Lightning Talk + Poster ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

The usp8 p703h germline mutation impairs 14-3-3 binding in a patient with cushing’s disease (144102)

Adwoa Sey 1 , Martyn Bullock 1 , Roderick Clifton-Bligh 1 2 , Christopher Muir 3 4
  1. Kolling Institute of Medical Research, St Leonards, NSW, Australia
  2. Department of Diabetes, Endocrinology & Metabolism , Royal North Shore Hospital, St Leonards, NSW, Australia
  3. Concord Repatriation General Hospital, Sydney, NSW, Australia
  4. St Vincent’s Hospital, Sydney, Australia, Sydney, NSW, Australia

The interaction between ubiquitin-specific protease 8 (USP8) and 14-3-3 proteins play a critical role in the pathogenesis of Cushing’s disease. As a deubiquitinase, USP8 prevents epidermal growth factor receptor (EGFR) degradation. Disruption of its binding to 14-3-3 enhances EGFR deubiquitination, leading to increased EGFR signalling and elevated production of adrenocorticotropic hormone [1]. We report a patient with Cushing’s disease carrying a germline USP8 mutation (c.2108C>A p.P703H). To date, only one other germline USP8 mutation (c.2155T>C, p.S719P) has been reported. This study aimed to determine whether the P703H mutation, which lies outside of the canonical 14-3-3 binding domain (residues 715-720), affects 14-3-3 binding.

COS7 cells were transfected with FLAG-tagged wild-type (WT) or mutant USP8 constructs (P703H and S719P). USP8/14-3-3 binding was assessed by co-immunoprecipitation followed by western blotting, using anti-14-3-3 and anti-FLAG antibodies. Band intensities were quantified by densitometric analysis. In parallel, AlphaFold-derived structures of USP8 and 14-3-3 were subjected to protein-protein docking with HADDOCK, followed by calculation of binding affinities using PRODIGY [2,3,4].

Western blot analysis demonstrated that the P703H mutant retained reduced but detectable binding to 14-3-3 relative to WT. In-silico analysis supported this finding: wildtype USP8 displayed stronger binding to 14-3-3 (ΔG = -9.2 kcal/mol) compared to the P703H mutant (ΔG = -8.4 kcal/mol) and the S719P mutant which exhibited negligible binding (ΔG = -7.4 kcal/mol).

In summary, our findings show that the USP8 P703H germline mutation reduces 14-3-3 binding, which may contribute to the increased cortisol production in the affected patient, consistent with the relatively mild Cushing phenotype observed clinically. This expands the spectrum of USP8 mutations implicated in the pathogenesis of Cushing’s disease.

  1. 1. Reincke, M., Sbiera, S., Hayakawa, A. et al. Mutations in the deubiquitinase gene USP8 cause Cushing's disease. Nat Genet 47, 31–38 (2015). https://doi.org/10.1038/ng.3166
  2. 2. Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). https://doi.org/10.1038/s41586-021-03819-2
  3. 3. R.V. Honorato, M.E. Trellet, B. Jiménez-García1, J.J. Schaarschmidt, M. Giulini, V. Reys, P.I. Koukos, J.P.G.L.M. Rodrigues, E. Karaca, G.C.P. van Zundert, J. Roel-Touris, C.W. van Noort, Z. Jandová, A.S.J. Melquiond and A.M.J.J. Bonvin. The HADDOCK2.4 web server: A leap forward in integrative modelling of biomolecular complexes. Nature Prot., In Press (2024).
  4. 4. Xue L., Rodrigues J., Kastritis P., Bonvin A.M.J.J.*, Vangone A.*, "PRODIGY: a web-server for predicting the binding affinity in protein-protein complexes", Bioinformatics, doi:10.1093/bioinformatics/btw514 (2016).