Abstract
E3 ubiquitin ligascs function in the ubiquitin proteasome pathway as a key component in the regulation of protein degradation. With their implications in a range of disease pathways, E3 ligases could be excellent drug targets. Previous research has shown that the mammalian E3 ligase, WWP I, degrades essential factors in skclctogcncsis, making it a strong drug target for osteoporosis treatment. In previous studies, potential inhibitors of the catalytic HECT domain in WWP I were identified and tested in vitro using polyubiquitination assays. In order to develop a high-throughput, physiologically relevant assay system for the inhibition of WWP 1 activity by drugs, a yeast model with a single allele deletion of the yeast E3 ligase orthologue, Rsp5, was selected for an in vivo functional analysis of the WWP I protein. We created domain deletion mutants and chimeric constructs of WWP 1 and assayed their effect on yeast growth. Our results demonstrate that WWPI is unable to functionally complement Rsp5 in the single RSP5 allele deletion yeast strain, and that expression of WWPI in this yeast strain results in a toxic phenotype. Furthermore, our results indicate that there are potentially two mechanisms of toxicity mediated by WWPl in this yeast model, as evidenced by the toxicity of various WWP I domain mutants and chimeric constructs. The dual mechanisms of toxicity ofWWPI provide the possibility of a powerful drug screen with an internal control for drug specificity. Further studies should elucidate HECT domain binding activity in order to develop more efficient drug screens.