Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.
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.
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.
Ling
L
Jiang
Amanda N
AN
Saavedra
George
G
Way
Jose
J
Alanis
Raphael
R
Kung
Jun
J
Li
Wensheng
W
Xiang
Jiayu
J
Liao
D000818:Animals; D001426:Bacterial Proteins; D057075:Enzyme Assays; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D013379:Substrate Specificity; D058207:Sumoylation; D025801:Ubiquitin / Q000737:chemistry; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C470301:UBA2 protein, human; D025801:Ubiquitin; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C470299:SAE1 protein, human; D044764:Ubiquitin-Activating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9