Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)
- Austral University of Chile, Chile
- HM Hospitales, Spain
- Universidad San Sebastian, Chile
- Technische Universität Berlin, Germany
- Universidad de Los Andes, Chile
- Universidad Mayor, Chile
- Universidad de Santiago de Chile, Chile
- University of Dundee, United Kingdom
Abstract
The p97 protein is a member of the AAA + family of ATPases. This protein is encoded by the VCP gene. It is a mechanoenzyme that uses energy from ATP hydrolysis to promote protein unfolding and segregation actively. The unfolded products are subsequently presented to the 26S proteasome for degradation. p97 substrate recognition is mediated by adaptors, which interact with substrates directly or indirectly through ubiquitin modifications, resulting in substrate funnelling into the central pore of the p97 hexamer and unfolding. Here, we engineered synthetic adaptors to target specific substrates to p97, using the extraordinary intracellular binding capabilities of camelid nanobodies fused to the UBX domain of the p97 adaptor protein Fas-associated factor-1 (FAF1). In such a way, we created a p97-directed proteolysis-targeting chimera (PROTAC), representing a novel and unique E3 ubiquitin ligase-independent strategy to promote specific proteolysis. All functional assays were performed in human cell lines to evaluate the system's efficacy and specificity in a physiologically relevant context.
Data availability
All data generated in this study are included in the manuscript and supporting files. Source data files for all gels and blots are provided for each main and supplementary figure, including original unprocessed images and labeled versions.
Article and author information
Author details
Pedro Chana-Cuevas
Alejandro Rojas-Fernandez
Funding
Fondo Nacional de Desarrollo Científico y Tecnológico (1200427)
- Alejandro Rojas-Fernandez
Fondo Nacional de Desarrollo Científico y Tecnológico (3220635)
- Guillermo Valenzuela-Nieto
Fondo Nacional de Desarrollo Científico y Tecnológico (13220075)
- Guillermo Valenzuela-Nieto
- Alejandro Rojas-Fernandez
Agencia Nacional de Investigación y Desarrollo (21170632)
- Constanza Salinas-Rebolledo
Centro Ciencia & Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia de ANID (FB210008)
- Alejandro Rojas-Fernandez
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2025, Salinas-Rebolledo et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)
eLife 14:e101496.
https://doi.org/10.7554/eLife.101496
