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E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1

Reuven Wiener1, Anthony T DiBello, Patrick M Lombardi

  • 11] Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. [2] Howard Hughes Medical Institute, Baltimore, Maryland, USA. [3] [4].

Nature Structural & Molecular Biology
|August 20, 2013
PubMed

Insights

OTUB1, a deubiquitinating enzyme, noncatalytically inhibits polyubiquitin accumulation. New findings reveal OTUB1 also cleaves Lys48 polyubiquitin chains, a process regulated by E2 enzyme interactions and ubiquitin levels.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biology

Background:

  • OTUB1 is a Lys48-specific deubiquitinating enzyme.
  • It interacts with E2 ubiquitin-conjugating enzymes like UBC13 and UBCH5.
  • OTUB1 noncatalytically inhibits polyubiquitin accumulation by binding E2~Ub thioester intermediates.

Purpose of the Study:

  • To investigate the dual roles of OTUB1 in ubiquitin regulation.
  • To elucidate the mechanism by which E2 enzyme interactions influence OTUB1 activity.
  • To understand how cellular conditions modulate OTUB1 function.

Main Methods:

  • In vivo complex formation studies.
  • Biochemical assays measuring deubiquitinating activity.
  • Structural studies (X-ray crystallography) of OTUB1-E2 complexes.
  • Analysis of substrate binding and enzyme kinetics.

Main Results:

  • OTUB1 possesses a second role: stimulating the cleavage of Lys48 polyubiquitin chains.
  • This stimulation is dependent on the ratio of charged to uncharged E2 enzymes and the concentrations of polyubiquitin and free ubiquitin.
  • Structural and biochemical data show E2 enzymes stabilize the OTUB1 N-terminal helix, enhancing polyubiquitin binding.
  • OTUB1-E2 complexes are dynamically regulated by cellular ubiquitin and E2 charging status.

Conclusions:

  • OTUB1-E2 interactions are critical for both noncatalytic inhibition and catalytic cleavage of polyubiquitin.
  • Cellular levels of E2 charging and free ubiquitin fine-tune OTUB1 activity.
  • These findings reveal a sophisticated regulatory mechanism for polyubiquitin chain dynamics involving OTUB1 and E2 enzymes.

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