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Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin

Shuai Fang1,2, Li Zhou2, Geng Chen2

  • 1Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.

Iscience
|July 21, 2025
PubMed

Insights

Parkin, an E3 ubiquitin ligase, has its substrates identified using a novel orthogonal ubiquitin transfer (OUT) cascade. This method reveals new roles for Parkin in cell cycle and metabolism, beyond mitophagy.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Parkin is an E3 ubiquitin ligase with a Ring-Between-Ring (RBR) domain crucial for ubiquitin transfer.
  • Mutations in Parkin are linked to cancer and Parkinson's disease.
  • Parkin's role in mitophagy is established, but its functions in cell cycle, metabolism, and neuronal synapses are less understood.

Purpose of the Study:

  • To develop a novel method for identifying Parkin substrates in living cells.
  • To elucidate the multifaceted cellular functions of Parkin beyond mitophagy.

Main Methods:

  • Engineered the Parkin RBR domain using phage display.
  • Assembled an orthogonal ubiquitin transfer (OUT) cascade for substrate profiling.
  • Verified identified substrates using biochemical and cellular assays.

Main Results:

  • The OUT cascade successfully profiled Parkin substrates in living cells.
  • Rab GTPases and CDK5 were identified as novel Parkin substrates.
  • Mitophagy stimulation enhanced Parkin-mediated ubiquitination of Rab proteins.

Conclusions:

  • The orthogonal ubiquitin transfer (OUT) cascade is an effective tool for identifying E3 ubiquitin ligase substrates.
  • This study expands the known functions of Parkin, highlighting its roles in regulating Rab GTPases and CDK5.
  • Further research using the OUT cascade can uncover additional Parkin substrates and functions.
Keywords:
BiochemistryBiomolecular engineeringMethodology in biological sciencesProteinStructural biology

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