Histone Citrullination by Protein Arginine Deiminase: Is Arginine Methylation a Green Light or a Roadblock?

Paul R. Thompson * and Walter Fast *

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A B S T R A C T Protein citrullination, a once-obscure post-translational modication (PTM) of peptidylarginine, has recently become an area of signicant interest because of its suspected role in human disease states, including rheumatoid arthritis and multiple sclerosis, and also because of its newfound role in gene regulation. One protein isozyme responsible for this modication, protein arginine deiminase 4 (PAD4), has also been proposed to reverse epigenetic histone modications made by the protein arginine methyltransferases. Here, we review the in vivo and in vitro studies of transcriptional regulation by PAD4, evaluate conicting evidence for its ability to use methylated peptidylarginine as a substrate, and highlight promising areas of future work. Understanding the interplay of multiple arginine PTMs is an emerging area of importance in health and disease and is a topic best addressed by novel tools in proteomics and chemical biology.

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, and Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712

he discovery of nonribosomally encoded citrulline in proteins was rst reported 40 years ago (1, 2), but the importance of this post-translational modication (PTM) to human physiology remained obscure until the 1990s, when two factors came together to bring this modication to prominence. The rst factor was the discovery that rheumatoid arthritis (RA) patients produce autoantibodies targeting citrulline-containing epitopes and that these autoantibodies are a highly specic predictor of the disease (3). The second factor was the determination that histones contain citrulline residues; this nding suggested that this modication could affect gene transcription as a part of the histone code hypothesis (46). In addition, alterations of protein citrullination have been tentatively tied to the etiology of multiple sclerosis, psoriasis, glaucoma, various adenocarcinomas, and even bacterial infections by Porphyromonas gingivalis (711). The contributions of protein citrullination to some of these disease states have been reviewed elsewhere (12, 13). Recent efforts at the molecular, cellular, and wholeorganism levels have begun to characterize the normal and pathophysiological roles of both protein citrullination and the enzymes responsible for catalyzing this modication: the protein (or peptidyl) arginine deiminases (PADs). In this review, we describe these efforts, placing particular emphasis on the role of one isozyme, protein arginine deiminase 4 (PAD4), in transcriptional regulation and its putative demethylimination activity. The PAD Family of Enzymes. The PADs, which are deiminating enzymes that hydrolyze guanidinium side chains to yield peptidylcitrulline and ammonia, belong to a larger group of guanidino-modifying enzymes called the amidinotransferase (AT) superfamily (14). Additional

*Corresponding authors, thompson@mail.chem.sc.edu, waltfast@mail.utexas.edu.

Received for review June 1, 2006 and accepted July 3, 2006. Published online August 18, 2006 10.1021/cb6002306 CCC: $33.50
2006 by American Chemical Society

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Intense efforts have been made to catalog the numbers and types of post-translational modications that occur to histones . . .

