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In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

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The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

Qi Zhang1, Zhongduo Wang2, Feng Hou1

  • 1Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

Biochimica Et Biophysica Acta. General Subjects
|November 7, 2016
PubMed
Abstract

Insights

Seven in absentia homologs (SIAHs) are crucial E3 ubiquitin ligases in cancer pathways. Menadione non-specifically destabilizes SIAH1 and SIAH2, challenging its use as a selective SIAH2 inhibitor.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Oncology

Background:

  • Seven in absentia homologs (SIAHs) are E3 ubiquitin ligases regulating tumorigenesis pathways like DNA damage repair and hypoxia.
  • SIAH1 and SIAH2 have opposing roles in tumors, despite similar substrate binding domains (SBDs).
  • Ubiquitin-specific protease USP19 interacts with SIAHs, counteracting their ligase activity.

Purpose of the Study:

  • To structurally and functionally characterize SIAH E3 ligases and their interactions.
  • To investigate the binding of USP19 and menadione to SIAHs.
  • To evaluate the specificity of menadione as a SIAH2 inhibitor.

Main Methods:

  • X-ray crystallography to solve SIAH1-USP19 peptide and apo SIAH2 structures.
  • Peptide array, bioinformatic, and biophysical analyses.
  • Phylogenetic analysis of SIAH/USP19 complex conservation.

Main Results:

  • Crystal structures revealed SIAH1-USP19 complex and apo SIAH2.
  • The SIAH/USP19 interaction is evolutionarily conserved.
  • Menadione non-specifically destabilizes both SIAH1 and SIAH2 via covalent modification.

Conclusions:

  • SIAH E3 ligase SBDs are similar, with subtle stability differences.
  • USP19 uniquely binds SIAHs within the substrate binding pocket.
  • Menadione lacks specificity for SIAH2 inhibition, necessitating caution in its application.
Keywords:
CrystallographyDeubiquitinaseE3 Ubiquitin ligaseMenadioneProtein-protein interactionSmall molecule inhibitors

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