Small Molecule Screen for the Identification of Novel Inhibitors of Endoplasmic Reticulum Associated Protein Degradation (ERAD)

Every protein must undergo quality control assurance before their transport to the appropriate site. If become misfolded, proteins can accumulate in insoluble deposits that can be harmful to the cell. A major site for protein quality control assurance is the endoplasmic reticulum (ER), Where misfolded proteins are targeted for elimination through ER-associated protein degradation (ERAD). The yeast ERAD system employs two E3 ubiquitin-protein ligase complexes, termed Hrd1 and Doa10, after their E3 components. Our previous studies have shown that Doa10 and Hrd1 operate in ERAD by distinct mechanisms (1,2).

In order to gain new insights on how these two enzymes operate in ERAD, we performed a high throughput small molecule screen, aimed at identifying pathway-specific inhibitors. This screen is based on a novel reporter system that we created, using distinct metabolic markers for each pathway, where cell growth is an indication for inhibition of ERAD components. After screening ~40,000 compounds, 13 of them demonstrated specific growth advantage for the Doa10 pathway while two compounds were nonselective. Currently, these findings are verified by in vivo protein degradation assays. Validated compounds will be tested in vitro. This will provide invaluable information about the distinct mechanism of action of the E3 ligases.

References:

Cohen et al. (2015). Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases. PNAS. , 625–632
Weber et al. (2016). Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase. Mol Cell. 63, 827-839