members of this superfamily include the arginine deiminases (ADIs), enzymes found in both prokaryotes and the primitive eukaryote Giardia intestinalis, that act on nonpeptidyl arginine and that are involved in energy production; the dimethylarginine dimethylaminohydrolases (DDAHs), enzymes found in both bacteria and mammals that convert nonpeptidyl methylarginines into citrulline; the ATs, enzymes involved in both creatine and streptomycin biosynthesis; and the dihydrolases, bacterial enzymes involved in arginine catabolism that catalyze two successive hydrolytic steps. Whereas most superfamily members act on nonpeptidyl amino acids, PADs are highly specic for peptidylarginine residues and require at least one additional residue N-terminal to the site of modication (15). Although PAD and PAD-like enzymes are not universally conserved, they are present in several species, from bacteria to humans. Interestingly, the bacterial PADs are composed of only the 40 kDa catalytic domain, whereas mammals have much larger multidomain enzymes ( 75 kDa) whose activity is regulated by calcium. The extra domains in the mammalian enzymes, present in the N-terminal half of the protein, include two immunoglobulin-like domains that are proposed to mediate proteinprotein interactions and/or substrate targeting (16). To date, ve human PAD homologues have been identied. For historical reasons, these isozymes are designated PAD14 and PAD6. Human PAD4 was initially named PAD5 but was later renamed PAD4 to reect the fact that it is a true ortholog of this isoform. PAD4 is distinguished by the insertion of a nuclear localization sequence and, in contrast to the cytoplasmic location of the other isoforms, is the only PAD localized to the cell nucleus (17). Role of PAD4 in Transcriptional Regulation. Since the seminal nding that the histone acetylases and deacetylases (18, 19) are transcriptional coregulators, intense and continuing efforts have been made to catalog the numbers and types of PTMs that occur to histones on the premise that other histone-modifying enzymes might also inuence gene transcription. Thus, the discovery that PAD4 is a nuclear enzyme that deiminates histones H2A, H3, and H4 (4) strongly suggested that it may also act as a transcriptional coregulator. A combination of proteomic techniques has determined that the in vivo sites of histone deimination occur at the N-terminal tails of histones H2A, H3, and H4 and, specically, H2 Arg3, H3 Arg2, H3 Arg8, H3 Arg17, H3 Arg26, and H4 Arg3 (2022). 434
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Several independent studies have subsequently conrmed transcriptional coregulation by PAD4. For example, Wang et al. (22) transfected increasing amounts of a PAD4-encoding construct into MCF7 cells, a mammary carcinoma or breast-cancer cell line, and monitored the effects of this treatment on the transcription of an estrogen-responsive luciferase reporter construct. In these experiments, PAD4 clearly acts as a transcriptional corepressor in a dose-dependent manner. The catalytic activity of PAD4 is essential for this function because the corepressor function of a catalytically inactive mutant is signicantly attenuated (22). Similarly, Cuthbert et al. (20) demonstrated that PAD4 fused to a zinc-nger DNA binding domain (PAD4-ZnDBD) could act as a transcriptional corepressor for the endogenous vascular endothelial growth factor-A (VEGF-A) promoter. For these experiments, the PAD4-ZnDBD was cotransfected with constructs encoding either the estrogen receptor or thyroid hormone receptor ligand binding domains fused to a different VEGF-A targeting ZnDBD, and transcription of the endogenous VEGF-A gene was monitored by real-time polymerase chain reaction (20). Importantly, the wild-type enzyme, but not a catalytically inactive mutant (a C-terminal truncation lacking amino acids 591663), could repress the levels of transcription afforded by the thyroid hormone receptor construct, an indication that citrullination by PAD4 is essential for this effect. Chromatin immunoprecipitation (ChIP) experiments further demonstrated a role for PAD4 in transcriptional regulation (20, 22). In these experiments, the levels of PAD4, various transcriptional cofactors, and specic histone modications on the endogenous estrogen receptor responsive pS2 promoter were monitored at specic time points before and after the addition of estradiol. MCF7 cells were used for these experiments because PAD4 protein is expressed in this cell line in an estrogen-dependent manner (20, 22). These ChIP experiments demonstrated that PAD4 is constitutively associated with the pS2 promoter; its levels increased slightly 2040 min after the addition of estradiol and then decreased thereafter to basal levels. Importantly, the amount of citrulline present in the N-terminal tails of histones H3 and H4 rose and then fell with similar kinetics. The levels of PAD4 and citrullinated histones on the pS2 promoter also correlated with its presumed function as a transcriptional corepressor because the levels of RNA polymerase II on this promoter were high when the
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H 2N NH2+ NH H N O H 2N O NH H N O H N H H N H Peptidyl-Arg CH3 NH2+ HN NH H N Peptidyl-Cit CH3 NH2+ N NH H N CH3 CH3 NH+ HN NH H N H 3C O Peptidyl-MMA H N H O Peptidyl-ADMA H N H O Peptidyl-SDMA H N H

Figure 1. Arginine modications.

