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Updated: Jul 1, 2025
Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Chemical Proteomics Reveals that the Anticancer Drug Everolimus Affects the Ubiquitin-Proteasome System
Anna A Lobas1, Amir Ata Saei2,3,4, Hezheng Lyu2
1V. L. Talrose Institute for Energy Problems of Chemical Physics, Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia.
Abstract:
Rapamycin is a natural antifungal, immunosuppressive, and antiproliferative compound that allosterically inhibits mTOR complex 1. The ubiquitin-proteasome system (UPS) responsible for protein turnover is usually not listed among the pathways affected by mTOR signaling. However, some previous studies have indicated the interplay between the UPS and mTOR. It has also been reported that rapamycin and its analogs can allosterically inhibit the proteasome itself. In this work, we studied the molecular effect of rapamycin and its analogs (rapalogs), everolimus and temsirolimus, on the A549 cell line by expression proteomics. The analysis of differentially expressed proteins showed that the cellular response to everolimus treatment is strikingly different from that to rapamycin and temsirolimus. In the cluster analysis, the effect of everolimus was similar to that of bortezomib, a well-established proteasome inhibitor. UPS-related pathways were enriched in the cluster of proteins specifically upregulated upon everolimus and bortezomib treatments, suggesting that both compounds have similar proteasome inhibition effects. In particular, the total amount of ubiquitin was significantly elevated in the samples treated with everolimus and bortezomib, and analysis of the polyubiquitination patterns revealed elevated intensities of the ubiquitin peptide with a GG modification at the K48 residue, consistent with a bottleneck in proteasomal protein degradation. Moreover, the everolimus treatment resulted in both ubiquitin phosphorylation and generation of a significant amount of semitryptic peptides, illustrating the increase in the protease activity. These observations suggest that everolimus affects the UPS in a unique way, and its mechanism of action is different from that of its close chemical analogs, rapamycin and temsirolimus.
Insights
Everolimus uniquely impacts the ubiquitin-proteasome system (UPS) by inhibiting proteasome activity, unlike rapamycin or temsirolimus. This study reveals everolimus
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- mTOR signaling regulates cell growth, but its interplay with the ubiquitin-proteasome system (UPS) is not fully understood.
- Rapamycin and its analogs (rapalogs) are known mTOR inhibitors, with some evidence suggesting direct proteasome inhibition.
- Understanding drug effects on cellular protein turnover pathways is crucial for drug development.
Purpose of the Study:
- To investigate the distinct molecular effects of everolimus, rapamycin, and temsirolimus on the A549 cell line using expression proteomics.
- To elucidate the specific impact of everolimus on the ubiquitin-proteasome system (UPS).
- To compare the mechanism of action of everolimus with its rapalogs and a known proteasome inhibitor.
Main Methods:
- Expression proteomics analysis of A549 cells treated with everolimus, rapamycin, temsirolimus, and bortezomib.
- Bioinformatic analysis including cluster analysis and pathway enrichment.
- Detailed analysis of ubiquitin levels, polyubiquitination patterns, and post-translational modifications.
Main Results:
- Everolimus treatment induced a cellular response distinct from rapamycin and temsirolimus, showing similarity to bortezomib.
- Proteasome inhibition was indicated by elevated ubiquitin levels and K48-linked polyubiquitination, specifically with everolimus and bortezomib.
- Everolimus uniquely caused ubiquitin phosphorylation and increased protease activity, suggesting a distinct UPS modulation mechanism.
Conclusions:
- Everolimus exhibits a unique mechanism of action by directly inhibiting the proteasome, differentiating it from rapamycin and temsirolimus.
- The study provides evidence for everolimus's significant impact on the ubiquitin-proteasome system (UPS).
- These findings highlight the differential effects of rapalogs and suggest novel therapeutic strategies targeting protein degradation pathways.
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