ABSTRACT
Filaggrin (FLG) is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it co-localized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis (RHEs) using benzyloxycarbonyl-Pro-Prolinal (ZPP) induced the accumulation of FLG monomers. Down-regulation of PREP expression in RHEs using RNA interference confirmed the impact of PREP on FLG metabolism, and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, Western blotting and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification like loricrin. Consequently, at the functional level, the trans-epidermal electric resistance was drastically reduced.
ABSTRACT
Deimination is a post-translational modification catalyzed by a family of enzymes named peptidylarginine deiminases (PADs). PADs transform arginine residues of protein substrates into citrulline. Deimination has been associated with numerous physiological and pathological processes. In human skin, three PADs are expressed (PAD1-3). While PAD3 is important for hair shape formation, the role of PAD1 is less clear. To decipher the main role(s) of PAD1 in epidermal differentiation, its expression was down-regulated using lentivirus-mediated shRNA interference in primary keratinocytes and in three-dimensional reconstructed human epidermis (RHE). Compared to normal RHEs, down-regulation of PAD1 caused a drastic reduction in deiminated proteins. Whereas proliferation of keratinocytes was not affected, their differentiation was disturbed at molecular, cellular and functional levels. The number of corneocyte layers was significantly reduced, expression of filaggrin and cornified cell envelope components, such as loricrin and transglutaminases, was down-regulated, epidermal permeability increased and trans-epidermal-electric resistance diminished drastically. Keratohyalin granule density decreased and nucleophagy in the granular layer was disturbed. These results demonstrate that PAD1 is the main regulator of protein deimination in RHE. Its deficiency alters epidermal homeostasis, affecting the differentiation of keratinocytes, especially the cornification process, a special kind of programmed cell death.
ABSTRACT
Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.
