title: Recognition mechanism of p63 by the E3 ligase Itch: novel strategy in the study and inhibition of this interaction.
abstract: The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer's or Huntington's diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly (13)C-(15)N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534-551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.
authors.affiliation: Dipartimento di Scienze e Tecnologie Chimiche, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Bellomaria
authors.forename: Gaetano
authors.initials: G
authors.lastname: Barbato
authors.forename: Gerry
authors.initials: G
authors.lastname: Melino
authors.forename: Maurizio
authors.initials: M
authors.lastname: Paci
authors.forename: Sonia
authors.initials: S
authors.lastname: Melino
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D003500:Cyclization; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D008670:Metals; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases