Nature Communications (Nov 2023)

BioE3 identifies specific substrates of ubiquitin E3 ligases

  • Orhi Barroso-Gomila,
  • Laura Merino-Cacho,
  • Veronica Muratore,
  • Coralia Perez,
  • Vincenzo Taibi,
  • Elena Maspero,
  • Mikel Azkargorta,
  • Ibon Iloro,
  • Fredrik Trulsson,
  • Alfred C. O. Vertegaal,
  • Ugo Mayor,
  • Felix Elortza,
  • Simona Polo,
  • Rosa Barrio,
  • James D. Sutherland

DOI
https://doi.org/10.1038/s41467-023-43326-8
Journal volume & issue
Vol. 14, no. 1
pp. 1 – 19

Abstract

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Abstract Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.