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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Published on: May 10, 2015

WDR20 regulates activity of the USP12 x UAF1 deubiquitinating enzyme complex

Younghoon Kee1, Kailin Yang, Martin A Cohn

  • 1Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

The Journal of Biological Chemistry
|February 12, 2010
PubMed

Insights

WDR20 protein binds USP12 and USP46, stimulating their deubiquitinating enzyme activity. This interaction does not impact the Fanconi anemia DNA repair pathway, suggesting WDR20 regulates a specific subset of UAF1 x USP complexes.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • UAF1 (Usp1-associated factor 1) protein interacts with deubiquitinating enzymes USP1, USP12, and USP46.
  • The USP1 x UAF1 complex is crucial for the Fanconi anemia (FA) DNA repair pathway.
  • The functions of USP12 x UAF1 and USP46 x UAF1 complexes are less understood.

Purpose of the Study:

  • To identify proteins interacting with USP12 and USP46 deubiquitinating enzyme complexes.
  • To elucidate the role of novel binding partners in the regulation of USP12 and USP46 activity.
  • To understand the functional significance of these complexes in cellular processes.

Main Methods:

  • Protein-protein interaction assays to identify binding partners.
  • Purification of protein complexes.
  • Enzymatic activity assays.
  • Small interference RNA (siRNA)-mediated gene silencing.
  • Assessment of DNA damage response pathways.

Main Results:

  • WDR20, a WD40-repeat protein, was identified as a common binding partner for UAF1, USP12, and USP46.
  • WDR20 exclusively associates with USP12 and USP46, not USP1.
  • A ternary complex of USP12 x UAF1 x WDR20 was purified.
  • WDR20 enhanced the enzymatic activity of the USP12 x UAF1 complex but not USP1 x UAF1.
  • Depletion of WDR20 did not affect the FA pathway or DNA damage responses.

Conclusions:

  • WDR20 acts as a specific stimulatory subunit for USP12 and USP46 deubiquitinating enzymes.
  • The USP12 x UAF1 x WDR20 complex regulates a distinct set of cellular processes separate from the FA pathway.
  • WDR20 is essential for preserving and modulating the activity of a subset of UAF1 x USP complexes.

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