Deimination is regulated at multiple levels including auto-deimination of peptidylarginine deiminases

Abstract

Peptidylarginine deiminases (PADs) catalyze deimination, converting arginyl to citrullyl residues. Only three PAD isotypes are detected in the epidermis where they play a crucial role, targeting filaggrin, a key actor for the tissue hydration and barrier functions. Their expression and activation depends on the keratinocyte differentiation state. To investigate this regulation, we used primary keratinocytes induced to differentiate either by increasing cell-density or by treatment with vitamin D. High cell-density increased PAD1 and 3, but not PAD2, at the mRNA and protein levels, and up-regulated protein deimination. By contrast, vitamin D increased PAD1-3 mRNA amounts, with distinct kinetics, but neither the proteins nor the deimination rate. Furthermore, auto-deimination was shown to decrease PAD activity, increasing the distances between the four major amino acids of the active site. In summary, deimination can be regulated at multiple levels: transcription of the PADI genes, translation of the corresponding mRNAs, and auto-deimination of PADs.

Fig. 1

Effect of Vit D on PADI1–3 transcript levels in NHKs at 1, 7, and 10 days. Quantitative RT-PCR analysis of the steady-state levels of PADI1–3 mRNAs was performed using NHKs treated for 24 h with either 10⁻⁷ M Vit D in DMSO or DMSO alone (control), harvested at the indicated time, and using the YWHAZ mRNA as the reference to normalize. The ratios (treated/control) are expressed as mean ± SEM for a representative experiment performed in duplicate from among at least three independent experiments (*P < 0.05)

Fig. 2

Effect of Vit D on PAD1–3 protein levels and PAD activity in NHKs. NHKs were treated with Vit D (with or without 1.5 mM calcium) or DMSO as the vehicle, as mentioned on the top of the panels, and harvested at the indicated time. a,c Proteins of the “TE-NP40” (a) and “Laemmli” (c) extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes and stained with Ponceau S. b Immunodetection of PAD1–3 and actin in “TE-NP40” extracts. d Immunodetection of deiminated proteins using the anti-citrulline AMC antibody in the “TE-NP40” (top) and “Laemmli” (bottom) extracts. Molecular masses are reported in kDa on the right. Positive controls (Co) correspond either to purified recombinant human PAD1, PAD2 and PAD3, and to human epidermal extracts (b) or to deiminated fibrinogen (d)

Fig. 3

Effect of cell density on PAD1–3 protein levels and PAD activity. a Proteins of the “TE-NP40” (left panel) and “Laemmli” (right panel) extracts of NHKs harvested at intermediate (Int.) and high cell density, as mentioned on the top of the panels, were stained with Ponceau S. b Immunodetections of PAD1–3 (arrowheads) and actin in the “TE-NP40” extracts. The positive controls (c) correspond to purified human recombinant PAD1 (top panel), PAD2 (middle panel), and PAD3 (bottom panel). c Immunodetection in the “Laemmli” extracts of deiminated proteins with the anti-citrulline AMC antibody (top panel), of involucrin (middle panel), and of actin (bottom panel). Data obtained for three independent experiments are shown. Molecular masses are reported in kDa on the right

Fig. 4

Calcium-dependent auto-deimination of PAD1–3 modulates their activity. a Purified PAD1–3 (as indicated) were incubated in the deimination buffer (Tris–HCl 50 mM pH 7.4, CaCl₂ 10 mM, DTT 5 mM) for 1 h at 50°C in the presence (+) or absence (−) of 10 mM EDTA. PAD1–3 were then immunodetected with their respective anti-PAD antibodies (top panel) and the anti-citrulline AMC antibody (bottom panel). b PADs (40 mU) were preincubated for 1 h in the deimination buffer at either 50°C to allow autodeimination (+) or 0°C as a negative control (−). To test for the residual PAD activity after auto-deimination, an equal amount of a recombinant filaggrin subunit was incubated for 5–1,140 min with the preincubated enzymes, as indicated. For each PAD isotype, the times of incubation with filaggrin were chosen according to their efficiency to deiminate this physiological substrate. Filaggrin was then immunodetected with AHF11, a monoclonal antibody recognizing the deiminated filaggrin. Fully deiminated filaggrin is observed at ~66 kDa, whereas partially deiminated forms migrate between ~45 and ~66 kDa. The more efficient the deimination of filaggrin, the closer was its migration to ~66 kDa

Fig. 5

Auto-deimination induces conformational changes of the PAD3 active site. Zoom, from topological models of PAD3, on the four amino acids (as indicated on the left panel) involved during catalysis in the active site: left panel, from “empty” PAD3 model (from WD8) with 39 Arg; middle panel, from “full” PAD3 model (from WDA) with 39 Arg; right panel, from “full” PAD3 model (from WDA) with Cit instead of the most solvent accessible Arg (accessibility ≥ 40%). Distances calculated between the atoms of the four major reactive amino acids, as indicated, are reported in Å