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Novel Quantitative FRET technology to Determine Protein Interaction Dissociation Constant and Enzymatic Kinetics in Solution for SUMOylation Cascade
Author(s) -
Liao Jiayu,
Xiong Zhehao,
Way George,
Madarha Vipul
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.819.11
Subject(s) - sumo protein , förster resonance energy transfer , chemistry , protein–protein interaction , isothermal titration calorimetry , enzyme kinetics , dissociation constant , biophysics , biochemistry , microbiology and biotechnology , ubiquitin , enzyme , biology , receptor , active site , fluorescence , physics , quantum mechanics , gene
The ubiquitin–proteasome system and ubiquitin‐like protein pathways, such as SUMOylation, are critical in protein homeostasis and activities in vivo and are emerging as a new strategy to treat many acute and chronic human diseases, such as cancers. Although various kinase inhibitors have been developed as target‐based therapy, solid tumors are still challenges in clinical therapy because various resistant are developed after kinase inhibitor treatments, and therapeutic agents with novel mechanisms are urgently needed. SUMO has been shown to modify various critical proteins, such as p53, MDM2, Estrogen receptor and androgen receptors. More recently, a genome‐wide siRNA screening shown that inhibition of SUMO E1 ligases can lead to synergistically lethality of c‐Myc overexpressed breast cancer cells. However, so far, specific inhibitor of SUMOylation is still not available for the community. By overcoming the shortages of tradition methodologies for protein interaction dissociation constant measurement, such as Surface Resonance Plasma (SPR), Isothermal titration calorimetry (ITC) and radio‐ligand binding assay, we developed a novel quantitative Förster resonance energy transfer (FRET) assays for both protein interaction dissociation constant and protease kinetics determinations in a high‐through format for overall SUMOylation cascade. The novel theoretical and experimental procedures for protein interactions affinity ( K d ) determinations in the SUMOylation cascade, including the interaction between SUMO1 and its E2 ligase, Ubc9, E1 heterodimers (Aos1 and Uba2), E1 and E2 interactions (Uba2 and Ubc9), and E2 and substrate interactions (Ubc9 and RanGap1c) and protease kinetics, K cat /K M of SENP1 endopeptidase activity have been developed in a systems biology manner. The data are in good agreement with traditional methods. This novel FRET technology provides not only robust methodology for the biochemical parameter determinations in solution from one sample but also powerful high‐throughput assay for quantitative systems biology in the future. 1Novel high‐throughput quantitative FRET technology for protein interaction dissociation and protease kinetics determination in SUMOylation cascade.