Structures of human peptidylarginine deiminase type III provide insights into substrate recognition and inhibitor design.

Peptidylarginine deiminase type III (PAD3) is an isozyme belonging to the PAD enzyme family that converts arginine to citrulline residue(s) within proteins. PAD3 is expressed in most differentiated keratinocytes of the epidermis and hair follicles, while S100A3, trichohyalin, and filaggrin are its principal substrates. In this study, the X-ray crystal structures of PAD3 in six states, including its complex with the PAD inhibitor Cl-amidine, were determined. This structural analysis identified a large space around Gly374 in the PAD3-Ca2+-Cl-amidine complex, which may be used to develop novel PAD3-selective inhibitors. In addition, similarities between PAD3 and PAD4 were found based on the investigation of PAD4 reactivity with S100A3 in vitro. A comparison of the structures of PAD1, PAD2, PAD3, and PAD4 implied that the flexibility of the structures around the active site may lead to different substrate selectivity among these PAD isozymes.

Deimination and Peptidylarginine Deiminases in Skin Physiology and Diseases.

Deimination, also known as citrullination, corresponds to the conversion of the amino acid arginine, within a peptide sequence, into the non-standard amino acid citrulline. This post-translational modification is catalyzed by a family of calcium-dependent enzymes called peptidylarginine deiminases (PADs). Deimination is implicated in a growing number of physiological processes (innate and adaptive immunity, gene regulation, embryonic development, etc.) and concerns several human diseases (rheumatoid arthritis, neurodegenerative diseases, female infertility, cancer, etc.). Here, we update the involvement of PADs in both the homeostasis of skin and skin diseases. We particularly focus on keratinocyte differentiation and the epidermal barrier function, and on hair follicles. Indeed, alteration of PAD activity in the hair shaft is responsible for two hair disorders, the uncombable hair syndrome and a particular form of inflammatory scarring alopecia, mainly affecting women of African ancestry.

Human S100A3 tetramerization propagates Ca(2+)/Zn(2+) binding states.

The S100A3 homotetramer assembles upon citrullination of a specific symmetric Arg51 pair on its homodimer interface in human hair cuticular cells. Each S100A3 subunit contains two EF-hand-type Ca(2+)-binding motifs and one (Cys)3His-type Zn(2+)-binding site in the C-terminus. The C-terminal coiled domain is cross-linked to the presumed docking surface of the dimeric S100A3 via a disulfide bridge. The aim of this study was to determine the structural and functional role of the C-terminal Zn(2+)-binding domain, which is unique to S100A3, in homotetramer assembly. The binding of either Ca(2+) or Zn(2+) reduced the α-helix content of S100A3 and modulated its affinity for the other cation. The binding of a single Zn(2+) accelerated the Ca(2+)-dependent tetramerization of S100A3 while inducing an extensive unfolding of helix IV. The Ca(2+) and Zn(2+) binding affinities of S100A3 were enhanced when the other cation bound in concert with the tetramerization of S100A3. Small angle scattering analyses revealed that the overall structure of the S100A3 tetramer bound both Ca(2+) and Zn(2+) had a similar molecular shape to the Ca(2+)-bound form in solution. The binding states of the Ca(2+) or Zn(2+) to each S100A3 subunit within a homotetramer appear to be propagated by sensing the repositioning of helix III and the rearrangement of the C-terminal tail domain. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminase (PAD) catalyzes the post-translational conversion of peptidylarginine to peptidylcitrulline in the presence of calcium ions. Among the five known human PAD isozymes (PAD1-4 and PAD6), PAD1 exhibits the broadest substrate specificity. Crystals of PAD1 obtained using polyethylene glycol 3350 as a precipitant diffracted to 3.70 Å resolution using synchrotron radiation. Two PAD1 molecules were contained in the asymmetric unit and the crystals belonged to space group P6(1), with unit-cell parameters a = b = 90.3, c = 372.3 Å. The solvent content was 58.2%.

Deimination of human filaggrin-2 promotes its proteolysis by calpain 1.

Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type III.

