Functional characterization of ubiquitome regulating genomic stability and proteostasis

Abstract

Covalent attachment of ubiquitin and ubiquitin-like modifiers (UBLs), which are share the β-grasp fold, to substrates is the key post-translational modifications (PTMs) that determine the fate, function, and turnover of most cellular proteins. Thus, ubiquitin- and UBLs-linked PTMs in the intracellular context precisely control various cellular processes such as DNA damage response, proteasomal proteolytic pathway and autophagy. Hence, stress-induced aberrations of ubiquitin and UBLs system cause multiple human diseases such as cancers, neurological disorders and aging disorders. In the physiological or pathophysiological context, however, it is unclear that how ubiquitin and UBLs differentially regulate versatile cellular process with high specificity through homologous and sometimes parallel modification pathways. In this study, to address this point, I selected ubiquitin and NEDD8 out of UBLs and analyzed whether and how they control DNA damage response and proteasomal proteolytic pathway. In the first study, I systematically screened 211 human E3 ubiquitin ligases (hE3s) related to DDR and identified 11 PARP1-dependent hE3s. Among them, drought-induced 19 (Di19) zinc-finger E3 ubiquitin ligases (DIZELs) translocate to DNA lesions in a PAR-dependent manner to generate unanchored K11 ubiquitin chains (UnK11Ubs). Notably, the conserved C2H2(I) domain of DIZELs RNF114 and RNF166 generates UnK11Ubs and interaction with PAR through C2H2(II) domain accelerates accumulation of UnK11Ubs. I also found that UnK11Ubs directly control PAR-seeded liquid demixing and enhance DOT1L-mediated H3K79 di-methylation on damaged chromatin, thereby facilitating recruitment of 53BP1 for DNA repair process. These findings demonstrate that DIZELs as a PAR-binding hE3s generate UnK11Ubs that control the PARP1-coupled DDR in an ATM-independent manner. In addition, accumulated evidence showed that crosstalk between NEDD8 and ubiquitin is important for the prompt elimination of misfolded proteins, which tends to form aggregates, through ubiquitin-proteasome system (UPS) or autophagy. However, it remains unclear how NEDD8 drives UPS or autophagy-mediated clearance of protein aggregates in responses to diverse stress conditions. Thus, in the second study, I screened NEDD8 binding proteins using a protein microarray system and identified HDAC6 as a direct NEDD8-binding protein. By systematic approach, I found that ubiquitin stress leads to accumulation of cytosolic NEDDylated protein aggregates, which form aggresome-like bodies (ALBs) in the perinuclear region. Intriguingly, HDAC6 colocalizes with stress-induced ALBs, and inhibition of HDAC6 activity suppresses ALBs formation, but not stress-induced NEDDylation, suggesting that HDAC6 carries NEDDylated proteins to generate ALBs. Then, I monitored the ALBs-associated proteostasis network and found that p62 directly controls ALBs formation as an acceptor of cytosolic NEDDylated aggregates. Interestingly, I also observed that ALBs are highly condensed in chloroquine-treated cells with impaired autophagic flux, indicating that ALBs rely on autophagy. Taken together, these data propose that NEDD8, HDAC6, and p62 are involved in the management of proteotoxic stress by forming cytosolic ALBs-coupled to the aggresome-autophagy flux. Collectively, all results in this study indicate functional roles of ubiquitin regulating DNA damage response to maintain genomic stability and of NEDD8 controlling proteostasis to protect cells from cellular stress.