In the normal human epidermis, filaggrin monomers are deiminated, mainly by PAD1, before their full degradation to free amino acids, a process also known as filaggrinolysis essential for the epidermal barrier functions. This proteolysis involves several peptidases, namely caspase-14, calpain-1 and bleomycin hydrolase. However, their combined activities do not explain the complete proteolysis of filaggrin, and other proteolytic enzymes should be implicated.
Mining several omics analyses we identified the prolyl-endopeptidase PREP as a candidate peptidase to act during filaggrinolysis. In normal human skin, PREP co-localizes with filaggrin in the upper epidermis as demonstrated by indirect immuno-fluorescence confocal microscopy. Tandem mass spectrometry and fluorescent quenching activity assays demonstrated that PREP cleaved several synthetic peptides derived from the filaggrin sequence, at the carboxyl side of internal proline residues. Deimination of an arginine close to the cleavage sites increased PREP enzymatic efficiency. In tridimensional reconstructed human epidermis, specific PREP inhibition using benzyl-oxy-carbonyl-prolin-prolinal (or ZPP) induced the accumulation of FLG monomers.
Since we, and others previously demonstrated that the deiminated form of filaggrin is more sensitive to the proteolytic activity of calpain-1 and bleomycin hydrolase, deimination appears as a major step to facilitate the full proteolysis of filaggrin in the epidermis. The decreased expression of PAD1 and deimination of filaggrin in the skin of atopic dermatitis patients may therefore participate to the pathogenesis of this frequent inflammatory skin disease.