levels of PAD4 and deiminated histones were low and vice versa. These results demonstrate that PAD4 is a transcriptional coregulator. The repressive effects of PAD4 on gene regulation are likely mediated by its ability to catalyze the deimination of specic residues present in the N-terminal tails of histones H3 and H4 (and possibly H2A) because histone modications are generally known to affect transcription by altering the local chromatin structure either directly or indirectly via the recruitment of additional transcriptional coregulators (5, 23, 24). PAD4 has been shown to catalyze the citrullination of the glucocorticoid receptor interacting protein 1 binding domain in p300 (and antagonize methylation of this domain by coactivator-associated arginine methyltransferase 1, CARM1) (25). This suggests that, in addition to histone citrullination, the deimination of other transcriptional coregulators can contribute to the transcriptional corepressor function of this enzyme. Does PAD4 Catalyze Demethylimination? In addition to citrullination, arginine residues in the N-terminal tails of histones H3 and H4 are alternatively monomethylated and asymmetrically dimethylated on their guanidinium side chains. These methylation events are catalyzed by protein arginine methyltransferase 1 (PRMT1) and CARM1 (CARM1/PRMT4), which are type I PRMTs that catalyze the sequential formation of both N -monomethylarginine (MMA) and asymmetric N ,N -dimethylarginine (ADMA) in an S-adenosylmethionine (SAM)dependent manner (Figure 1). Note that PRMTs are generally classied as either type I or II enzymes. Type I
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PRMTs (e.g., PRMT1 and CARM1) catalyze the formation of peptidyl MMA and ADMA, whereas type II enzymes (e.g., PRMTs 5 and 7) catalyze the formation of peptidyl MMA and symmetric N ,N =-dimethyl-L-arginine (SDMA). Notably, the sites of deimination by PAD4 overlap with the sites of arginine methylation. For example, CARM1 methylates arginines 2, 17, and 26 in histone H3 (2628), and PRMT1 methylates arginine 3 in histone H4 (29, 30). These PRMTs act as transcriptional coactivators for numerous transcription factors (e.g., the estrogen receptor (31)), and their methyltransferase activity is required for this function. Generally, asymmetric dimethylation, not monomethylation, is associated with transcriptional activation (26, 27, 29, 30). This overlap of methylation and deimination sites provided further support for the initial suggestion by Bannister et al. (6) that PAD4 might act to reverse the transcriptional coactivator function of the PRMTs by hydrolyzing methylated arginines to citrulline. PADcatalyzed hydrolysis of methylated arginines, dubbed a demethylimination reaction, would require the recognition of methylated arginines as substrates and would not truly reverse the modication but instead leave peptidylcitrulline as a nal product. True reversibility by an arginine demethylase has not been discovered yet, but if PAD4 can remove the methyl mark introduced by the PRMTs, then it might serve an analogous function. Several lines of evidence reported by Cuthbert et al. (20) and Wang et al. (22) suggest that, in addition to its established deiminase activity, PAD4 may also catalyze demethylimination in vivo. For example, ChIP assays on the pS2 promoter show that the appearance of citrulline on both histone H3 and histone KEYWORDS H4 correlates with decreasing Activity-based protein proling: A technique that levels of ADMA. Similarly, the uses small-molecule probes to detect enzyme activities in complex mixtures. levels of methylated histones S-Alkylthiouronium: A cationic S-alkylated thioH3 Arg17 and H4 Arg3 are urea functional group [(R-S(NR=2)ANR 2)] reduced in HL 60 granulocytes, found as a covalent intermediate in the amidinotransferase superfamily. a cell line known to express Amidinotransferase: An enzyme capable of transPAD4, after treatment of this ferring an amidino group (C(NH2)ANH2) from cell line with a calcium ionoa donor substrate to an acceptor substrate. Also, a superfamily of enzymes that can phore. The disappearance of catalyze group transfer or hydrolysis reactions these epitopes correlates with at the iminium carbon of selected guanidines. the appearance of citrulline on Autoantibodies: Antibodies produced by an organism, such as humans, that recognize histones H3 and H4. Although host antigens and can elicit an inappropriate these results appear to supimmune response. port an in vivo PAD4 demethVOL.1 NO.7 433441 2006

