Activation of PAD4 in NET formation

1The mammalian genome encodes 20 natural amino acids; however, this diversity is greatly increased by posttranslational modification of the original set to yield more than one hundred unique amino acids. Citrullination, or deimination, is the posttranslational modification of an arginine to a citrulline residue. Hydrolysis of the guanidino group of the arginine yields a ureido group and the loss of an ammonia (Figure 1). Citrullination is catalyzed by the peptidyl arginine deiminase family of enzymes, or PADs. This process results in the loss of positive charge and an approximately 1 Da increase in mass. While this modification seems quite modest, the loss of positive charge, and hydrogen bond acceptors, can have dramatic effects on cell signaling because these types of interactions are critical for stabilizing protein–protein, protein–DNA, and protein–RNA interactions. Additionally, this PTM may disrupt intra-molecular interactions, which could trigger major conformational changes in a protein, potentially altering intermolecular interactions and decreasing protein stability.

2Five PAD enzymes are expressed in humans and mice, and the major difference between these isozymes appears to be tissue localization. PADs 1, 3, and 6 are expressed in the skin and uterus, hair follicles, and egg, ovary and embryo, respectively. PAD4 expression has been reported in granulocytes, as well as some cancerous cell lines and tumors, and most recently in mammalian oocytes and the preimplantation embryo. PAD2 has a much broader tissue expression profile and can be found in the CNS, skeletal muscle, and cells of the immune system. PADs 1, 2, and 4 are the only PADs expressed in the hematopoietic lineage, and, thus, are especially of immunological interest.

3PADI genes, encoding the PAD enzymes, are located in a single gene cluster on chromosome 1p36.1 in humans and chromosome 4pE1 in mice. The regions encoding the PADI locus in humans and mice span 334.7 and 230.4 kb, respectively. All PAD enzymes are highly conserved, sharing at least 50% sequence homology among isozymes and 70% or greater homology of each vertebrate ortholog. Eighteen per cent sequence identity is shared among all PADs. Catalysis by all of these enzymes is calcium dependent, and, at least in vitro, requires calcium concentrations that are higher than that available in homeostatic cytoplasm, indicating calcium flux or a calcium-producing event is necessary to induce activity. Alternatively, a PTM or interacting protein may decrease the calcium concentration required for activation to physiologic levels.

4Of all of the PADs, PAD4 is of specific interest because of its importance in innate immunity and its putative role in a variety of pathogenic states, including autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis (MS), ulcerative colitis (UC), and systemic lupus erythematosus (SLE), and other inflammatory conditions, such as sepsis and thrombosis. Many autoantibodies in RA are directed against citrullinated proteins. In fact, the presence of anti-citrullinated protein antibodies (ACPA) is a better predictor of RA than rheumatoid factor, and, in 2011, ACPA were included in the new classification criteria for RA. A genome-wide association study identified a PAD4 haplotype that is associated with RA in a Japanese population, albeit with a low odds ratio (OR = 1.14; ). The mutations in the PAD4 gene appear to confer prolonged stability to the transcript, leading to a model where increased expression of PAD4 in these populations would favor the generation of citrullinated self-epitopes to prime the autoimmune response. Though this association has been confirmed in other Asian populations, it has not been replicated in studies using patients from all Western European populations, indicating that the RA-associated PAD4 haplotype found in Asian RA patients can not explain the presence of ACPA’s in all ethnicities. Interestingly, the PAD4 RA-associated disease haplotype has also been found in some Japanese patients with UC. MS patients have increased levels of the citrullinated form of myelin basic protein, and both PAD2 and PAD4 are overexpressed in the brains of MS patients. Finally, as will be discussed later in this review, in response to microbes, neutrophils can extrude their nuclear contents to form antimicrobial neutrophil extracellular traps (NETs; ). Since PAD4 is essential for the formation of NETs, PAD4 has also been implicated in NET-related pathologies, such as SLE and thrombosis, where NETs presumably promote deleterious inflammatory responses. Thus, PAD4 may be a relevant target for several disease indications.

5PAD4 is a 74 kDa protein that exists as a head-to-tail dimer. Each monomer consists of two N-terminal immunoglobulin (Ig) domains, formed by Ig subdomain 1, which contains nine β-sheets, and Ig subdomain 2, which contains 10 β-sheets and four short α-helices. The C-terminal catalytic domain adopts the α/β propeller fold that is characteristic of the deiminase superfamily. The C-terminal catalytic domain is the most highly conserved area of the molecule, suggesting that the active sites are likely quite similar among PADs. While a high degree of conservation exists among PADs, PAD4 is the only family member to contain a classic nuclear localization sequence (56-PPAKKKST-63), found in Ig1 near the N-terminus, and, thus, is trafficked to the nucleus. However, it is worth noting that recent data indicates that other PADs, most notably PAD2, are localized to the nucleus.

