteins and ∼400 oxidized proteins that were modified during oxidative stress, and around half of which contain both modifications. These proteins were highly abundant and function in translation and energy metabolism. Innovation and Conclusion: Our work showed for the first time that K48 ubiquitin modifies a large fraction of oxidized proteins, demonstrating that oxidized proteins can be targeted by the ubiquitin/proteasome system. We suggest that oxidized proteins that rapidly accumulate during stress are subsequently ubiquitinated and degraded during the late phase of the response. This delay between oxidation and ubiquitination may be necessary for reprogramming protein dynamics, restoring proteostasis, and resuming cell growth.

Keywords: oxidation; oxidative stress; protein degradation; proteomics; ubiquitin.

FIG. 1.

Ubiquitin is required for the degradation of oxidized proteins during oxidative stress. (A) Schematic overview of the workflow used in this analysis. Yeast was permeabilized by incubation with 0.003% of SDS in MPD medium (40). When specified, cells were preincubated for 15 min with inhibitors before stress induction. Oxidative stress was induced for 45 min with 0.6 mM H₂O₂, and cells were transferred to fresh media and allowed to recover for 8 h. The equivalent concentrations of inhibitors were added to the recovery medium where applicable. (B) Whole-cell extract was immunoblotted (IB) and probed using distinct antibodies to monitor the dynamics of protein oxidation through carbonyl formation (anti-DNP), pan-ubiquitin, K48 ubiquitin, and the 20S proteasome (20SPT). MG-132 (75 μM) was used to inhibit proteasome activity, PYR-41 (75 μM) to inhibit E1 ubiquitin activation enzyme, and CHX (50 μg/mL) to inhibit translation. Anti-actin was used as a loading control. (C) Whole-cell extract from yeast cells was immunoblotted and probed using a K63 ubiquitin antibody. Anti-GAPDH was used as a loading control. (D) Proteasomal activity in cell lysates from yeast treated with the indicated inhibitors. Activity was measured following incubation of cellular lysate with 100 μM suc-LLVY-AMC fluorogenic substrate. Error bars, standard deviation. CHX, cycloheximide; DNP, dinitrophenyl; H₂O₂, hydrogen peroxide; MPD, minimum proline dextrose; SDS, sodium dodecyl sulfate; Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin.

FIG. 2.

Oxidized proteins are K48 polyubiquitinated. (A) Schematic overview of the workflow selected for oxidized and ubiquitinated protein enrichment and mass spectrometry analysis. Untreated (Control), H₂O₂ stress (Stress), and cells that recovered in fresh media for 2 h (Recovery) were used as representative samples of different stages of oxidative stress. The TUBE system was used to isolate ubiquitinated proteins, anti-DNP was used to isolate oxidized (carbonylated) proteins for blots, and biotin hydrazide was used to isolate oxidized proteins for mass spectrometry. (B) Ubiquitinated and (C) oxidized proteins were isolated by TUBE affinity purification and by anti-DNP immunoprecipitation, respectively. The input, the TUBE/DNP unbound and eluted fractions were immunoblotted against pan-ubiquitin (Ub) and oxidized proteins (DNP). In (C), beads were saturated with cell lysate to normalize the amount of oxidized proteins retrieved from each condition. Samples were immunoblotted against pan-ubiquitin, K63, K11, and K48. Anti-DNP blotting was used as loading control. *Immunoblotting for high-molecular-weight range (>50 kDa) was subject to longer exposure times. DNPH, 2,4-dinitrophenylhydrazine; LC-MS/MS, liquid chromatography with tandem mass spectrometry; TUBE, tandem ubiquitin binding entities.

FIG. 3.

Mass spectrometry identified proteins that are oxidized and ubiquitinated. (A) Matrix depicting the logarithmic ratio of expression changes for ubiquitin and oxidation during stress and recovery phases relative to the untreated cells. Yeast strain GMS413 containing the K63R ubiquitin mutation was cultivated in SILAC medium labeled with heavy isotopes for lysine and arginine (Control) and light isotopes for Stress and Recovery. Each row denotes one quantified protein. Hierarchical clustering was performed in Perseus, and the four largest clusters were analyzed for function enrichment using the DAVID annotation tool (FDR <0.02) (28). (B) Venn diagram displaying the overlap between populations of ubiquitinated and oxidized proteins. (C) Immunoblotting of selected proteins after ubiquitin (TUBE) and oxidation enrichment (biotin-hydrazide). Representative proteins Htb2 (14 kDa) from cluster B and Rpt1 (52 kDa) from cluster D were analyzed by immunoblot during the stress response. FDR, false discovery rate; Htb2, histone H2B; SILAC, stable isotopic-labeled amino acid in cell culture.

FIG. 4.

Protein interaction network for proteins ubiquitinated during oxidative stress. EBprot analysis of differentially expressed ubiquitinated (left) and oxidized proteins (right). (A) Identification of the null component to be fitted (green) by EBprot algorithm. Dotted lines specify the range of ratios for the nondifferentially expressed proteins. (B) Histogram of fitted mixture model of peptide ratio distribution for differentially (solid red lines) and nondifferentially modified peptides. (C) Probability scores from peptide-level EBprot scores versus the median of logarithmic peptide ratios.

FIG. 5.

Protein interaction network for proteins ubiquitinated during oxidative stress. Interaction maps for ubiquitinated protein data sets determined by EBprot association analysis were determined by STRING and visualized with Cytoscape (58). The top four clusters of protein association (translation, UPS, ATP binding/mitochondria, vacuole/Golgi apparatus) were determined by the ClusterONE tool (49).

FIG. 6.

Protein interaction network for proteins oxidized during oxidative stress. Interaction maps for oxidized protein data sets determined by EBprot association analysis were determined by STRING and visualized with Cytoscape (58). The top four clusters of protein association (translation, metabolism, tRNA synthetase, proteasome) were determined by the ClusterONE tool (49).

FIG. 7.

Model of the dynamics of protein oxidation and ubiquitin linkages during the stress response. In response to stress, cells accumulate oxidized proteins, K48 and K63 ubiquitins. During the early phase of the response, a second wave of K48 ubiquitin accumulates modifying oxidized proteins, while K63 ubiquitin is removed by deubiquitinating enzymes. Later, K48 ubiquitinated proteins are degraded by the proteasome. Gray arrows indicate the directions of the molecules.