Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin
Summary: 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 ubiquitina...
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| Language: | English |
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Elsevier
2025-07-01
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| Series: | iScience |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004225011745 |
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| author | Shuai Fang Li Zhou Geng Chen Jing Zhang Xiaoyu Wang In Ho Jeong Savannah E. Jacobs Bradley R. Kossmann Wei Wei Shu Liu Geon H. Jeong Yayun Xie Duc Duong Nicholas T. Seyfried Ivaylo Ivanov Angela M. Mabb Hiroaki Kiyokawa Bo Zhao Jun Yin |
| author_facet | Shuai Fang Li Zhou Geng Chen Jing Zhang Xiaoyu Wang In Ho Jeong Savannah E. Jacobs Bradley R. Kossmann Wei Wei Shu Liu Geon H. Jeong Yayun Xie Duc Duong Nicholas T. Seyfried Ivaylo Ivanov Angela M. Mabb Hiroaki Kiyokawa Bo Zhao Jun Yin |
| author_sort | Shuai Fang |
| collection | DOAJ |
| description | Summary: 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. |
| format | Article |
| id | doaj-art-a3e8d2c8cc0b4523af2e4b1e1fa27c22 |
| institution | Matheson Library |
| issn | 2589-0042 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj-art-a3e8d2c8cc0b4523af2e4b1e1fa27c222025-07-03T04:40:11ZengElsevieriScience2589-00422025-07-01287112913Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 ParkinShuai Fang0Li Zhou1Geng Chen2Jing Zhang3Xiaoyu Wang4In Ho Jeong5Savannah E. Jacobs6Bradley R. Kossmann7Wei Wei8Shu Liu9Geon H. Jeong10Yayun Xie11Duc Duong12Nicholas T. Seyfried13Ivaylo Ivanov14Angela M. Mabb15Hiroaki Kiyokawa16Bo Zhao17Jun Yin18Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USANeuroscience Institute and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USADepatrment of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USADepatrment of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USADepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USANeuroscience Institute and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302, USADepartment of Pharmacology, Northwestern University, Chicago, IL 60611, USA; Corresponding authorEngineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Corresponding authorDepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2589004225011745BiochemistryProteinBiomolecular engineeringStructural biologyMethodology in biological sciences |
| spellingShingle | Shuai Fang Li Zhou Geng Chen Jing Zhang Xiaoyu Wang In Ho Jeong Savannah E. Jacobs Bradley R. Kossmann Wei Wei Shu Liu Geon H. Jeong Yayun Xie Duc Duong Nicholas T. Seyfried Ivaylo Ivanov Angela M. Mabb Hiroaki Kiyokawa Bo Zhao Jun Yin Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin iScience Biochemistry Protein Biomolecular engineering Structural biology Methodology in biological sciences |
| title | Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin |
| title_full | Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin |
| title_fullStr | Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin |
| title_full_unstemmed | Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin |
| title_short | Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin |
| title_sort | engineering a cell based orthogonal ubiquitin transfer cascade for profiling the substrates of rbr e3 parkin |
| topic | Biochemistry Protein Biomolecular engineering Structural biology Methodology in biological sciences |
| url | http://www.sciencedirect.com/science/article/pii/S2589004225011745 |
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