6PAD4 binds five calcium molecules, designated Ca1–Ca5, in a cooperative manner. Ca1 and Ca2 bind in the C-terminal catalytic domain, and their binding induces major conformational changes that move several active site residues into positions that are competent for catalysis. This calcium-induced formation of the active site is unique to the PADs, and represents a novel mechanism of enzyme activation. Calcium binding also induces large structural changes in the N-terminus of the protein. For example, binding of Ca3, Ca4, and Ca5, along with two water molecules, induces the formation of the a1 α-helix, which is disordered in the apoenzyme. These conformational changes may provide, or remove, docking sites for other proteins, which may serve to further regulate PAD activity.

7While it is unknown whether all PAD enzymes are capable of multimerizing, the dimer has been suggested to be required for PAD4 activity. However, the effects on enzyme activity and the calcium dependence of the enzyme are relatively minor (approximately twofold), and we routinely see robust enzyme activity at concentrations of protein that are an order of magnitude below the Kd of the dimer. Nevertheless, dimer formation may represent a possible regulatory mechanism. Dimerization is mediated by both hydrophobic interactions and salt bridges between adjacent monomers.

8The PADs display limited substrate specificity and citrullinate many proteins in vitro, preferring to modify arginine residues present in unstructured regions; the rate of substrate turnover is inversely proportional to the structural order of the substrate. Structurally, PAD4 interacts with the backbone atoms surrounding the site deimination, i.e., R-2, R-1, R0, and (R + 1), with few, if any, contacts with the side chains. The only requirement appears to be a small side chain at the R-2 position so as to avoid steric contacts with the active site. Upon binding to PAD4 the backbone of the substrate adopts a β-turn-like conformation within the substrate binding cleft, thereby explaining why the enzymes show such a high level of substrate promiscuity. In contrast to the situation in vitro, the PADs are believed to show greater substrate specificity in vivo. Presumably, interacting proteins modulate the substrate specificity of the enzyme or spatially target the enzyme to specific regions of the cell. For example, PAD4 is present in the nucleus and may be targeted to chromatin where it citrullinates a number of nuclear proteins, including the histones and protein arginine methyltransferase 1 (PRMT1;; ). Although PAD4 was reported to convert monomethylated arginine residues to citrulline, this modification occurs at rates that are 102- to 103-fold slower than an unmodified arginine, suggesting that the, so-called “demethylimination” reaction is not physiologically relevant and that citrullination simply antagonizes arginine methylation as originally suggested by.

9In addition to the aforementioned protein substrates, PAD4, as well as the other PADs, autocitrullinate at several sites on the enzyme. Although autocitrullination has been reported to directly modulate PAD4 activity, in our hands, this self-modification has no direct effect on enzyme activity, but it does appear to modulate protein–protein interactions. For example, demonstrated that citrullination of PAD4 reduces its ability to interact with PRMT1 and histone deacetylase (HDAC) 1, perhaps modulating its ability to alter gene transcription.

10Given the substrate promiscuity of the PADs, it is unsurprising that the PADs also citrullinate a number of small molecule arginine mimics, including benzoyl arginine ethyl ester (BAEE) and benzoyl arginine amide (BAA). In fact, these compounds have served as important mechanistic probes of PAD4 catalysis and provided the molecular scaffold for the construction of the first highly potent PAD4 inhibitors. Below we highlight key mechanistic insights that guided the design of these inhibitors.