In the presence of calcium ions, human peptidylarginine deiminase (PAD) converts arginine residues in proteins to citrulline. Of the five known human PAD enzymes, the type III isozyme (PAD3) exhibits the highest specificity for synthetic and natural substrates. This study aimed to determine the structure of PAD3 in order to elucidate its selective citrullination mechanism. Crystals of PAD3 obtained using polyethylene glycol 400 as a precipitant diffracted to 2.95 Å resolution using synchrotron radiation. They belonged to space group R3, with unit-cell parameters a = b = 114.97, c = 332.49 Å (hexagonal axes). Assuming two molecules were contained in an asymmetric unit, the calculated Matthews coefficient was 2.83 Å(3) Da(-1), corresponding to a solvent content of 56.6%. Initial phases were determined using PAD4 as a molecular-replacement model.

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations.

Peptidylarginine deiminases (PADs) are enzymes that catalyze the Ca2+-dependent conversion of arginine residues into proteins to citrulline residues. Five PAD isozymes have been identified in mammals. Several studies have shown that the active-site pockets of these isozymes are formed when Ca2+ ions are properly bound. We previously characterized the structures of PAD3 in six states. Among these, we identified a "nonproductive" form of PAD3 in which the active site was disordered even though five Ca2+ ions were bound. This strange structure was probably obtained as a result of either high Ca2+ concentration (∼260 mM)-induced denaturation during the crystallization process or high Ca2+-concentration-induced autocitrullination. While autocitrullination has been reported in PAD2 and PAD4 for some time, only a single report on PAD3 has been published recently. In this study, we investigated whether PAD3 catalyzes the autocitrullination reaction and identified autocitrullination sites. In addition to the capacity of PAD3 for autocitrullination, the autocitrullination sites increased depending on the Ca2+ concentration and reaction time. These findings suggest that some of the arginine residues in the "nonproductive" form of PAD3 would be autocitrullinated. Furthermore, most of the autocitrullinated sites in PAD3 were located near the substrate-binding site. Given the high Ca2+ concentration in the crystallization condition, it is likely that Arg372 was citrullinated in the "nonproductive" PAD3 structure, the structure was slightly altered from the active form by citrulline residues, and probably inhibited Ca2+-ion binding at the proper position. Following Arg372 citrullination, PAD3 enters an inactive form; however, the Arg372-citrullinated PAD3 are considered minor components in autocitrullinated PAD3 (CitPAD3), and CitPAD3 does not significantly decrease the enzyme activity. Autocitrullination of PAD3 could not be confirmed at the low Ca2+ concentrations seen in vivo. Future experiments using cells and animals are needed to verify the effect of Ca2+ on the PAD3 structure and functions in vivo.

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

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.

Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3.

Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated, the development of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies, which suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors ever described.

Transcriptional regulation of peptidylarginine deiminase expression in human keratinocytes.

Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzyme catalyzes the conversion of arginine residues to citrulline residues in the presence of calcium ion, which is an elaborate post-translational modification on the target protein. Recently, five isoforms have been identified in mammals. Among them, three isoforms (type I, II, III) are expressed in the human epidermis, and involved in several skin physiological and pathological processes. In the past few years, several researches concerning the transcriptional regulation of three human PADI type genes (PADI1, PADI2 and PADI3) in the epidermis have been carried out. In this review, we describe an overview of the current outcomes about these studies with their significance. It is anticipated that these investigations will provide novel therapeutic and prophylactic targets for future approaches to the treatment or prevention of severe psoriasis and bullous congenital ichthyosiform erythroderma.

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress.

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four alpha-helixes and 16 beta-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2h after initiation of the stress and was highly induced between 18-72h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.

Peptidylarginine Deiminase Inhibitor Cl-Amidine Attenuates Cornification and Interferes with the Regulation of Autophagy in Reconstructed Human Epidermis.

