Biophysical and structural insight into the USP8/14‐3‐3 interaction

Abstract

The ubiquitin-specific protease 8 (USP8)/14-3-3 protein-protein interaction has recently been shown to exert a significant role in the pathogenesis of Cushing's disease (CD). USP8 is a deubiquitinase that prevents epidermal growth factor receptor (EGFR) degradation. Impairment of 14-3-3 binding leads to a higher deubiquitination of EGFR and results in a higher EGFR signaling and an increased production of adrenocorticotropic hormone. Here we report the high-resolution crystal structure of the 14-3-3 binding motif of USP8 surrounding Ser718 in complex with 14-3-3ζ and characterize the interaction with fluorescence polarization and isothermal titration calorimetry. Furthermore, we analyze the effect of USP8 mutations identified in CD on binding to 14-3-3.

Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms

“…6), Conversely, phosphopeptides, even very similar in sequence, can sample much wider ranges of binding affinities for a given 14-3-3 isoform. For instance, for 14-3-3γ, the K D ratio between the strongest and the weakest binding phosphopeptide is almost 625-fold in the present work, and 39,000-fold when taking into account other reports 42,43,60,61 (Fig. 6).…”

“…This principle needs to be further examined for all kinds of phosphorylation-regulated interactions as charge-clamp formation might be an often ignored biochemical property of practically all phosphorylation sites. A. Affinity maps of 14-3-3 interactions based on experimentally determined dissociation constants against the 14-3-3ome, as obtained in the current work and in refs 42,43,60,61 . B.…”

“…Therefore, the motif-to-motif affinity differences are expected to be much higher than the 14-3-3 isoform-to-isoform affinity differences. Binding motifs that are analyzed in other studies are highlighted with a grey background 42,43,60,61 . The color scale is either based on affinity values or in K D ratios.…”

14-3-3: A Case Study in PPI Modulation

“…Recently, the role of 14-3-3 in other pathologies has emerged; for example, cystic fibrosis, through the regulation of the trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein [ 35 ]. The PPI interaction between 14-3-3 and the ubiquitin specific protease 8 (USP8) has recently been shown to exert a significant role in the pathogenesis of Cushing’s disease through the regulation of USP8 enzymatic activity [ 22 , 36 ]. Another relevant example is the GTPase activity of Ras, which is modulated through its protein–protein interaction with SOS1.…”

“…Higher side: 14-3-3σ (white surface) and the peptide ERα (red sticks); lower side: Polar contacts (black dashed lines) and hydrophobic interactions (wireframe white spheres) between the residues of 14-3-3σ (red sticks and white-transparent cartoon) and the pSer binding site of ERα (violet sticks). Mode I for the complex ubiquitin specific protease 8 (USP8)/14-3-3ζ (PDB ID: 6F09) [ 22 ] ( C ). Higher side: 14-3-3ζ (white surface) and the peptide USP8 (red sticks); lower side: Polar contacts (black dashed lines) and hydrophobic interactions (wireframe white spheres) between the residues of 14-3-3ζ (red sticks and white-transparent cartoon) and the pSer binding site of USP8 (violet sticks).…”