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

作者：Shuai Fang, Li Zhou, Geng Chen, Jing Zhang, Xiaoyu Wang, In Ho Jeong, Savannah E. Jacobs, Bradley R. Kossmann, Wei Wei, Shuming Liu, Geon Jeong, Yayun Xie, Duc M. Duong, Nicholas T. Seyfried, Ivaylo Ivanov, Angela M. Mabb, Hiroaki Kiyokawa, Bo Zhao, Jun Yin · 发表于：iScience · 年份：2025 · DOI：10.1016/j.isci.2025.112913 · 被引用次数：3 · 研究领域：Ubiquitin and proteasome pathways、Autophagy in Disease and Therapy、Protein Degradation and Inhibitors

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.