USP8 Mutations Associated with Cushing's Disease Alter Protein Structure Dynamics

Abstract

The adenomas in Cushing's disease frequently exhibit mutations in exon 14, within a binding motif for the regulatory protein 14-3-3 located between the catalytic domain (DUB), responsible for ubiquitin hydrolysis, and the WW-like domain that mediates autoinhibition, resulting in constantly active USP8. The exact molecular mechanism of deubiquitinase activity disruption in Cushing's disease remains unclear. To address this, Sanger sequencing of USP8 was performed to identify mutations in corticotropinomas. These mutations were subjected to computational screening, followed by molecular dynamics simulations to assess the structural alterations that might change the biological activity of USP8. Eight different variants of the USP8 gene were identified both within and outside the "hotspot" region. Six of these had previously been reported in Cushing's disease, while two were detected for the first time in our patients with CD. One of the two new variants, initially classified as benign during screening, was found in the neighboring SH3 binding motif at a distance of 20 amino acids. This variant demonstrated pathogenicity patterns similar to those of known pathogenic variants. All USP8 variants identified in our patients caused conformational changes in the USP8 protein in a similar manner. The identified mutations, despite differences in annotation results-including evolutionary conservation assessments, automated predictor data, and variations in localization within exon 14-exhibit similar patterns of protein conformational change. This suggests a pathogenic effect that contributes to the development of CD.

Keywords: 14-3-3 protein; Cushing’s disease; USP8 mutations; USP8 protein; docking; molecular dynamics; pituitary adenoma; protein modeling.

Figure 1

Schematic representation of the protein structure, highlighting functional domains (MIT, Rhod, WW−like, DUB), 14-3-3 and SH3 binding motifs, and the localization and spectrum of detected mutations in our patient cohort. Newly identified mutations (P720_D721delinsR and T739A) are marked with an asterisk (*).

Figure 2

Evolutionary analysis and conservation assessment of positions of interest: (a) Phylogenetic tree of USP8, USP50, and USP2. (b) Alignment segment of positions 708–740. The alignment uses the “Turn” color scheme from iTOL, where the gradient from red to cyan indicates the propensity to form turns. Red represents the highest propensity, while cyan represents the lowest. Gray shades indicate intermediate propensities.

Figure 3

Structural and functional analysis of the USP8 protein using molecular dynamics illustrates the differences in protein behavior between WT and mutants. (a) Root mean square deviation (RMSD) of the protein backbone; (b) radius of gyration; (c) root mean square fluctuation (RMSF) plot comparing WT and mutant USP8 proteins, with the shaded areas highlighting regions of interest, including the catalytic domain (DUB), which demonstrates reduced flexibility in mutants, shaded in pink.