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D473 O H 2N S C645 R [P] + [S] O NH2 NH

+ +

H471 HN NH

O S

O NH

O O

D350

+ HN NH2 NH2 O NH O R

NH4+ + H2O

ylimination activity, alternative explaO O O O HOH N nations exist. For HN NH NH NH NH S O example, deiminaSH O NH O NH tion might antagoO O R R nize methylation (substrate depleFigure 2. Proposed chemical mechanism for PAD4. See the tion), and deiminatext for details. tion of other sites within histones H3 and H4 might prevent antibody recognition (epitope occlusion). These alternatives would not necessarily require PAD4 to deiminate methylated arginine residues. Although the in vivo evidence described above supporting a physiologically relevant demethyliminase activity is compelling, the in vitro studies of this putative activity are less so and, in many cases, are contradictory. For example, recombinant histones H3 and H4 methylated by CARM1 and PRMT1, respectively, with [3H]-SAM used as the methyl donor and presumably bearing MMA, were demethyliminated by PAD4 with the concomitant production of methylamine (22). In sharp contrast, several independent laboratories have provided compelling evidence that PAD4 cannot catalyze efcient demethylimination in vitro. For example, Kearney et al. (15) synthesized several small-molecule and peptide substrates containing MMA, ADMA, and SDMA and then evaluated their ability to act as PAD4 substrates. While these compounds were demethyliminated by PAD4, the rates at which they are processed are much slower than the rates of deimination observed for the corresponding peptidylarginines, with observed rate reductions on the order of 100- to 10,000-fold (15). The in vitro rates of PAD4-mediated demethylimination are 1000-fold slower than the rate at which arginine residues in histone H3 are methylated by CARM1 (28); this makes PAD4 a relatively inefcient demethyliminase. Similar results have been reported by three groups: Hidaka et al. (32) utilized HPLC and mass spectrometry (MS)-based assays to demonstrate that benzoylated methyl arginine derivatives are neither good substrates nor good inhibitors of PAD4; Cuthbert et al. (20) showed that ADMA- and SDMA-containing peptides were not demethyliminated by PAD4; and Wang et al. (22) established that histone H4-based peptides methylated by PRMT1 (and presumably bearing MMA) were poor substrates for PAD4 in vitro. These in vitro studies strongly suggest that PAD4 does not possess a physi+ 2 2 +

ologically relevant demethylimination activity and are in apparent conict with the in vivo data. Structure and Mechanism behind PAD Activity and Specicity. Recent structural and mechanistic studies of PAD4 and other members of the AT superfamily provide a framework for understanding how PAD4 catalyzes deimination and how this enzyme can achieve a 100- to 10,000-fold kinetic discrimination between substrates that differ by as little as one methyl group. One possible chemical mechanism for PAD4catalyzed deimination is proposed below (Figure 2). The substrate guanidinium is held in place by an intricate hydrogen-bonding network formed between this group and two active-site aspartyl groups (Asp350 and Asp473; PAD4 numbering is used unless otherwise indicated) that are conserved in both PADs and ADIs. These interactions help to position the guanidinium for nucleophilic attack by a conserved active-site cysteine nucleophile (Cys645 in PAD4), resulting in formation of an initial tetrahedral adduct between Cys645 and the guanidinium carbon. Collapse of this adduct to form a covalent planar S-alkylthiouronium intermediate then proceeds with the concomitant release of the rst product, ammonia. Cleavage of this CN bond likely requires concomitant protonation, and His471 is well-positioned to serve as a general acid for this step. The resulting planar S-alkylthiouronium intermediate is subsequently hydrolyzed to release the nal product, citrulline. The hydrolytic water required for this step is potentially deprotonated to hydroxide by a general base such as His471, which is well-positioned to serve this function. Alternatively, the participation of ammonia (product-assisted catalysis) has been proposed to play a role in hydroxide formation because PAD4 does not possess a suitably positioned amino acid to improve the basicity of His471, whereas other AT superfamily members do possess such a residue (15). A substrate-assisted mechanism has also been proposed (33). Although detailed studies of PAD4 catalysis have been limited, key aspects of the proposed mechanism have been conrmed, including the identity and stoichiometry of the reaction products (15, 32), the incorporation of solvent 18O into citrulline (15, 32), the structural conservation of the active site, and the essential nature of conserved active site residues, for example, Cys645, Asp350, Asp473, and His471, for activity (16). This proposed mechanism is also supported by structural and mechanistic studies on PAD4 and similarities
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Figure 3. Structures of putative deiminase reaction intermediates. a) Human C645S PAD4 complexed with a protected arginine substrate mimicking the proposed Michaelis complex. b) Mycoplasma arginini ADI complexed with a proposed covalent tetrahedral reaction intermediate. c) M. arginini ADI complexed with an sp2-hybridized covalent reaction intermediate showing an ordered water (red sphere) poised for the subsequent hydrolytic step. PDB structures 1WDA, 1LXY, and 1S9R, respectively, were used to create the gure panels.

