A new FRET-based platform to track substrate ubiquitination by fluorescence
| Autor: | Zhen-Qiang Pan, Robert A. Chong, Kevin Ching, Kenneth Wu |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
CRL4 Cullin-RING E3 ubiquitin ligase 4 HTS high-throughput screening Skp2 S phase kinase-associated protein 2 Biochemistry NF-KappaB Inhibitor alpha E1 E1 ubiquitin-activating enzyme Fluorescence Resonance Energy Transfer IκBα nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha ROC1 RING-box protein 1 beta Catenin chemistry.chemical_classification Stem Cell Factor biology Drug discovery Chemistry Myc myelocytomatosis CUL1 Cullin1 Fbxw7 F-box/WD repeat-containing protein 7 Aux auxin Ubiquitin ligase E3 ubiquitin ligase protein degradation CUL1 Target protein Research Article UB ubiquitin TCEP tris(2-carboxyethyl)phosphine Recombinant Fusion Proteins Ubiquitin-Protein Ligases Protein degradation ubiquitination Cdc34 cell division cycle 34 03 medical and health sciences SCF Skp1–Cullin1–Fbox protein E3 ligase GST glutathione-S-transferase Humans Amino Acid Sequence CRBN cereblon Molecular Biology Fluorescent Dyes DNA ligase 030102 biochemistry & molecular biology Ubiquitin E2 E2 ubiquitin-conjugating enzyme Substrate (chemistry) Cell Biology 030104 developmental biology Förster resonance energy transfer chemoenzymatic ligation kinetics βTrCP beta-transducin repeats-containing protein Ubiquitin-Conjugating Enzymes Biophysics biology.protein FRET TIR1 transport inhibitor response 1 Peptides Protein Processing Post-Translational E3 E3 ubiquitin ligase |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences. |
| Databáze: | OpenAIRE |
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