Abstract
E3 ubiquitin ligases 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, WWP1, degrades essential factors in skeletogenesis, making it a strong drug target for osteoporosis treatment. In previous studies, potential inhibitors of the catalytic HECT domain in WWP1 were identified and tested in vitro using polyubiquitination assays. In order to develop a high-throughput, physiologically relevant assay system for the inhibition of WWP1 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 WWP1 protein. We created domain deletion mutants and chimeric constructs of WWP1 and assayed their effect on yeast growth. Our results demonstrate that WWP1 is unable to functionally complement Rsp5 in the single RSP5 allele deletion yeast strain, and that expression of WWP1 in this yeast strain results in a toxic phenotype. Furthermore, our results indicate that there are potentially two mechanisms of toxicity mediated by WWP1 in this yeast model, as evidenced by the toxicity of various WWP1 domain mutants and chimeric constructs. The dual mechanisms of toxicity of WWP1 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.