Deimination, a post-translational modification catalyzed by a family of enzymes called peptidylarginine deiminases (PADs), is the conversion of arginine into citrulline residues in a protein. Deimination has been associated with numerous physiological and pathological processes. Our aim was to study its implication in the homeostasis of human epidermis, where three PADs are expressed, namely PAD1, 2, and 3. Three-dimensional reconstructed human epidermis (RHEs) were treated for 2 days with increased concentrations (0-800 µM) of Cl-amidine, a specific PAD inhibitor. Cl-amidine treatments inhibited deimination in a dose-dependent manner and were not cytotoxic for keratinocytes. At 800 µM , Cl-amidine was shown to reduce deimination by half, alter keratinocyte differentiation, decrease the number of corneocyte layers, significantly increase the number of transitional cells, induce clustering of mitochondria and of heterogeneous vesicles in the cytoplasm of granular keratinocytes, and upregulate the expression of autophagy proteins, including LC3-II, sestrin-2, and p62/SQSTM1. LC3 and PADs were further shown to partially co-localize in the upper epidermis. These results demonstrated that Cl-amidine treatments slow down cornification and alter autophagy in the granular layer. They suggest that PAD1 and/or PAD3 play a role in the constitutive epidermal autophagy process that appears as an important step in cornification.

Molecular characterization of a novel salt-inducible gene for an OSBP (oxysterol-binding protein)-homologue from soybean.

Oxysterol-binding protein (OSBP) and its homologues constitute a protein family in many eukaryotes from yeast to humans, which are involved in cellular lipid metabolism, vesicle transport and signal transduction. In this study, we characterized a novel salt-inducible gene for an OSBP-homologue from soybean (Glycine max [L.] Merr.). The soybean OSBP-homologous gene, denoted as G. max OSBP (GmOSBP), encoded a 789 aa putative protein with two characteristic domains; the pleckstrin homology (PH) domain and the ligand-binding (LB) domain, in the N- and C-terminus, respectively. The GmOSBP-PH domain showed localization into/around the nucleus in a transient subcellular localization assay. The phylogenetic relationship of the GmOSBP-LB domain to those in other OSBP-homologues suggested that GmOSBP might bind a lipid molecule(s) different from the ligand-candidates found for the human/yeast OSBP-homologues. The GmOSBP gene was constitutively transcribed in all of the soybean organs examined--root, stem and trifoliate leaf--at low levels and was highly induced in all these organs by high-salt stress (300 mM NaCl). Interestingly, gene expression of GmOSBP was also markedly induced in the senesced soybean cotyledon, which contains high levels of a variety of cellular lipids utilized for energy for germination and as membrane components. Therefore, we suggest that GmOSBP may be involved in some physiological reactions for stress-response and cotyledon senescence in the soybean.

Estrogen-enhanced peptidylarginine deiminase type IV gene (PADI4) expression in MCF-7 cells is mediated by estrogen receptor-alpha-promoted transfactors activator protein-1, nuclear factor-Y, and Sp1.

Human peptidylarginine deiminase type IV (PAD4), a Ca(2+)-dependent enzyme known to convert arginine residues to citrulline residues in histones, has been shown to be associated with the development of rheumatoid arthritis. Recently, it was noted that the human peptidylarginine deiminase type IV gene (PADI4) regulates the expression of estrogen-responsive genes by modifying the methylated arginine sites in histones H3 and H4. In this study, we demonstrated that PADI4 was expressed in MCF-7 cells and was responsive to estrogen at the transcriptional level. Using the luciferase reporter gene fused to wild-type or mutated 5'-flanking region of PADI4, we characterized that as few as 348 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Chromatin immunoprecipitation and small interfering RNA assays revealed that activator protein-1, Sp1/Sp3, and nuclear factor-Y were cis-acting factors bound to the minimal promoter of PADI4 and that they regulated gene expression in a cooperative manner. Moreover, it was indicated that estrogen stimulated PADI4 expression through binding of estrogen receptor (ER)-alpha to the upstream of the PADI4 gene and ERalpha-mediated enhancement of activator protein-1, Sp1, and nuclear factor-Y levels. These findings indicated that estrogen stimulated PADI4 expression through both of the classical and nonclassical ER-mediated pathways.

Purification and characterization of three neutral extracellular isoperoxidases from rye leaves.

