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<p>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.</p>

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Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月26日

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  • Pavana Suresh,
  • Sun Zhen-Yu Jim,
  • Ishani Sahay,
  • Allison Y. Tang,
  • Charlotte Wijne,
  • Jin Gan,
  • Novalia Pishesha,
  • Jose L. Avalos,
  • Hidde Ploegh

摘要

Four nanobodies (VHH1-4SMT3) that target the yeast SUMO protein Smt3p were isolated and characterized. VHH1-4SMT3 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 VHH1SMT3 showing complete inhibition. The use of immobilized VHH2SMT3 enabled efficient purification of Smt3p-tagged proteins, while VHH1SMT3 can be used for immunoblotting and detects both Smt3p-tagged and free Smt3p. When expressed in yeast, VHH1SMT3 causes significant growth defects, particularly when targeted to the nucleus or fused with GFP, indicative of interference with essential SUMOylation-dependent processes.