Structure insights into the autoinhibitory mechanism of the deubiquitinating enzyme usp25 and into the sumo e1-e2 protein-protein recognition

• Autores: Bing Liu
• Directores de la Tesis: David Reverter Cendrós (dir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2018
• Idioma: español
• Tribunal Calificador de la Tesis: Salvador Ventura Zamora (presid.), Francesc Xavier Gomis-Rüth (secret.), Travis H. Stracker (voc.)
• Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en:  TESEO  TDX 
• Resumen

  • Ubiquitination and SUMOylation are of the most studied post-translational modifications (PTMs). Here, we focus on USP25, USP28, and the SUMO E1-E2 protein-protein recognition in these two PTM pathways. USP25 and USP28 have important roles in cellular processes, and their enzymatic activities are regulated by diverse PTMs including SUMOylation, ubiquitination, and phosphorylation. SUMO E1-E2 protein-protein interaction is a major discrimination step in the conjugation pathway. In this thesis, the main goals include the elucidation of the structural basis for the activity regulation of USP25 and USP28, as well as to decipher the structural determinants for the specificity provided by the E1 UFD-E2 interaction.

    We have solved the crystal structure of human USP25 (18 – 714). Unexpectedly, USP25 displays a homotetrameric quaternary assembly that is directly involved in the inhibition of its enzymatic activity, revealing a novel tetramerization/inhibition mechanism. In vitro biochemical and kinetic assays with dimer, tetramer and truncation constructs of USP25 support this mechanism, displaying in all cases a higher catalytic activity in the dimer assembly. Moreover, the strong stabilization of tankyrases in cultured cells by the ectopic expression of the USP25 dimer verifies the biological relevance of this novel tetramerization/inhibition mechanism.

    Regarding to the E1 UFD-E2 interaction, we have solved the crystal structure of the E1 UFD-E2 complex in both human and A. thaliana. Despite the low sequence homology displayed by the UFD binding interface, structural comparison between complexes reveals common determinants in the interfaces between human, yeast, and A. thaliana. Structural comparison also reveals a strong conservation in the E2 binding interface across species, despite the strong specificity displayed in SUMO conjugation assays for each organism. Interestingly, E2 residues outside the UFD interface had impact on SUMO conjugation, suggesting the contribution of determinants other than the primary UFD binding interface in the specificity of the conjugation system.