11Briefly, there are four key catalytic residues, Asp350, His471, Asp473, and Cys645. Asp473 binds to both ω-nitrogens and Asp350 coordinates to one ω-nitrogen and the δ-nitrogen (Figure 2A). Cys645 and His471 lie on opposite sides of the guanidinium group and are appropriately positioned to promote catalysis via nucleophilic attack on the guanidinium carbon (Cys645) and protonation of the developing tetrahedral intermediate (His471; Figure 2B). Collapse of this intermediate leads to the loss of ammonia and the formation of the stable S-alkyl thiouronium intermediate that is subsequently hydrolyzed via a second tetrahedral intermediate to form citrulline; His471 likely activates the water molecule for nucleophilic attack (Figure 2B). Mechanistic studies, including mutagenesis, pH rate profile, solvent isotope effects, and solvent viscosity effects, as well as crystal structures of PAD4 bound to several substrates (i.e., BAA; ), and histone H3 and histone H4 tail analogs;, ), and inhibitors (i.e., F-amidine, Cl-amidine, o-F-amidine, o-Cl-amidine, and TDFA;;; ), provide strong support for the above mechanism and helped drive our thoughts about inhibitor design. For example, the presence of a reactive active site Cys prompted us to consider the synthesis of irreversible inhibitors. Additionally, the fact that mono-methylated arginine residues were exceptionally poor PAD substrates, as well as the steric restraints of the active site, told us that the reactive moiety would have to be relatively isosteric with respect to the substrate guanidinium. Furthermore, mutagenesis studies on the two active site aspartyl groups in PAD4, Asp350 and Asp473, showed that these residues are critical for catalysis, indicating that proper positioning, hydrogen bonding, and electrostatic interactions between these residues and the substrate guanidinium are critical determinants for efficient substrate turnover, and would have to be maintained for efficient enzyme inactivation. As such, we initially focused our efforts on synthesizing F-amidine and Cl-amidine (Figure 2C), two haloacetamidine-containing compounds that we hypothesized, and later confirmed, would inactivate the PADs by alkylating Cys645. These initial inhibitors, F-amidine and Cl-amidine, as well as our second generation compounds o-F-amidine, o-Cl-amidine, and TDFA, which show enhanced potency and selectivity, are bioavailable and have been used to show that the PADs play important roles in controlling gene transcription, fertility, differentiation, and NET formation. Additionally, Cl-amidine, or a Cl-amidine analog, decrease disease severity in animal models of RA, UC, nerve damage, and cancer. Specifically, we were the first to show that the PAD inhibitor, Cl-amidine dose dependently decreased inflammation by up to 55% in the mouse collagen-induced arthritis (CIA) model of RA. Concomitant with the decreased severity there were significant decreases in the levels of citrullinated proteins, complement deposition, and epitope spreading. Similar dose-dependent effects were observed in the dextran sodium sulfate (DSS) model of UC where dosing of up to 75 mg/kg after the onset of disease led to significant reductions in weight loss, inflammation score, and colon lengthening. The effects of Cl-amidine on nerve damage was examined in a chick embryo model of spinal cord injury where treatment with Cl-amidine reduced the abundance of deiminated histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at embryonic day 15. Finally, showed that a Cl-amidine analog, YW3-56, decreased tumor growth in a mouse sarcoma S-180 cell-derived solid tumor model and that additive effects on growth inhibition were observed when this compound was combined with the histone deacetylase inhibitor SAHA. We have observed similar effects with Cl-amidine in xenografts model of ductal carcinoma in situ.

12Neutrophils are terminally differentiated granulocytes, which differentiate from hematopoietic stem cells in the bone marrow, and make up to 75% of white blood cells in the circulation. Mature neutrophils are released into circulation, where they have a very short life span of several hours to one day before undergoing apoptosis. Neutrophils are an important component of the innate immune system and form the first line of defense against invading pathogens, such as bacteria and fungi. Neutrophils contain an arsenal of antimicrobial proteins and peptides in primary (or azurophilic), secondary (or specific), and tertiary granules. Primary granules are mostly composed of proteases, such as myeloperoxidase and neutrophil elastase (NE), and antimicrobial peptides, such as defensins. Secondary and tertiary granules contain lactoferrin and gelatinase, respectively. Secretory granules also harbor stores of membrane proteins, such as the NADPH oxidase machinery (Nox; ), which can be trafficked to the surface of the cell quickly when necessary. In response to chemoattractants, neutrophils are guided to areas of infection, where they respond with several effector mechanisms to invading pathogens, including phagocytosis, release of bactericidal products, and ROS production. Neutropenia, or the state of having too few neutrophils, leads to extreme immunosuppression and susceptibility to bacterial infections, which can be fatal.

13In 2004, recognized the formation of NET structures, which were extruded by neutrophils in response to bacteria. NETs are composed of nuclear DNA that are decorated with a variety of nuclear and granular proteins, actively thrown out into the extracellular space, and result in death of the NET-producing cell. Cell death by this mechanism is unique from apoptosis and necrosis and has been termed “NETosis”. NETs ensnare extracellular bacteria, which are killed by the inherent antimicrobial properties of NET-associated proteins, such as histones, NE, and lactoferrin. These structures represent a novel method for pathogen killing, independent of both degranulation and phagocytosis, and have been shown to effectively kill a variety of pathogens, including bacteria, fungi, and protozoa. NETs have also been reported to occur in response to viral infection; however, they not appear to show any observable anti-viral capabilities. NETs may represent a killing mechanism for pathogens too large for the neutrophil to phagocytose, such as fungal hyphae or helminthes. Interestingly, bacteria have adapted defense mechanisms to NET formation. For example, the Group A Streptococcus express a DNase enzyme that can degrade NETs, and Pseudomonas aeruginosa expresses surface molecules that can prevent neutrophil activation and NET formation.

