System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes

Mol Cell. 2016 Apr 7;62(1):121-36. doi: 10.1016/j.molcel.2016.02.005. Epub 2016 Mar 3.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Catalytic Domain
  • Cell Line
  • Cell Movement
  • Dogs
  • HCT116 Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Models, Molecular
  • Organoids / cytology
  • Organoids / metabolism
  • Peptide Library
  • Ubiquitin / chemistry
  • Ubiquitin / genetics
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Peptide Library
  • Ubiquitin
  • Ubiquitin-Protein Ligases