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Tytuł pozycji:

Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades

Tytuł :
Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades
Autorzy :
Fenteany, Gabriel
Gaur, Paras
Sharma, Gaurav
Pintér, Lajos
Kiss, Ernő
Haracska, Lajos
Pokaż więcej
Temat :
Methodology Article
Ubiquitination
Ubiquitin-like proteins
Post-translational modifications
E1 ubiquitin-conjugating enzymes
E2 ubiquitin-conjugating enzymes
E3 ubiquitin ligases
Step-specific assays and evaluation
High-throughput screening
Źródło :
BMC Molecular and Cell Biology
Wydawca :
BioMed Central, 2020.
Rok publikacji :
2020
Oryginalny identyfikator :
pmc: PMC7106726
pmid: 32228444
Język :
English
ISSN :
2661-8850
DOI :
10.1186/s12860-020-00262-5
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.
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