with other hydrolytic members of the AT superfamily, such as ADI and DDAH (3339). For example, the extensive hydrogen-bonding network of the substrate guanidinium with Asp350 and Asp473 can easily be seen in the structure of the PAD4(C645S)N -benzoyl-L-arginine amide complex, which mimics the initial enzyme substrate complex (Figure 3, panel a). A snapshot of the initial tetrahedral species has also been observed in structures of the ADIarginine complex (Figure 3, panel b) (33, 34). While this tetrahedral adduct was originally assigned as a covalent complex formed by the back reaction with citrulline (34), later functional analyses suggested that this structure most likely depicts the species preceding formation of the planar thiouronium intermediate (37). The lifetime of this tetrahedral species does not appear to contribute signicantly to the kinetic mechanism of ADI (38), but its structure does strongly suggest that a conserved active-site histidine is appropriately positioned to serve as a general acid to donate a proton to the leaving group. This proposed role is consistent with the elevated pKa (7.9) assigned to His471 of PAD4 (15) and also with studies of ADI and DDAH (16, 33, 3537). Collapse of the tetrahedral adduct and the elimination of ammonia lead to the formation of a planar sp2-hybridized thiouronium intermediate that is known to have a signicant lifetime in the kinetic mechanisms of both ADI and DDAH (33, 3537). Structures of the thiouronium intermediate bound to ADI are available (Figure 3, panel c) and demonstrate that the tight hydrogen-bonding network between the substrate and the two active-site aspartyl groups is conserved throughout the reaction (33, 34). The structure of
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the ADIthiouronium intermediate also reveals an ordered water molecule that is poised to attack the thiouronium intermediate, passing through a similar tetrahedral species (not shown) and generating citrulline during the hydrolytic half-reaction. Both His471 and Asp473 are well-placed to assist this step by deprotonating the water to provide a nucleophilic hydroxide, but additional studies will be required to determine the actual general base for this step. It should also be emphasized that further study will be required to determine the subtle differences in mechanism between ADI, DDAH, and PAD that result in their drastically different pH-rate proles (15, 36, 37). While the molecular details regarding PAD4 catalysis are still being deciphered, the structural and mechanistic data obtained to date for PAD4 and the related enzymes ADI and DDAH provide insight into how PAD4 is able to discriminate between substrates that differ by only one methyl group. First, it is unlikely that methylation contributes intrinsically to the stability of these residues KEYWORDS toward hydrolysis. N -MethylCitrullination: A post-translational protein modication ation only has small effects on that converts arginine side chains to citrulline. Also the overall charge, electrophicalled deimination. Covalent intermediate: A reaction intermediate that licity, and leaving-group stabilcontains a transient covalent bond formed with an ity of these substrates, as demenzymes active-site residue or with an enzyme-bound onstrated by the small variance cofactor. Deiminase: An enzyme that can hydrolyze an imine bond in pKa values (13.413.6) (R1R2CANR3R4). reported for guanidine, N-methyl- Demethyliminase: A deiminase capable of hydrolyzing an guanidine, and N,N-dimethylN-methylated imine substrate (R1R2CANR3CH3). Epigenetic information: Heritable information that is not guanidine (40). The fact that directly encoded by the genome. N -methylated substrates are
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Figure 4. Structural overlay of an arginine analogue complexed with human PAD4 (red) and citrulline complexed with Pseudomonas aeruginosa DDAH (green). Ligands are shown as ball-andstick models, and the active-site surfaces are color-coded to match the parent protein. The positioning of Asp residues interacting with the N and N nitrogens of the ligands are highly conserved, but the terminal Asp473 in PAD4, which makes a bidentate complex to each of the substrates N guanidino nitrogens, is not conserved in DDAH and serves to narrow the binding pocket and exclude N -methylated ligands. The gure was created from PDB structures 1WDA and 1H70.

