Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (p97-PROTAC)
The p97 protein is a member of the AAA+ family of ATPases. This protein is encoded by the <i>VCP</i> 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.
Authors
- Guillermo Valenzuela (ORCID: https://orcid.org/0000-0002-3012-8289)
- Natalia López‐González del Rey
- Pedro Chaná‐Cuevas (ORCID: https://orcid.org/0000-0001-9946-3299)
- Alejandro Rojas‐Fernández (ORCID: https://orcid.org/0000-0002-6004-5425)
- Ignacio Pérez Catalán (ORCID: https://orcid.org/0000-0002-2146-378X)
- Maxs Méndez-Ruette (ORCID: https://orcid.org/0000-0001-7588-4531)
- Gopal P. Sapkota (ORCID: https://orcid.org/0000-0001-9931-3338)
- Carlos León (ORCID: https://orcid.org/0000-0001-8302-0995)
- Natalia Salvadores (ORCID: https://orcid.org/0000-0001-8700-8520)
- David Schwefel (ORCID: https://orcid.org/0000-0002-2945-0908)
- Jose Angel Obeso (ORCID: https://orcid.org/0000-0002-6996-8613)
- Constanza Salinas-Rebolledo (ORCID: https://orcid.org/0000-0002-6220-504X)
- Ronald Jara (ORCID: https://orcid.org/0009-0005-2861-3911)
- Luis Federico Bátiz (ORCID: https://orcid.org/0000-0002-3043-8797)
- Javier Blesa (ORCID: https://orcid.org/0000-0002-4257-1325)
- Janine Burkhalter
- Elizabeth Carrazana
- Francisca Díaz-Tejeda
Institutions
- Universidad de Santiago de Chile (CL)
- Universidad Mayor (CL)
- Austral University of Chile (CL)
- University of Dundee (GB)
- German Center for Neurodegenerative Diseases (DE)
- San Sebastián University (CL)
- Bioscience Research (US)
- Bioanalytica (Switzerland) (CH)
- MRC Protein Phosphorylation and Ubiquitylation Unit (GB)
- Centro de Investigación Biomédica en Red (ES)
- Institute for Biomedicine (IT)
- HM Hospitales (ES)
- University Hospital HM Puerta del Sur (ES)
- Universidad de Los Andes (BO)
- Technische Universität Berlin (DE)
Publication Details
- Journal
- eLife
- Published
- 2025-11-25
- DOI
- https://doi.org/10.7554/elife.101496
- Primary Topic
- Protein Degradation and Inhibitors
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Comunidad de Madrid
- Deutsche Forschungsgemeinschaft
- Swedish Foundation for International Cooperation in Research and Higher Education
- Max-Planck-Gesellschaft
- Agencia Nacional de Investigación y Desarrollo
- Instituto de Salud Carlos III