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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
09:45

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Published on: January 29, 2018

Ubc9 sumoylation regulates SUMO target discrimination.

Puck Knipscheer1, Annette Flotho, Helene Klug

  • 1The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Molecular Cell
|August 12, 2008
PubMed
Summary

Autosumoylation of Ubc9, a key enzyme in small ubiquitin-related modifier (SUMO) conjugation, regulates its target specificity. This modification enhances SUMOylation of Sp100 by creating a novel binding interface, distinct from E3 ligase activity.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biology

Background:

  • Posttranslational modification by SUMO is crucial for protein function regulation.
  • The SUMOylation pathway involves a limited number of enzymes, including a single E1, E2 (Ubc9), and several E3 ligases.
  • Understanding how specificity is achieved with limited enzymes is key to SUMOylation research.

Purpose of the Study:

  • To investigate the role of Ubc9 autosumoylation in regulating SUMOylation target specificity.
  • To elucidate the mechanism by which Ubc9 modification affects its activity towards different substrates.
  • To determine the structural basis for altered substrate recognition upon Ubc9 sumoylation.

Main Methods:

  • Biochemical assays to assess Ubc9 activity on various substrates.
  • Site-directed mutagenesis to investigate the role of Lys14 in Ubc9 autosumoylation.
  • Co-immunoprecipitation and Western blotting to detect protein interactions.
  • X-ray crystallography to determine the structure of sumoylated Ubc9.

Main Results:

  • Autosumoylation of Ubc9 at Lys14 was identified as a regulatory mechanism.
  • Sumoylated Ubc9 showed altered activity, impairing RanGAP1 SUMOylation while enhancing Sp100 SUMOylation.
  • Sp100's SUMO-interacting motif (SIM) was found to mediate enhanced SUMOylation via interaction with conjugated SUMO on Ubc9.
  • Crystal structure revealed a new binding interface on sumoylated Ubc9, contributing to substrate affinity.

Conclusions:

  • Ubc9 autosumoylation is a critical determinant of SUMOylation target specificity.
  • A novel mechanism involving Ubc9-SUMO interaction, mediated by Sp100's SIM, enhances specific substrate SUMOylation.
  • The structural insights into sumoylated Ubc9 provide a basis for understanding how E2 modification can substitute for E3 ligase function.