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Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.

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Year: 2014 Volume: 10 Issue: 3

Year: 2014 Volume: 10 Issue: 2

Year: 2014 Volume: 10 Issue: 1

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Jiang, Ling Saavedra, Amanda N. Way, George Alanis, Jose Kung, Raphael Li, Jun Xiang, Wensheng Liao, Jiayu
Description	Country affiliation: United States Author Affiliation: Jiang L ( Department of Bioengineering, Center for Bioengineering Research, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA. jiayu.liao@ucr.edu.)
Abstract	Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.
File Format	HTM / HTML
ISSN	1742206X
Issue Number	4
Volume Number	10
e-ISSN	17422051
Journal	Molecular BioSystems
Language	English
Publisher	Royal Society of Chemistry
Publisher Date	2014-04-01
Publisher Place	Great Britain (UK)
Access Restriction	Subscribed
Subject Keyword	Discipline Molecular Biology Discipline Biochemistry Fluorescence Resonance Energy Transfer Methods Sumoylation Ubiquitin-activating Enzymes Chemistry Ubiquitin-conjugating Enzymes Ubiquitin-protein Ligases Animals Bacterial Proteins Enzyme Assays Green Fluorescent Proteins Humans Luminescent Proteins Substrate Specificity Ubiquitin Metabolism Ubiquitination Physiology Journal Article Research Support, N.i.h., Extramural
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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Systematic determinations of SUMOylation activation intermediates and dynamics by a sensitive and quantitative FRET assay

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.

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Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.