A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants

Mol Cell. 2017 Oct 19;68(2):456-470.e10. doi: 10.1016/j.molcel.2017.09.027.

Abstract

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

Keywords: RING E3 ligase; U-box E3 ligase; activation; inhibition; ubiquitin variant.

MeSH terms

  • Drug Discovery / methods*
  • Enzyme Activators* / chemistry
  • Enzyme Activators* / pharmacology
  • Enzyme Inhibitors* / chemistry
  • Enzyme Inhibitors* / pharmacology
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Protein Multimerization / drug effects*
  • Tumor Suppressor Proteins* / agonists
  • Tumor Suppressor Proteins* / antagonists & inhibitors
  • Tumor Suppressor Proteins* / genetics
  • Tumor Suppressor Proteins* / metabolism
  • Ubiquitin-Protein Ligase Complexes* / antagonists & inhibitors
  • Ubiquitin-Protein Ligase Complexes* / genetics
  • Ubiquitin-Protein Ligase Complexes* / metabolism
  • Ubiquitin-Protein Ligases
  • X-Linked Inhibitor of Apoptosis Protein* / agonists
  • X-Linked Inhibitor of Apoptosis Protein* / antagonists & inhibitors
  • X-Linked Inhibitor of Apoptosis Protein* / genetics
  • X-Linked Inhibitor of Apoptosis Protein* / metabolism

Substances

  • Enzyme Activators
  • Enzyme Inhibitors
  • Tumor Suppressor Proteins
  • X-Linked Inhibitor of Apoptosis Protein
  • XIAP protein, human
  • Ubiquitin-Protein Ligase Complexes
  • UBE4B protein, human
  • Ubiquitin-Protein Ligases