Agonist stimulation of integrin receptors, composed of transmembrane alpha and beta subunits, leads cells to regulate integrin affinity ('activation'), a process that controls cell adhesion and migration, and extracellular matrix assembly. A final step in integrin activation is the binding of talin to integrin beta cytoplasmic domains.
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Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large “head” on two “legs,” with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains.
These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.
The name “integrin” was suggested for an integral membrane protein complex first characterized in 1986. The name was devised because the protein identified linked the extracellular matrix to the cytoskeleton (early developments in this field have been well described ).
In the 25 years since that first characterization, a vast amount of work has been performed, with consequent increased understanding. The essential role of integrins in tissue organization and cell development, their signal transduction mechanisms (from outside to in and inside to out!), and their potential as therapeutic targets is now established. In this article, we provide an overview of the structure of integrins, the conformational changes that determine activation state, and the mechanisms of ligand binding.
Overall StructureIntegrins are heterodimers of non-covalently associated α and β subunits. In vertebrates, there are 18 α and 8 β subunits that can assemble into 24 different receptors with different binding properties and different tissue distribution (; ). The α and β subunits are constructed from several domains with flexible linkers between them.
Each subunit has a single membrane-spanning helix and, usually, a short unstructured cytoplasmic tail. The size varies but typically the α- and β-subunits contain around 1000 and 750 amino acids, respectively. Numerous reviews on integrin structure and function have been published (;;; ) so here we concentrate mainly on the implications of recent structural work that includes studies of intact ectodomains, membrane spanning regions, cytoplasmic tails and their ligands. The EctodomainsThe breakthrough crystal structure of αVβ3 started a deluge of structural information about integrin ectodomains. Structures of αVβ3, with and without ligand (, ), αIIbβ3 , and αxβ2 are all now available.
These crystal structures are all in a similar overall “bent” conformation that would place the ligand binding site near the membrane surface. The overall topology and structure of integrin ectodomains is illustrated in for the case of αxβ2 , which has an inserted α-I domain. Integrin structure. The Structure of α Subunit EctodomainsThe α-chain consists of four or five extracellular domains: a seven-bladed β -propeller, a thigh, and two calf domains.
Nine of the 18 integrin α chains have an α -I domain of around 200 amino-acids, inserted between blades 2 and 3 of the β-propeller. The I domain, a copy of which also appears in the β-chain, has five β-sheets surrounded by seven α helices; it is similar to von Willebrand A domains. The last three or four blades of the β-propeller contain domains that bind Ca 2+ on the lower side of the blades facing away from the ligand-binding surface. Ca 2+ binding to these sites has been shown to influence ligand binding (; ).The thigh and calf domains have similar, immunoglobulin-like, β-sandwich folds. They have 140–170 residues with more β-strands than typical Ig-like domains (∼100 residues).
There are two main regions of interdomain flexibility. One is the linker between the β-propeller and the thigh; the other is the “genu” or knee at the bend between the thigh and the calf-1 domain. The α-subunit genu is located close to the similar bend in the β subunit, thereby allowing extension by a hinging at the knees.
The α-I domain in αxβ2 is inserted in the β-propeller domain with flexible linkers (C). Unlike the other four α-leg domains, which have relatively rigid structures, I domains show conformational changes within the domain that are important for regulating binding affinity (see below and ). The Structure of β Subunit EctodomainsThe β-leg has seven domains with flexible and complex interconnections. A β -I domain is inserted in a hybrid domain, which is, in turn, inserted in a plexin-semaphorin-integrin (PSI) domain. These domains are followed by four cysteine-rich epidermal growth factor (EGF) modules and a β -tail domain. The hybrid domain in the upper β-leg has a β-sandwich construction.
The β-I domain, which is homologous to the α-I domain, is inserted into the hybrid domain. The small PSI domain, with an α/β fold, is also split into two portions (; ) connected, in β3, by a long-range Cys-13 to Cys-435 disulfide bond.Unusual EGF module boundaries were first proposed in the αVβ3 structure , but recent crystal structures suggest that each EGF module has an even number of eight cysteines, bonded in a C1-C5, C2-C4, C3-C6, and C7-C8 pattern except for EGF1, which lacks the C2-C4 disulfide. The αIIbβ3 structure shows that all 56 cysteines in the integrin β3 subunit are disulfide bonded.The β-tail domain has an α + β fold.
The weak electron density of this domain observed in the αIIbβ3 crystal structure was taken to suggest a flexible connection to other regions of the β-leg by a mobile “ankle”. A contact between the CD loop of the β-tail domain and the α7 helix of the β-I domain has been proposed to inhibit integrin activation—the “deadbolt” model. This contact is, however, small and no such contact is observed in the αIIbβ3 or αxβ2 structures.In general, the β-leg seems to be more flexible than the α-leg. Evidence for this comes from the ten different structures of αXβ2 observed in three different crystal lattices. The EGF domain region is relatively plastic, especially between EGF1 and EGF2, the β knee, and at the PSI/hybrid and hybrid/I-EGF1 junctions. There is evidence for important conformational changes occurring in the β-I/hybrid region. A transition from a “closed” to an “open” conformation of the β-I domain has been observed when the β-I α7-helix moves toward the hybrid domain.
The connecting rodlike motion of the α7-helix causes the hybrid domain to swing-out by ∼60 o (see ). Cation Binding SitesAs described below, ligand binding in α-I less integrins takes place at the largest interface between the two subunits (the β-propeller/β-I domain interface); binding is dependent on the cations Mg , Ca 2+, and Mn.