14Histone citrullination is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA coated with antimicrobial molecules. In fact, chemical inhibition of PAD4 activity significantly reduces histone decondensation and NET formation in response to either the calcium ionophore ionomycin or the bacterium Shigella flexneri. Our group and the Wang group have independently created PAD4-deficient mice. Neutrophils isolated from PAD4-deficient mice are unable to citrullinate histones, decondense chromatin, and generate NETs. Because of their inability to form NETs, PAD4 KO mice were shown to be more susceptible to bacterial infection, and, thus, PAD4 is an important mediator of innate immunity.

15Neutrophil elastase, a neutrophil serine protease, resides in the azurophilic granules and is a component of NETs. The cleavage of microbial virulence factors by NE is essential for the clearance of specific Gram-negative bacteria. NE also cleaves histones to drive chromatin decondensation during NET formation. Indeed, NE is essential for the process of NETosis, and it is interesting to speculate that histone citrullination, by PAD4, promotes a relaxing of the chromatin structure, allowing NE to gain access. Thus, the activity of NE and PAD4 may converge upon the chromatin decondensation process and NET formation. Neutrophils isolated from PAD4-deficient mice will be useful to delineate the hierarchy between PAD4 and other molecules, like NE, that are required for NETosis.

16A number of stimuli, including live and heat-killed bacteria, fungi, protozoa, and chemokines have been reported to induce NET formation. Because NET formation is PAD4-dependent, these same stimuli likely also induce PAD4 activation. However, the activity of PAD4 in relation to each stimuli must be assessed by looking for citrullination of histones, which is both a hallmark of PAD activity and a component of NETs. Only a handful these, including live bacteria, the Gram-negative bacterial cell wall component lipopolysaccharide (LPS), the Gram-positive bacterial cell wall component lipoteichoic acid (LTA), the fungal cell wall component zymosan, the proinflammatory cytokine TNFα, and H2O2 have been shown to induce PAD4 activity and histone citrullination. As discussed earlier, PAD4 is calcium-dependent, and it is thought that PAD4 requires calcium levels higher than are found in the homeostatic cell to be active. Not surprisingly, the calcium ionophore ionomycin activates PAD4 to induce histone citrullination and elicit NET formation. Table 1 catalogs the variety of stimuli reported to stimulate NET formation.

17Although little is known about the downstream signaling pathways required for PAD4 activation in neutrophils, cytoskeletal activity may be involved in PAD4 activation. Pretreatment of cells with nocodazole or cytochalasin D, which inhibit microtubule polymerization, prior to LPS stimulation leads to a reduction of histone citrullination and NET formation. Additionally, blockade of integrin signaling through Mac-1 and cytohesin-1 impeded PAD4 activity and NET formation. How cytoskeletal signaling impacts PAD4 is unknown; however, it has been proposed that the same receptors establish whether a cell will undergo phagocytosis or NET formation. Indeed, studies have indicated that neutrophils initiate NET formation when phagocytosis of a large particle fails. Perhaps cytoskeletal activity and PAD4-mediated citrullination are linked because the initiation of NET formation represents a back-up killing mechanism following unsuccessful phagocytosis.

18The generation of reactive oxygen species (ROS) is initiated by a wide variety of neutrophil stimuli, including phagocytosis of pathogens and signaling by LPS and TNF, which are also NET-inducing stimuli. Indeed, ROS generation is required for NET formation, and, thus, it is likely that ROS generation is required for PAD4 activation as well. In neutrophils, superoxide is generated by the NADPH complex (Nox) and by the electron transport chain in mitochondria. is then converted to H2O2 spontaneously or by the enzyme superoxide dismutase (SOD; ). H2O2 acts directly on target cells and is converted to additional effectors by enzymes such as myeloperoxidase (MPO). Interestingly, the addition of H2O2 to primary murine or human neutrophils induces PAD4-dependent histone citrullination. ROS molecules are highly cytotoxic and act as antimicrobial agents, but they can also play a dual role as reversible signal transduction mediators to regulate redox-sensitive target proteins.

