Deimination, Intermediate Filaments and Associated Proteins

Abstract

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

Keywords: citrullination; cytoskeleton; filaggrin; keratin; peptidylarginine deiminase; post-translational modification.

Figure 1

(a) Schematic representation of keratin structure, keratin major post-translational modifications and deamination reaction. P, phosphorylation, yellow dots; G, O-GlcNAcylation, green squares; Cit, citrullination or deimination, orange diamonds. Blue rectangles, 1A, 1B, 2A, 2B helicoidal domains constituting the α-helix rod domain; black boxes, helix initiation and termination motifs (Type I keratins do not contain any helix termination motifs); black triangles, caspase cleavage sites in Type I keratins; grey box, stutter region, variable amino-acid region inducing an imperfect coiled-coil arrangement [37]. L1, L12, L2, non-helicoidal linkers; Head and Tail, non-helicoidal terminal domains; V1/V2, domains highly variable in size. (b) Schematic representation of the irreversible reaction of deimination (or citrullination). AA, amino acid; Arg, arginine; Cit, citrulline (unconventional amino acid not encoded by a tRNA). (c) Deiminated epidermal keratins and filaggrin. Lane 1, Ponceau staining of a total protein extract of a human reconstructed epidermis. Note the keratin migration between 50 and 70 kDa, as expected. Lane 2, immunodetection of human (pro)filaggrin with the AHF3 specific monoclonal antibody [38], (filaggrin monomer, black arrow; filaggrin precursor, profilaggrin, black vertical line). Lane 3, deiminated proteins immunodetected with the anti-modified citrulline specific AMC antibody [17]. (d) Human recombinant His-Filaggrin deiminated by recombinant human PAD1, -2 or -3 (Lanes 1–3, respectively) or non-deiminated (control, Lane 4), as previously described [39]. A huge differential shift can be observed in the migration between the non-modified (Lane 4) and the deiminated (Lane 1–3) filaggrin. Filaggrin was immunodetected with AHF11 monoclonal antibody as previously described [39].

Figure 2

Localization of PAD1 (a–c) and PAD3 (d–e) in human epidermis. (a,d) Immunofluorescence and (b,c,e) immuno-electronic microscopy detection on normal human skin. Bar, 200 nm. The white dashed lines indicate either the dermo-epidermal junction (a) or the top of the Stratum corneum (a,d). White arrowheads point to the immunogold labeling of PAD1 on keratin IF (b) and PAD3 on keratohyalin granules (e).

Figure 3

Multiple alignment of 13 serpin reactive center loops (RCL). Multiple alignment (Multalin) of the full primary sequence of 13 serpins was performed (available at http://multalin.toulouse.inra.fr/multalin/). At the top of the figure, RCL region is delimited by a thick black line and surrounded by beta-sheets (black arrows). Amino acids in red are conserved in all sequences and those in blue are partially conserved. Uniprot accession numbers of the 13 human serpins used are: A12 (Q8IW75), B1 (P30740), B2 (P05120), B3 (P29508), B5 (P36952), B6 (P35237), B7 (O75635), B8 (P50452), B12 (Q96P63), B13 (Q9UIV8), C1 (P01008), G1 (P05155) and F2 (P8697). The positively charged arginine (R) at position P1 of the RCL is indicated by a red triangle for serpin F2 and is highlighted in yellow for the others. Other arginines of the RCL are highlighted in grey, and lysine (K) in green.

Figure 4

Schematic representation of the major involvements of PADs.