not effective PAD4 inhibitors (32) supports the idea that PAD4s preference for unmethylated substrates is due to steric exclusion rather than intrinsic stability of methylated guanidiniums. A structural comparison with the related DDAH enzymes is instructive. DDAH is very selective for N -methylated arginine substrates, and against unmodied arginine residues. This selectivity is essential for the proper physiological function of DDAH because it ensures that DDAH only hydrolyzes N -methylated arginines, which are endogenous inhibitors of nitric oxide synthase, and does not hydrolyze arginine, which is a nitric oxide synthase substrate. A structural overlay of the PAD4 and DDAH active sites (Figure 4) shows very similar positioning of the substrates side chains, and a side-on bidentate ligation of N and N by Asp350. Also conserved is a visible water channel that extends to the surface of the protein, presumably allowing the rst product, ammonia, to diffuse away, and providing solvent access for the hydrolytic step. The most notable divergence between these two structures is the binding pocket for the two terminal N nitrogens. PAD4 (and ADI) interacts with these nitrogens through a bidentate interaction with Asp473, whereas DDAH has a lysine at this position. In DDAH, the hydrophobic portion of this lysine residue helps to form a pocket for the N -methyl groups. DDAH does possess a residue that is the functional equivalent of Asp473 (Glu65 in DDAH), but this residue comes from a different loop of the enzyme, forms only a monodentate interaction with the unsubstituted N -nitrogen, and helps to direct the substituted N -methyl group into its binding pocket. Although these particular substitutions are the most obvious differences between these two proteins, more extensive remodeling of the active-site environment (33, 37), including the positioning of Asn588, Val469, and Glu474, likely help to occlude N -methyl group binding in PAD4 and underlie the preference of this enzyme toward unmethylated arginine residues. Future Perspectives. Protein citrullination is emerging as an important PTM for both human disease and for gene regulation. However, signicant questions about 438
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PAD activity, specicity, and regulation remain unanswered. These questions represent excellent opportunities for the application of novel tools in proteomics and chemical biology and will likely be areas of intense future research. Of primary interest is reconciling the in vitro and in vivo data for the demethylimination activity of PAD4: are the in vivo results due to substrate depletion or epitope occlusion, or can PAD4 be transformed into an efcient demethyliminase in vivo? Several reasonable explanations for the latter possibility have been proposed, including the presence of accessory proteins or PTM of PAD4 that might increase its activity toward methylated substrates. The recent design and synthesis of covalent inactivators that either broadly target the AT superfamily (41) or specically target the active form of the PADs (42) will be very useful in designing activitybased protein proling reagents (43) that should be able to directly address these and other related issues. The sequence specicities of PAD isozymes are also poorly dened, and recent crystal structures of PAD4 bound to short peptides (44) have not fully explained how the PADs, and in particular PAD4, regiospecically modify particular arginine residues in vitro and in vivo (2022). Although interactions between substrates and either the immunoglobulin-like domains or PAD binding proteins likely contribute to the substrate selectivity of these enzymes, it should be possible to determine a consensus sequence for this modication by analysis of a citrullinated proteome. Tools for detecting protein citrullination in the proteome include immunodetection of chemically modied citrulline residues (45) and MS analysis of either 16O/18O PAD-labeled products (46) or chemically modied citrulline residues. These residues can be derivatized with 2,3-butanedione alone or in combination with antipyrine to generate characteristic mass shifts in citrulline-containing peptides (47). The regulation of PAD isoforms also raises important unanswered questions. PAD4 is a calcium-dependent enzyme that requires millimolar amounts of calcium to deiminate its protein substrates in vitro (15), yet the in vivo concentrations of calcium typically do not rise
