In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers

Abstract

Background: Loss of function mutation in FLG is the major genetic risk factor for atopic dermatitis (AD) and other allergic manifestations. Presently, little is known about the cellular turnover and stability of profilaggrin, the protein encoded by FLG. Since ubiquitination directly regulates the cellular fate of numerous proteins, their degradation and trafficking, this process could influence the concentration of filaggrin in the skin.Objective: To determine the elements mediating the interaction of profilaggrin with the ubiquitin-proteasome system (i.e., degron motifs and ubiquitination sites), the features responsible for its stability, and the effect of nonsense and frameshift mutations on profilaggrin turnover.Methods: The effect of inhibition of proteasome and deubiquitinases on the level and modifications of profilaggrin and processed products was assessed by immunoblotting. Wild-type profilaggrin sequence and its mutated variants were analysed in silico using the DEGRONOPEDIA and Clustal Omega tool.Results: Inhibition of proteasome and deubiquitinases stabilizes profilaggrin and its high molecular weight of presumably ubiquitinated derivatives. In silico analysis of the sequence determined that profilaggrin contains 18 known degron motifs as well as multiple canonical and non-canonical ubiquitination-prone residues. FLG mutations generate products with increased stability scores, altered usage of the ubiquitination marks, and the frequent appearance of novel degrons, including those promoting C-terminus-mediated degradation routes.Conclusion: The proteasome is involved in the turnover of profilaggrin, which contains multiple degrons and ubiquitination-prone residues. FLG mutations alter those key elements, affecting the degradation routes and the mutated products’ stability.

Excess filaggrin in keratinocytes is removed by extracellular vesicles to prevent premature death and this mechanism can be hijacked by Staphylococcus aureus in a TLR2‐dependent fashion

“…Our modelling determined that PRR and vesicle networks converge at the level of HspB1 and HSG, which seem to link the exposure to S. aureus with the ubiquitination process (Katz et al., 2002 ; Parcellier et al., 2003 ). On this note, we have recently found that profilaggrin undergoes ubiquitin‐proteasome system (UPS)‐mediated turnover, with relevance to the disease, especially with the context of FLG mutations, which alter predicted stability of this protein (Paul et al., 2023 ). Given the enrichment in the ubiquitination marks, it is hence plausible that ubiquitination could mediate profilaggrin/filaggrin trafficking to the endocytic system (Ageta & Tsuchida, 2019 ) and sorting into exosomes at the level of MVBs, beyond its role in proteasomal degradation (Liao et al., 2022 ); it could also participate in gene regulation by promoting nuclear localisation (Liao et al., 2022 ).…”