System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.
Publication Type:Journal Article
Source:Mol Cell, Volume 62, Issue 1, p.121-36 (2016)
Keywords:Animals, Catalytic Domain, Cell Line, Cell Movement, Dogs, HCT116 Cells, Humans, Madin Darby Canine Kidney Cells, Models, Molecular, Organoids, Peptide Library, Ubiquitin, Ubiquitin-Protein Ligases
<p>HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.</p>