Investigations of the conserved deubiquitinase Ubp12 in Saccharomyces cerevisiae and Candida albicans in response to oxidative stress

Abstract

Ubiquitination is a dynamic post-translational modification that regulates many cellular processes. Ubiquitin is attached to proteins using conjugation machinery and removed by deubiquitinases (DUBs), and the coordination of the activities of conjugation/deconjugation proteins determines the effects of ubiquitination on the functions of substrates. Many of the ubiquitin conjugation/deconjugation enzymes utilise catalytic cysteine residues, which renders them potentially susceptible to oxidation by reactive oxygen species (ROS). Low levels of ROS play essential functions in intracellular signalling processes, however high levels of ROS can cause oxidative damage to cellular components. Hence, ROS-induced damage is linked with numerous common age-related diseases. It is therefore essential that cells can recognise the specific ROS present in the cell, and distinguish between low levels and high levels of ROS, in order to elicit an appropriate response. Work from our lab and others revealed that ubiquitin and ubiquitin-like (UBL) conjugation and deconjugation proteins can be regulated by ROS, which consequently influences ubiquitin and UBL regulation of substrates. For example, our lab found that a conserved DUB in the model eukaryote Saccharomyces cerevisiae, Ubp12, forms a H2O2-specific intramolecular disulphide complex in a H2O2 concentration dependent manner. Interestingly, further work showed that the catalytic cysteine residue of Ubp12 is essential for the formation of this complex and that the cytosolic thioredoxin reductase, Trr1, may influence the cellular abundance of Ubp12. To gain insights into the regulation and functions of Ubp12 in S. cerevisiae a main aim of this study was to assess whether other cysteine residues present in Ubp12 are involved in the oxidation of the DUB and, moreover, to examine whether the thioredoxin system regulates the formation and/or reduction of the Ubp12 intramolecular disulphide complex. In addition, very little is known about the regulation and functions of the Ubp12 homologue present in the human fungal pathogen Candida albicans and hence studies were initiated to investigate the functions and regulation of this uncharacterised DUB. Excitingly, this study is the first to establish a role for the thioredoxin system in the oxidation of ubiquitin/UBL conjugation/deconjugation enzymes. For example, the cytosolic thioredoxins, Trx1 and Trx2, were found to regulate the previously identified Ubp12 disulphide complex, and other previously unidentified modified Ubp12 complexes. In addition, Trr1, Trx1 and Trx2 were shown 7 to influence the abundance of Ubp12. This study also revealed that specific conserved cysteine residues, in addition to the catalytic cysteine residue, present in homologues of Ubp12 are important in the oxidation of Ubp12 in S. cerevisiae. Interestingly, the cysteine residues in conserved CXXC motifs present in Ubp12 are important for oxidation of Ubp12 and raises the possibility, together with the analyses of the effects of other cysteine mutations and mutations of the thioredoxin system components, that disulphide shuffling may occur within Ubp12 in response to H2O2. Previous studies from our lab and others revealed that different H2O2 signalling pathways regulate H2O2 responses in two different strain backgrounds of S. cerevisiae. The results presented here revealed that, although Ubp12 is important for ROS responses in both of these strain backgrounds, Ubp12 may also have different functions and be regulated differently in each strain. Finally, studies to investigate the regulation and functions of the Ubp12 homologue in C. albicans, revealed that, like Ubp12 in S. cerevisiae, Ubp12 in C. albicans is important for oxidative stress responses. Moreover, oxidative stress induces the formation of an oxidised Ubp12 complex(es) which appeared similar in mobility to the oxidised Ubp12 complex observed in S. cerevisiae. However, interestingly, loss of Ubp12 had different effects on the sensitivity of C. albicans and S. cerevisiae cells to oxidative stress suggesting different functions for the DUB in each yeast. Thus, Ubp12 is important in oxidative stress responses in both S. cerevisiae and C. albicans, but specific roles and regulation appear to be different. Consistent with these observations, cells lacking Ubp12 displayed significantly reduced levels of HMW ubiquitinated proteins in C. albicans but not S. cerevisiae, and Ubp12 was found to be important for cell responses to DNA damage and DNA replication stress in C. albicans which but not in S. cerevisiae. Finally, this work also suggested that Ubp12 may have roles in morphological changes of C. albicans cells in response to environmental signals including H2O2. Given the potential roles of Ubp12 in processes linked to pathogenicity in C. albicans it is possible that Ubp12 is important for virulence. Collectively, this thesis provides new insights into the functions and regulation of Ubp12 in S. cerevisiae and in C. albicans. Dysregulation of the human homologue of Ubp12, USP15, is linked to several diseases and hence these studies may further understanding of the normal functions of USP15 an important step in the development of drug treatments for the benefit of human health.