Perturbation of Protein Ubiquitination and Mechanisms of Arsenic Toxicity

Arsenite exposure in drinking water and food has long been a health concern for decades because of its cellular toxicity and carcinogenic effects in inducing various cancers and cardiovascular diseases. This research focuses on exploring underlying mechanism accounting for arsenite toxicity, in the aspect of targeting E3 ubiquitin ligases to suppress ubiquitination of substrate proteins, which serve as important signals in multiple functional pathways and proteasome-mediated degradation of proteins. In chapter 1, I briefly described some background of arsenite toxicity and the role of ubiquitination in cells. I also introduced the application of LC-MS for the analysis of proteome, typically ubiquitin-modified proteome in this study. By employing LC-MS/MS couple with SILAC labeling and affinity enrichment, I was able to identify ~1500 ubiquitinated peptides, of which ~40% peptides were decreased upon arsenite exposure. I did a KEGG pathway analysis and functional categories of the identified proteins and was able to find some interested protein targets that were studied in chapter 2 and chapter 3, including FANCD2 and HMGCR, respectively.The LC-MS/MS results showed a 50% decrease of FANCD2-K561 ubiquitination upon arsenite treatment. I further demonstrated, for the first time, that arsenite targets the RING finger domain of the E3 ubiquitin ligase, FANCL, to suppress FANCD2 ubiquitination, using MALDI-TOF mass spectrometry and biotin-As pull down coupled with Western blot. This interaction was also found to impair chromatin localization of FANCD2, which is crucial for recruiting downstream repair factors for DNA interstrand crosslink repair. Therefore, for the first time, I revealed a role of arsenite in perturbing the Fanconi anemia pathway for DNA interstrand crosslink repair by targeting the RING finger of FANCL E3 ubiquitin ligase and inhibit FANCD2 monoubiquitination.The LC-MS/MS results also detected a 70% decrease of HMGCR-K248 upon arsenite treatment, which is important for HMGCR degradation and maintain cholesterol homeostasis in mammalian cells. Indeed I found that arsenite could stabilize HMGCR protein level by inhibiting its ubiquitination. This finding suggests a role of arsenite in affecting the cholesterol biosynthesis pathway.In chapter 4, I further expanded the role of arsenite in targeting E3 ubiquitin ligase by measuring the interaction between arsenite and the E3 ubiquitin ligase RBX1, which is responsible for Nrf2 ubiquitination. This provides a novel insight in arsenite-mediated activation of antioxidant response