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above the low micromolar levels. Additionally, the presence of an active-site cysteine nucleophile and the sensitivity of the PADs to oxidation raise the possibility of redox regulation through reaction of this group with reactive oxygen or nitrogen species. An understanding of the physiologically relevant regulation mechanisms is essential for building a complete picture of how protein citrullination is controlled. From a perspective of systems biology, the interplay of other enzymes and reactive metabolic compounds with residues targeted by PAD4 is also of signicant interest. For example, if PAD4 cannot hydrolyze methylated substrates, then the possibility exists that other enzymes could serve as demethylases to truly reverse this modication. Interestingly, recent reports indicate that lysine-specic demethylase 1 (LSD1), a avinadenine dinucleotide-dependent amino oxidase, and members of the JmjC domain-containing histone demethylase (JHDM) family of enzymes, which are nonheme Fe(II), O2, and -ketoglutarate-dependent dioxygenases, can reverse histone lysine methylation (4850). The presence of large numbers of LSD1 and JHDM homologues ( 10 and 28, respectively) in an individual species (49) makes it reasonable to expect that a true arginine demethylase might be found among these coding sequences and would help to determine whether these enzymes act alone or possibly in combination with a modied form of PAD4 to control the levels of methylarginine. Also, the reaction of peptidylarginine and peptidyllysine with metabolically derived dicarbonyl reagents, such as methylglyoxal, results in PTMs collectively called advanced glycation end (AGE) products, which are well-studied hallmarks of diabetes and other diseases (5153). Dimethylation or citrullination would be expected to substantially change the susceptibility of peptidylarginine to these modications. Finally, the demonstration that citrulline levels in the N-terminal tails of histones H3 and H4 rise and then fall suggests that citrullination itself may be reversible. These results could be explained by epitope occlusion, histone tail clipping (54), or nucleosome displacement by chromatin remodeling enzymes; however, a more interesting proposal would be that the transitory nature of this PTM is due to the actions of an enzyme or enzymes that convert citrulline back into arginine. The existence of such a decitrullinase, as suggested by Bannister et al. (6), is a distinct possibility because precedents exist for the conversion of nonpeptidyl citrulline to arginine in the urea cycle by argininosuccinate synthetase and argininosuccinase. In summary, a complete understanding of the interplay of citrullination with other PTMs of arginine (e.g., methylation, AGE) will greatly inform our ideas about RA, gene regulation, and other aspects of human physiology. The characterization of these activities promises to be a fullling avenue of future research. Reconciling the results obtained from in vitro and in vivo studies of the PADs and PRMTs can best be achieved through the development and application of novel tools for studying the proteomics and chemical biology of arginine modication.
Acknowledgment: We thank the University of South Carolina Research Foundation (P.R.T.), the American Heart Association (0565409U; P.R.T.), the Arthritis National Research Foundation (W.F.), the American Cancer Society (RSG-05-061-01-GMC; W.F.), and the Robert A. Welch Foundation (F-1572; W.F.) for support.

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