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Related Experiment Video

Updated: Dec 25, 2025

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay
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Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

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Ddi1 is a ubiquitin-dependent protease.

Matthew C J Yip1,2, Nicholas O Bodnar1,2, Tom A Rapoport3,2

  • 1Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115.

Proceedings of the National Academy of Sciences of the United States of America
|March 21, 2020
PubMed
Summary

The protein Ddi1 is a ubiquitin-dependent protease that cleaves proteins tagged with long ubiquitin chains. Its activity is crucial for preventing the accumulation of polyubiquitinated proteins in yeast cells.

Keywords:
Ddi1Nrf1proteaseproteasomeubiquitin

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • * Saccharomyces cerevisiae Ddi1 and its homologs are proposed shuttling factors for proteasome delivery.
  • * Ddi1 possesses UBA, UBL, helical domain of Ddi1 (HDD), and retroviral-like protease (RVP) domains.
  • * The precise function of Ddi1 and the enzymatic activity of its RVP domain remain poorly understood.

Purpose of the Study:

  • * To elucidate the enzymatic activity and function of the Saccharomyces cerevisiae Ddi1 protein.
  • * To investigate the role of Ddi1's domains in its proteolytic activity.
  • * To understand Ddi1's contribution to protein homeostasis.

Main Methods:

  • * Biochemical assays to determine Ddi1's proteolytic activity on ubiquitinated substrates.
  • * Domain analysis to assess the contribution of UBL, HDD, and RVP domains to Ddi1 function.
  • * Yeast cell experiments to observe the effects of compromised Ddi1 activity on protein ubiquitination.

Main Results:

  • * Ddi1 functions as a ubiquitin-dependent endoprotease, cleaving substrates with long polyubiquitin chains (≥8 ubiquitins).
  • * The RVP domain is inactive in isolation, requiring the HDD domain for proteolytic activity.
  • * The UBL domain enhances Ddi1 activity by mediating high-affinity polyubiquitin chain interaction.
  • * Impairing Ddi1 activity in yeast leads to the accumulation of polyubiquitinated proteins.

Conclusions:

  • * Ddi1 is a unique ubiquitin-dependent protease acting on polyubiquitinated substrates, distinct from the proteasome.
  • * Ddi1's proteolytic function is dependent on its HDD domain and stimulated by its UBL domain.
  • * Ddi1 plays a significant role in managing polyubiquitinated protein levels, especially when proteasome function is impaired.