19The link between ROS and NET formation was first recognized by the fact that patients with chronic granulomas disease (CGD), who are missing the Nox2 protein essential for NADPH assembly and, thus, cannot form ROS. Neutrophils isolated from CGD patients do not make NETs in response to S. aureus or phorbol myristate acetate (PMA; ). This phenotype is rescued by addition of H2O2 or exogenous glucose oxidase, which generates H2O2, indicating that the ROS production facilitated by Nox2 is necessary for NETs. Catalase removes intracellular H2O2 by reduction to water and oxygen, and catalase inhibition increases intracellular H2O2 leading to increased NET production in healthy neutrophils. Subsequent studies have demonstrated that ROS generation is upstream of chromatin decondensation, suggesting that NADPH oxidase activation may also be a prerequisite for PAD4 activation. Indeed, LPS-induced citrullination of histone H4 is decreased when cells are pre-incubated with the NADPH oxidase inhibitor apocynin. Although, to our knowledge, the activity of PAD4 in CGD neutrophils has not yet been directly explored, since chromatin decondensation is not observed in CGD neutrophils, we would predict PAD4-mediated histone citrullination is also impaired. Since H2O2 treatment can activate PAD4-mediated histone deimination in primary murine and human neutrophils, and since NADPH activation seems to be upstream of NET formation, we speculate that PAD4 activation may be downstream of NADPH (Figure 3).

20The contribution of specific ROS molecules to NET formation has also been examined. NADPH oxidase or mitochondrial ROS selective inhibitors established a requirement for NADPH oxidase generated, but excluded a need for mitochondrial ROS in PMA-induced NET generation. MPO catalyzes the oxidation of Cl- ions to generate hypochlorous acid (HOCl) and other reactive products using H2O2 as a cosubstrate. In the absence of MPO activity, NET generation is absent, but this phenotype can be rescued by addition of HOCl. In fact, the HOCl product of MPO has also been found to be both necessary and sufficient for NET formation, and in CGD neutrophils, the addition of HOCl is also sufficient to initiate NET formation. Taurine is a cellular antioxidant capable of reducing HOCl and H2O2 to promote cell survival. Accordingly, preincubation of neutrophils with taurine prior to PMA or HOCl stimulation reduces NET formation. Additionally, while SOD inhibition does not impede NET formation, addition of exogenous SOD does seem to increase NET production, perhaps owing to the increase in available H2O2. These studies indicate a model in which NADPH oxidase activity generates, which then dismutates to H2O2 either spontaneously or with the help of SOD, and is then used by MPO to generate HOCl, which is necessary and sufficient to induce NET formation. It will be interesting to determine whether HOCl can also directly regulate PAD4 activation.

21Like ROS, autophagy has been shown to be required for chromatin decondensation during NET generation; however, these two processes seem to be independent of each other. Blockade of PI3K with wortmannin inhibits autophagy, and pretreatment of PMA stimulated neutrophils with wortmannin prevented chromatin decondensation. However, no direct role between autophagy, PI3K and citrullination has been shown. Recently, newly developed PAD4 inhibitors were found to reduce autophagy processes in an osteosarcoma cell line, further providing evidence of a link between PAD4 activity and autophagy.

22Neutrophil extracellular traps have been reported in several pathological scenarios, including SLE, sepsis, thrombosis, and infectious disease, and may induce or exacerbate inflammation through prolonged inflammatory response, tissue damage, and presentation of neo-antigens. Indeed, our group recently described the PAD4-dependent formation of NETs in a murine model of the effector phase of RA; however, PAD4 was dispensable for disease in this model. Pathology caused or exacerbated by NETs is an expanding field of research. Because PAD4 is required for NET formation, inhibition of PAD4 activity may improve clinical outcomes in patients experiencing these inflammatory diseases. In fact, PAD inhibitors have demonstrated efficacy in a variety of immune pathologies. Of course, because PAD4 is required for microbial-induced NET formation, the side effects of PAD4-targeted therapeutics may also include increased susceptibility to infectious diseases.

23PAD4 is an important component in the innate immune system, however its activity has been linked to a wide variety of disease states, including cancer, autoimmunity, and other inflammatory conditions. Despite its significance, little is understood about how the PAD4 enzyme becomes active in order to impart its helpful and harmful effects. At the protein level, calcium binding, dimerization, and autocitrullination may help regulate its activity. ROS may also play a role in regulating PAD4 activation (see model in Figure 3), and recently associations between PAD4 activity and autophagy have been proposed. Despite these efforts, much is left to understand about PAD4 enzyme regulation. Anti-PAD4 therapies have been proposed for inflammatory conditions and cancer, thus a more comprehensive understanding of the pathways that activate PAD4 in neutrophils will be important.

24The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.