Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies
Four nanobodies (VHH1-4<sub>SMT3</sub>) that target the yeast SUMO protein Smt3p were isolated and characterized. VHH1-4<sub>SMT3</sub> bind to Smt3p and Smt3p-tagged proteins with high affinity (Kd: low nM). NMR analysis shows that the four nanobodies all bind near the C-terminus of Smt3p, partially overlapping with the binding site for the SUMO protease Ulp1p. Binding of Smt3p-specific nanobodies impairs Ulp1-mediated cleavage of Smt3p-tagged proteins, with VHH1<sub>SMT3</sub> showing complete inhibition. The use of immobilized VHH2<sub>SMT3</sub> enabled efficient purification of Smt3p-tagged proteins, while VHH1<sub>SMT3</sub> can be used for immunoblotting and detects both Smt3p-tagged and free Smt3p. When expressed in yeast, VHH1<sub>SMT3</sub> causes significant growth defects, particularly when targeted to the nucleus or fused with GFP, indicative of interference with essential SUMOylation-dependent processes.
Authors
- Novalia Pishesha (ORCID: https://orcid.org/0000-0002-8124-1265)
- Allison Y. Tang (ORCID: https://orcid.org/0000-0003-0602-7063)
- Pavana Suresh (ORCID: https://orcid.org/0000-0003-1597-6818)
- Charlotte Wijne
- Gan Jin (ORCID: https://orcid.org/0000-0002-8785-3035)
- José L. Avalos (ORCID: https://orcid.org/0000-0002-7209-4208)
- Hidde L. Ploegh (ORCID: https://orcid.org/0000-0002-1090-6071)
- Ishani Sahay
- Sun Zhen-Yu Jim
Institutions
- Boston Children's Hospital (US)
- Harvard University (US)
- Princeton University (US)
- Dana-Farber Cancer Institute (US)
Publication Details
- Journal
- Scientific Reports
- Published
- 2025-05-26
- DOI
- https://doi.org/10.1038/s41598-025-02380-6
- Citations
- 2
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 2.72
Funders
- National Institutes of Health