UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN
Sungjin Park1, Peter K Foote2, David T Krist2
1From the Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
The Journal of Biological Chemistry
|July 16, 2017
Summary
Researchers developed UbFluor, a novel chemical probe, to easily measure the activity of Ring-between-ring E3 ligases like PARKIN. This tool aids in studying these enzymes involved in diseases and in screening for new drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ring-between-ring (RBR) E3 ligases are crucial in cellular processes but their complex regulation and function, especially in diseases like autoimmune and neurodegenerative disorders, remain poorly understood.
- Studying RBR E3 ligase activity is challenging due to the intricate nature of ubiquitination, requiring multiple enzymes and cofactors.
Purpose of the Study:
- To develop a simplified method for assessing RBR E3 ligase activity.
- To create a tool for real-time, quantitative analysis of PARKIN, a key RBR E3 ligase.
- To enable high-throughput screening for PARKIN activators and inhibitors.
Main Methods:
- Development of novel ubiquitin C-terminal fluorescein thioester chemical probes (UbMES and UbFluor).
- Utilizing fluorescence polarization to monitor RBR E3 ligase activity in a simplified experimental setup.
- Investigating PARKIN's ubiquitination mechanism, including its independence from E2 enzymes and the role of post-translational modifications.
Main Results:
- Confirmed PARKIN's ability to perform substrate ubiquitination, lysine selection, and polyubiquitin chain formation without an E2 enzyme.
- Demonstrated that UbFluor accurately quantifies PARKIN activation states, including those induced by Ser65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb).
- Elucidated the contributions of pUb and ubiquitin-accepting substrates to PARKIN's turnover, showing pUb enhances transthiolation and substrates clear the thioester intermediate.
- Validated UbFluor's capability to quantify the effects of structural mutations on PARKIN activity.
Conclusions:
- UbFluor provides a robust and quantitative method for studying RBR E3 ligase biochemistry.
- This chemical probe facilitates real-time assessment of PARKIN activity and its regulatory mechanisms.
- UbFluor is a valuable tool for basic research and for high-throughput screening of therapeutic modulators targeting PARKIN and other RBR E3 ligases.
