The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations

Abstract

Deimination, a post-translational modification catalyzed by peptidylarginine deiminases (PADs), appears as a crucial Ca(2+)-dependent event in the last steps of epidermal differentiation. In normal human epidermis, where the deiminated proteins are filaggrin and keratins, PAD1, 2 and 3 are expressed but their relative role is unknown. The three PADs, produced as active recombinant forms, showed distinct synthetic-substrate specificities, various efficiencies to deiminate filaggrin and particular calcium and pH sensitivities. Immunoelectron microscopy demonstrated that PAD1 and PAD3 are co-located with filaggrin within the filamentous matrix of the deeper corneocytes where the protein is deiminated. This result strongly suggests that both isoforms are involved in the deimination of filaggrin, an essential step leading to free amino acid production necessary for epidermal barrier function. Moreover, PAD1 was shown to persist up to the upper corneocytes where it deiminates keratin K1, a modification supposed to be related to ultrastructural changes of the matrix.

Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer

“…The granules with PAD1 immunoreactivity were clearly discriminated from the nuclear and cytoplasmic distribution of S100A3. PAD1 in the differentiating cortical cells appeared to be associated with hair keratin fiber, as has been described for the epidermis (18,38). PAD2 was observed as granular patches from the early differentiation stages of all hair epithelial matrix daughter cells; however, as each cellular differentiation advanced, PAD2 appeared in the cytoplasm and partially colocalized with S100A3 in the cuticular and cortical cells.…”

“…In Vitro Modification of S100A3-Active recombinant human PAD1, PAD2, and PAD3 enzymes were prepared according to the previously reported procedures (18,35). Conventionally, 1 g of recombinant S100A3 was reacted with 25 milliunits of PAD enzymes in 20 l of 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl 2 and 5 mM DTT at 37°C.…”

“…All three PAD isozymes were able to catalyze arginine citrullination at lower Ca 2ϩ levels in S100A3 (Fig. 4C) than needed for synthetic substrates and recombinant filaggrin subunit (18). PAD2 and PAD3 enzymes that conduct S100A3 citrullination in vivo required higher Ca 2ϩ concentrations for half-maximal activation (0.35 mM for PAD2 and 0.53 mM for PAD3) than needed by PAD1 enzyme (0.18 mM).…”

“…Although this biochemical process is believed to be precisely regulated by the intracellular Ca 2ϩ concentration, there remains controversy regarding the mechanism by which PAD and transglutaminase could be activated in vivo, since both require a nearly millimolar level of Ca 2ϩ to exhibit their full activities in vitro (12,18). Several epithelial protein barrier components containing Ca 2ϩ -binding domains have been reported to be natural substrates of PAD (the mature filaggrin subunit, proteolytically processed from profilaggrin (13)) or of transgutaminase (S100A7, S100A10, and S100A11 (14,15)) in skin epidermis or of both these enzymes (trichohyalin (16,17)) in hair follicles.…”

La désimination ou citrullination

“…Nous avons montré que PAD1 et PAD3 sont les isotypes qui interviennent dans le catabolisme de la filaggrine, protéine-clé de la différenciation terminale des kératinocytes (cellules majoritaires souris ou de rat (72 à 94 %). Pourtant nous avons démontré que les isotypes 1 à 3 des PAD présentent des spécificités de substrat différentes et des sensibilités au calcium et au pH distinctes [7] et, plus récemment, qu'elles sont régulées de façon différentielle et à de multiples niveaux [8][9][10][11]. Des tests utilisant l'activité luciférase, réalisés dans des kéra-tinocytes humains en culture primaire, ont montré que les gènes PADI1, 2 et 3 sont contrôlés au niveau transcriptionnel par des facteurs ubiquistes comme Sp1, Sp3, NF-Y et MZF1 qui se lient à leurs promoteurs minimaux, longs respectivement de 195, 132 et 129 paires de bases (pour revue voir [8]).…”