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.

Arachidonate 12-Lipoxygenase Inhibitors Promote S100A3 Citrullination in Cultured SW480 Cells and Isolated Hair Follicles.

The human hair shaft is covered with multiple scale-like cuticular layers. During the terminal differentiation stage of immature cuticular cells within the hair follicle, cysteine-rich calcium binding S100A3 protein is predominantly translated, and its arginine residues are converted to citrullines by peptidylarginine deiminases (PADI). In this study, we found several naturally occurring compounds (e.g., hinokitiol, escletin, and quercetin) elevate S100A3 citrullination in a human colorectal adenocarcinoma cell line (SW480). Selected compounds similarly promoted cuticular differentiation within isolated human hair follicles. Their promotive activities correlated with the previously reported inhibitory activities of arachidonate 12-lipoxygenase (ALOX12) in vitro. Microarray analysis revealed that ALOX12 inhibitor remarkably up-regulated heparin-binding epidermal growth factor-like growth factor (HBEGF). ALOX12 inhibitor and recombinant HBEGF similarly regulated expression of PADI genes in SW480 cells. In isolated hair follicles, arachidonic acid strongly promoted S100A3 citrullination along with elevation of HBEGF. These results suggest that ALOX12 inhibition efficiently triggers hair cuticle maturation by modulating arachidonate metabolism in concert with HBEGF.

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%.

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.

Age-dependent damage of hair cuticle: contribution of S100A3 protein and its citrullination.

BACKGROUND: There are two types of damage pattern of human hair cuticle: type L, where the cell membrane complex is split and the cuticle lifts up, and type E, where the fragile substructure of the cuticle cell (endocuticle) is broken. In our previous paper, it was reported that the dominant damage pattern shifts from type L to E with the subjects' age around the 40s. Loss of the cuticle due to daily grooming stresses increases with the subjects' age and is related to the level of type E damage. It is supposed that deterioration of endocuticle advances the loss of cuticle. S100A3 protein, located at the endocuticle, was found to be citrullinated and transformed into tetramer to improve its Ca(2+) -binding ability. It is postulated that this biochemical property affects the maturation of cuticle and contributes to its reinforcement. AIMS: This study aims to elucidate the role that S100A3 plays in age-dependent cuticle damage. METHODS: Hair fibers collected from Japanese females were evaluated for the content and citrullination rate of S100A3, incidence of type E damage, and resistance of cuticle. RESULTS: In the aged hair, the content of S100A3 was positively correlated with the level of type E damage and low resistance to stress. Hair fibers in which S100A3 is highly citrullinated, however, showed low levels of type E damage and high resistance of cuticle, even in the aged hair as well as at younger ages. CONCLUSIONS: S100A3 and its citrullination process are related to rigidity of endocuticle of aged hair.

Monomeric Form of Peptidylarginine Deiminase Type I Revealed by X-ray Crystallography and Small-Angle X-ray Scattering.

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca(2+)-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca(2+) at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.

Characterization of epithelial cells in the hair follicle with S100 proteins.

S100 proteins are the largest subgroup of Ca2+ binding proteins with the EF-hand structural motif. A unique feature of this protein family is that individual members are localized in specific cellular compartments. For example, various S100 proteins are expressed in very restricted regions of the hair follicle: S100A3 and S100A6 in distinct postmitotic differentiated epithelial cells and S100A4 and S100A6 in the epithelial stem cell compartments. Characterization of epithelial cells by their S100 protein expression profiles is therefore useful for a better understanding of the dynamic cellular events associated with hair follicle development and regeneration. This chapter presents our protocols for probe preparations and histochemical analyses of S100 proteins in hair follicle tissue, including simultaneous detection procedures for pulse-labeled proliferating cells.

Dissimilar effect of perming and bleaching treatments on cuticles: advanced hair damage model based on elution and oxidation of S100A3 protein.

Hair treatment chemicals induce sudden and severe hair damage. In this study, we examined cuticles from untreated, permed, and bleached hair that were mechanically discriminated by shaking in water. Both perming and bleaching treatments are prone to easily delaminate cuticles. Confocal microscopy revealed that the cuticles of permed hair were delaminated with larger pieces than untreated ones. On the other hand, the cuticles of bleached hair tend to fragment into small peptides. At the minimum concentration of thioglycolate required to elute S100A3 protein from the endocuticle into the reductive permanent waving lotion, enlarged delaminated cuticle fragments were observed. Although S100A3 is retained in bleached hair, S100A3 is irreversibly oxidized upon bleaching treatment. It is likely that the oxidative cleavage of disulfide bonds between cuticle-constituting proteins, including S100A3, results in the fragile property of cuticles. Here we present a more comprehensive model of hair damage based on a diverse mechanism of cuticle delamination.

Label-retaining cells in the bulge region are directed to cell death after plucking, followed by healing from the surviving hair germ.

Hair plucking is the most frequently used method of anagen induction within hair follicles. In this study, we found that plucking leads to the entire renewal of the follicular stem cell region of the mouse pelage follicle. Comparative histochemical analysis revealed that S100A4 protein was specifically distributed in the outer layer of the epithelial sac, which has been identified as the stem cell region of the pelage follicle, whereas the slow cycling cells that retained 5-bromo-2'-deoxyuridine label for 8 wk were located in the epithelial sac and also in the hair germ. Combined terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling method and immunohistochemistry revealed that positive cells were detected in the outer layer of the epithelial sac possessing both bromo-2'-deoxyuridine and S100A4 labels 4.5 h after plucking. No terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling signal, however, was observed in the hair germ. Serial inspection of the plucked follicle revealed that almost all regions of the epithelial sac became terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling positive 12 h after plucking. Terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling-positive cells ultimately degenerated without forming apoptotic bodies. Subsequently, the surviving label-retaining cells in the hair germ migrated upward to re-epithelialize the damaged portion. These results indicate that follicular stem cells in the epithelial sac underwent cell death after plucking. It is likely that the hair germ is responsible for the reconstruction of the stem cell region of the hair follicle.

Labile proteins accumulated in damaged hair upon permanent waving and bleaching treatments.

We previously found that certain hair proteins were soluble by means of a partial extraction method. In this study, we demonstrate that the amount of soluble proteins internally formed in permed and bleached hair, labile proteins, is a useful index for hair damage assessment. Compared to tensile property changes, this index rose in widely dynamic ranges as the time of either permanent waving or bleaching treatments increased. The amount of labile proteins was much larger than that of proteins eluted into perming and bleaching lotions. However, the labile proteins showed electrophoretic profiles similar to those of the eluted proteins. These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.

Practical use of labile protein as an index of hair.

Because of small fluctuations, it is difficult to evaluate hair damage caused by bleaching using previously utilized hair damage indexes. Application of commercial bleaching products elevates partially extractable labile hair protein amounts in the range of 0.4-1.2 mg/g of hair. Within this range, the level of labile protein fluctuates greatly, depending on the extent of bleaching. In the current study, it was found that the effects of alkaline constituents and various peptides contained in bleaching lotions on hair damage could be evaluated by measuring labile protein amounts without employing harsher bleaching conditions.

Purification and characterization of the human cysteine-rich S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

High quantity and quality of recombinant Ca(2+)-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca(2+)-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca(2+)-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn(2+)-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles.

Epithelial Ca(2+)-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca(2+)-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca(2+)-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca(2+)-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn(2+)-pocket, and may transfer Ca(2+) ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca(2+)-cycling occurred as a result of a skin adaptation in terrestrial mammals.

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges.

S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3.

Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca2+ modulator matures human hair cuticle.

S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.

Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen.

The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles.

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.