Rye (Secale cereale L.) seedlings; contain two major flavone glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (L3GlcUA) (1) and luteolin 7-O-diglucuronide (L2GlcUA) (2) in abundance in the apoplast of primary leaves; express a large number of peroxidase isoenzymes; and release H(2)O(2) into the apoplast during primary leaf development. We purified and characterized three neutral extracellular peroxidase isoenzymes (rPOXs N1, N2, and N3) that can oxidize L2GlcUA as a natural substrate. The isoelectric points and molecular weights of rPOXs N1, N2, and N3 were 6.1, 7.2, and 6.3, and 42, 37, and 51 kDa, respectively. The optimum pH of the rPOXs N1, N2, and N3 were 5.5, 5.5, and 8.5, respectively, and their optimum temperatures ranged from 45 to 50 degrees C for all isoenzymes. rPOXs N1, N2, and N3 recognized flavonoids with 3', 4'-OH groups as potential substrates, but not flavonoids with a glycosylated 4'-OH group or those without a 3'-OH group. The activities on phenol-type substrates were high in the order of guaiacol>catechol>o-cresol for all isoenzymes. rPOXs N1, N2, and N3 exhibited broad reactivity with endogenous hydrogen donors including luteolin glucuronides derived from the apoplast of rye primary leaves.

NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes.

Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.

Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown.

BACKGROUND: Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. OBJECTIVE: To test whether external humidity plays a role, and investigate the molecular mechanisms involved. METHODS: Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30-50% relative humidity) were compared. RESULTS: RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. CONCLUSION: These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation.

Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases.

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated in vivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNRHis) deiminated in vitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNRHis was shown to be cleaved by calpain-1 in vitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism.

Proteomics implicates peptidyl arginine deiminase 2 and optic nerve citrullination in glaucoma pathogenesis.

PURPOSE: Proteomic analyses of normal and glaucomatous human optic nerve were pursued for insights into the molecular pathology of primary open-angle glaucoma (POAG). Peptidyl arginine deiminase 2 (PAD2), an enzyme that converts protein arginine to citrulline, was found only in POAG optic nerve and was probed further for a mechanistic role in glaucoma. METHODS: Protein identification used liquid chromatography-tandem mass spectrometry. Northern, Western, and immunohistochemical analyses measured PAD2 expression and/or protein citrullination and arginyl methylation in human and mouse optic nerve and in astrocyte cultures before and after pressure treatment. Proteins were identified after anticitrulline immunoprecipitation. In vitro translation of PAD2 was monitored in polyA RNA depleted optic nerve extracts. PAD2 shRNA transfections were evaluated in pressure-treated astrocytes. RESULTS: Western and immunohistochemical analyses confirmed elevated PAD2 and citrullination in POAG optic nerve and decreased arginyl methylation. PAD2 was also detected in optic nerve from older, glaucomatous DBA/2J mice, but not in younger DBA/2J or control C57BL6J mice. Myelin basic protein was identified as a major citrullinated protein in POAG optic nerve. Pressure-treated astrocytes exhibited elevated PAD2 and citrullination without apparent change in PAD2 mRNA. Addition of exogenous polyA RNA to depleted optic nerve extracts yielded increased PAD2 expression in POAG but not in control extracts. Transfection with shRNA restored PAD2 and citrullination to control levels in pressure-treated astrocytes. CONCLUSIONS: Current results support translational modulation of PAD2 expression and a possible role for the enzyme in POAG optic nerve damage through citrullination and structural disruption of myelination.

Modulation of peptidyl arginine deiminase 2 and implication for neurodegeneration.

PURPOSE: To demonstrate that elevated pressure increases the peptidyl arginine deiminase 2 (PAD2) expression in cultured astrocytes in vitro that can be modulated by pharmacological agents modulating intracellular calcium. METHODS: Isolated rat brain astrocytes were subjected to pressure treatment. Western and immunohistochemical analyses detected PAD2 protein expression. Calcium measurements were achieved employing fluorescence-based microscopic imaging and quantification system. Experiments were repeated with human optic nerve head-derived astrocytes. RESULTS: PAD2 has recently been shown to be associated with glaucomatous optic nerve. Astrocytes subjected to pressure (25-100 mmHg) show elevated level of PAD2, increased intracellular calcium, and concomitant citrullination but not significant cell death. PAD2 expression in response to elevated pressure may play a role in glaucomatous neurodegeneration. Pressure-treated astrocytes were also subjected to thapsigargin (50-250 nM) treatment, but it is unclear whether this had any further effect in increasing PAD2 expression. Conversely, treatment with calcium chelating agent BAPTA-AM (50-250 nM) results in decreased intracellular calcium concentration and PAD2. CONCLUSIONS: These results suggest calcium modulation could be exploited as therapeutic strategy to modulate pressure-induced PAD2 expression and citrullination.