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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
Stability of thioester intermediates in ubiquitin-like modifications
Jing Song1, Jianghai Wang, Agnieszka A Jozwiak
1Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Protein Science : a Publication of the Protein Society
|September 29, 2009
Summary
Ubiquitin-like protein thioester intermediates are surprisingly stable, with a half-life of 3.6 hours. This intrinsic stability suggests enzymatic regulation is crucial for controlling cellular processes involving these key modification pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Regulation
Background:
- Ubiquitin-like modifications are vital for cellular regulation.
- These modifications involve thioester conjugates as reaction intermediates.
- The stability of these crucial intermediates has not been well-characterized.
Purpose of the Study:
- To investigate the intrinsic stability of ubiquitin-like protein thioester intermediates.
- To understand the influence of structural context and E3 ligase binding on thioester stability.
Main Methods:
- Utilized a model compound and the Ubc9 approximately SUMO-1 thioester conjugate.
- Measured hydrolysis rates under various conditions, including denaturing conditions and E3 ligase binding.
Main Results:
- The Ubc9 approximately SUMO-1 thioester intermediate exhibited a half-life of approximately 3.6 hours.
- Stability was only moderately affected by denaturing conditions and binding to active or inactive E3 ligases.
- Hydrolysis rate constants (k) were determined for different conditions.
Conclusions:
- Ubiquitin-like protein thioester intermediates possess significant intrinsic stability.
- This stability implies that enzymatic mechanisms, such as thioesterases, are essential for regulating their abundance and cellular trafficking.
- Understanding thioester stability is key to controlling ubiquitin-like modification pathways.
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