| Arsenic is a potent toxicant and known human carcinogen. Arsenite [As (III)], a major inorganic form of arsenic, can stimulate oxidative stress, decrease DNA repair, alter gene expression, modulate stress signaling, and damage proteins in numerous model systems. This dissertation focuses on the effects of low-level arsenite on the ubiquitin-proteasome pathway. Ubiquitin is a small protein that acts as a post-translational modification of other proteins within the cell. In most cases, modification of a protein by ubiquitin acts as a signal for its degradation by the proteasome. Studies performed in rabbit renal cortical slices and HEK293 cells demonstrated that 24 hr exposure to 0.5–10 μM As (III) caused a dose dependent accumulation of ubiquitin-protein conjugates within cells. This accumulation was shown to correlate with decreases in cellular 20S proteasomal activity. As (III) did not increase ubiquitin-conjugating activity. Depletion of cellular glutathione exacerbated the effects of As (III) on ubiquitin-protein conjugates in HEK293 cells. To characterize this perturbation of the ubiquitin pathway within As (III) exposed cells, a proteomics method was developed to purify and identify the specific proteins modified by ubiquitin. A cell line expressing epitope tagged His(6X)-Ub-GFP was developed by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using Ni-affinity chromatography and digested into peptides. Complex mixtures of peptides were separated by reverse phase chromatography and analyzed using the LCQ ion-trap mass spectrometer. In purified samples from transfected cells, ubiquitin and 22 other proteins were confidently identified using Sequest. These proteins included many of the expected carriers of ubiquitin including ubiquitin-conjugating enzymes and histone proteins. In samples from transfected cells treated with 10 μM As (III) (24 hr), a number of unique proteins were identified, including the DNA repair protein, and known ubiquitin substrate, PCNA. This proteomics method, developed for the analysis of ubiquitinated proteins in As (III) treated cells, will allow for large-scale characterization of ubiquitin-protein conjugates in numerous physiological and pathological states. These results suggest that low-level arsenic may facilitate its detrimental health effects through perturbation of the ubiquitin-proteasome pathway, and that future investigations into interactions between arsenic, ubiquitin and protein homeostasis are warranted. |
