Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin
The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate UB transfer to substrate proteins, and mutations affecting Parkin catalysis promote cancer and are associated with Parkinson's disease. An essential role of Parkin is to initiate mitophagy by ubiquitinating mitochondrial proteins. Still, it is unclear how Parkin carries out other cellular functions, such as the regulation of the cell cycle, metabolism, and the neuronal synapse. Here, we used phage display to engineer the RBR domain of Parkin and assembled an orthogonal ubiquitin transfer (OUT) cascade to profile Parkin substrates in living cells. Guided by the substrate profile from the OUT screen, we verified a panel of Rab GTPases and CDK5 as Parkin substrates. We also showed mitophagy stimulation enhanced Parkin-mediated ubiquitination of Rab proteins. Our work demonstrates that the OUT cascade can be an empowering tool for identifying Parkin substrates to elucidate its multifaceted cellular functions.
Authors
- Hiroaki Kiyokawa (ORCID: https://orcid.org/0000-0002-7942-6455)
- In Ho Jeong (ORCID: https://orcid.org/0009-0001-6836-2708)
- Bo Zhao (ORCID: https://orcid.org/0000-0002-9525-0541)
- Nicholas T. Seyfried (ORCID: https://orcid.org/0000-0002-4507-624X)
- Jun Yin (ORCID: https://orcid.org/0000-0002-4803-7510)
- Bradley R. Kossmann
- Ivaylo Ivanov (ORCID: https://orcid.org/0000-0002-5306-1005)
- Angela M. Mabb (ORCID: https://orcid.org/0000-0002-3000-8690)
- Duc Minh Duong (ORCID: https://orcid.org/0000-0001-5880-1888)
- Shuai Fang (ORCID: https://orcid.org/0000-0001-9155-5797)
- Savannah E. Jacobs
- Xiaoyu Wang (ORCID: https://orcid.org/0000-0001-5477-1570)
- Geng Chen (ORCID: https://orcid.org/0000-0002-3452-9434)
- Shuming Liu (ORCID: https://orcid.org/0000-0002-4949-4318)
- Geon Jeong
- Wei Wei
- Jing Zhang (ORCID: https://orcid.org/0009-0003-8777-8754)
- Li Zhou
- Yayun Xie
Institutions
- Northwestern University (US)
- Emory University (US)
- Georgia State University (US)
- Shanghai Jiao Tong University (CN)
Publication Details
- Journal
- iScience
- Published
- 2025-06-17
- DOI
- https://doi.org/10.1016/j.isci.2025.112913
- Citations
- 2
- Primary Topic
- Ubiquitin and proteasome pathways
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- National Science Foundation
- Foundation for the National Institutes of Health
- National Natural Science Foundation of China
- Science and Technology Commission of Shanghai Municipality
- National Institutes of Health