Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades
| Autor: | Lajos Haracska, Lajos Pintér, Gaurav Sharma, Gabriel Fenteany, Ernő Kiss, Paras Gaur |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
E1 ubiquitin-conjugating enzymes
0301 basic medicine DNA damage Ubiquitin-Protein Ligases High-throughput screening E3 ubiquitin ligases 03 medical and health sciences 0302 clinical medicine Ubiquitin Ubiquitin-like proteins Proliferating Cell Nuclear Antigen lcsh:QH573-671 Ubiquitins Molecular Biology biology lcsh:Cytology Chemistry Drug discovery Methodology Article Mutagenesis Ubiquitination E2 ubiquitin-conjugating enzymes Step-specific assays and evaluation 01.06. Biológiai tudományok Cell Biology UBA1 Protein ubiquitination 3. Good health Ubiquitin ligase Cell biology 030104 developmental biology 030220 oncology & carcinogenesis Ubiquitin-Conjugating Enzymes biology.protein Protein Processing Post-Translational DNA Damage Post-translational modifications |
| Zdroj: | BMC Molecular and Cell Biology, Vol 21, Iss 1, Pp 1-16 (2020) BMC Molecular and Cell Biology |
| DOI: | 10.1186/s12860-020-00262-5 |
| Popis: | Background Ubiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development. Results We developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones. Conclusions These approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades. |
| Databáze: | OpenAIRE |
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