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, which has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional K63-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae subjected to peroxides in a highly sensitive and regulated manner. We demonstrated that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2 leading to accumulation of K63 conjugates assembled by the Rad6-Bre1 ubiquitin conjugase and ligase, respectively. Using linkage-specific isolation methods and SILAC-based quantitative proteomics, we identified >100 new K63 polyubiquitinated targets, which were significantly enriched in ribosomal proteins. Finally, we demonstrated that impairment of K63 ubiquitination during oxidative stress impacts polysome stability and protein expression, rendering cells more sensitive to stress, revealing a new redox-regulatory role for this modification.

Purpose Pheochromocytomas and paragangliomas (PPGLs) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown. Experimental Design We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was used for microarray-based transcription, protein expression and histone methylation analysis by western blot or immunohistochemistry. In vitro analysis of mutants was performed in cell lines. Results We detected mutations in chromatin remodeling genes, including histone-methyltransferases, histone-demethylases and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3.3 gene, H3F3A. Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes mutations were also detected in the validation group. Additionally, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor. Conclusions This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes.

The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin-and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. Aims: The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. Results: Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal a5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. Innovation: We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. Conclusion: S-glutathiolation is a posttranslational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption.

Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein–protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.