Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome

Arsenic contamination in food and groundwater constitutes
a public
health concern for more than 200 million people worldwide. Individuals
chronically exposed to arsenic through drinking and ingestion exhibit
a higher risk of developing cancers and cardiovascular diseases. Nevertheless,
the underlying mechanisms of arsenic toxicity are not fully understood.
Arsenite is known to bind to and deactivate RING finger E3 ubiquitin
ligases; thus, we reason that a systematic interrogation about how
arsenite exposure modulates global protein ubiquitination may reveal
novel molecular targets for arsenic toxicity. By employing liquid
chromatography-tandem mass spectrometry, in combination with stable
isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation
of di-glycine-conjugated lysine-containing tryptic peptides, we assessed
the alterations in protein ubiquitination in GM00637 human skin fibroblast
cells upon arsenite exposure at the entire proteome level. We observed
that arsenite exposure led to altered ubiquitination of many proteins,
where the alterations in a large majority of ubiquitination events
are negatively correlated with changes in expression of the corresponding
proteins, suggesting their modulation by the ubiquitin-proteasomal
pathway. Moreover, we observed that arsenite exposure confers diminished
ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis,
HMGCR, at Lys248. We also revealed that TRC8 is the major
E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and
the arsenite-induced diminution of HMGCR ubiquitination is abrogated
upon genetic depletion of TRC8. In summary, we systematically characterized
arsenite-induced perturbations in a ubiquitinated proteome in human
cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination
in HEK293T cells involves TRC8.