From a structure of an α-I domain it was suggested that integrin ligand binding involves a Mg ion, a “metal-ion-dependent adhesion site” (MIDAS) ; a crystal form with bound Mn also showed considerable movement of the α7-helix on activation. In the recent αIIbβ3 structure strong electron densities were ascribed to cations at three sites formed by loops in the β-I domain ; Mg was assigned to the central MIDAS site with Ca 2+ at the two flanking sites. One of these adjacent sites (ADMIDAS) binds an inhibitory Ca 2+ ion; binding of Mn here results in a structural change that gives an active integrin. The second Ca 2+-binding site has been called the synergistic metal ion binding site (SyMBS). Mutational studies show that the SyMBS site is responsible for Ca 2+ synergy (; ). Three β-I domain metal-binding sites in αXβ2 ; aspartate ligands to the metal ions are shown in cyan (from PDB:3FCS); figure drawn using PyMOL (DeLano Scientific).As mentioned above, the β-I domain has distinct closed and open conformational states, involving movement of the α7 helix, in α-I-less integrins (; ). Similar conformational changes are seen in α-I-domains.
When integrin ligands, such as Arg-Gly-Asp, bind the open state, the MIDAS Mg ion coordinates the Asp side chain of the ligand. In α-I integrins it has been suggested that the β-I MIDAS may bind an intrinsic ligand, an invariant Glu, Glu-318 in αX. Support for this model comes from Glu mutations that abolish integrin activation (; ); the observed flexibility of the αx α-I domain would also facilitate such interdomain interactions. The Membrane Spanning HelicesThe current view is that association of integrin α and β transmembrane (TM) segments, results in an inactive resting receptor. Evidence for this includes experiments using EM , disulfide cross-linking , activating mutations , and FRET of labeled cytoplasmic tails. Recent studies have given new insight into the structure of the resting state. The structures of β3 and αIIb TM segments in phospholipid bicelle model membranes have been solved by NMR separately (,) and in complex.
A similar structure was obtained for the TM region in intact αIIbβ3 using disulfide-based distance restraints combined with protein modeling. A recent structure of a complex in organic solvents has been described but the relevance of a system without a phospholipid/water interface is questionable. A bacterial reporter system has been used to define the sequence motif required for TM helix-helix interactions in β1 and β3 integrin subfamilies. Several modeling studies of the TM regions have also been published (;; ). Note that whereas most evidence supports the heterodimeric form, there is also evidence that homomeric TM oligomers can form in vitro (; ); nevertheless, the evidence for homodimeric forms mainly comes from experiments or simulations performed without the ectodomains, whose presence would be expected to favor the heterodimeric form.The NMR structure of the αIIbβ3 TM complex is shown in. The αIIb helix is perpendicular to the membrane whereas the β3 helix is tilted.
There are glycines at the helix-helix interface in the membrane and an unusual αIIb backbone reversal that packs a consecutive pair of Phe residues against the β3 TM helix, promoting electrostatic interactions between αIIb(D723) and β3(R995). The two TM segments have essentially the same structure when studied separately suggesting that the topological features of the TM segments will remain unchanged in the separated, active state. ( A) NMR structure of the complex between the αIIb (blue) and β3 (red) TM domains (PDB: 2K9J). The approximate position of the membrane glycerol backbone is shown by gray lines. ( B) The talin F2 (blue)/F3 (yellow) domain pair in complex with a β integrin tail (red). The salt bridge that forms between K324 on F3 and D723 in the tail is shown; some key Lys and Arg residues are indicated in blue near the putative membrane interface with the F2 domain.
B was constructed from a composite of coordinates of the talin/β complex (PDB: 3G9W; ) and the membrane complex (PDB:2K9J ). Images made using PyMOL (DeLano Scientific).The ectodomain-TM linkers seem to be flexible (; ) and are thus unlikely to constrain the orientation between the ectodomain and the membrane. Although αXβ2 is bent in a similar way to integrins without an α-I domain, the terminal domains of the α-legs and β-legs, calf-2, and β-tail domains, are oriented differently.
These observations are all consistent with the flexible transmembrane domain separation model of activation rather than stiff pistons or levers. The TM complex is also likely to be stabilized by the resting ectodomain. The Cytoplasmic TailsSeveral NMR studies of cytoplasmic tails have been published, although there is little agreement among them. Some studies could not detect an interaction between α and β tails, whether they were connected by a coiled coil construct or inserted in a membrane with TM segments. A study of isolated mixed peptides found two distinct structures of the αβ complex , both significantly different from the most detailed published structure of the αβ tail complex. The latter structure does not seem to be consistent with the recently solved structures of the TM regions (; ). The most obvious explanation for these discrepancies is that the tails are rather flexible, only forming transient structures in solution in the absence of a protein interaction partner and that complexes between the tails and their binding proteins are likely to provide the most significant insights into transduction events.
Cytoplasmic Tail LigandsBecause they are extended and flexible, the cytoplasmic tails, especially β, can “flycast” and reach out to form “hub” interactions with a number of proteins (; ). In particular, PTB domains bind to one of the two conserved NPXY motifs in β-tails. Especially important for activation of integrins from inside the cell are the proteins talin and kindlin. The talin PTB domain (the head F3 subdomain) binds the first β-NPXY and the membrane proximal helix whereas kindlin binds the second NPXY. It has been proposed that the F2.F3 subdomains of talin make a defined contact with the membrane surface via numerous lysine and arginine residues especially in the F2 domain (B; ). Binding of F3 to the β-tail promotes tail dissociation by breaking the salt bridge between the α and β tails (αIIbD723-β3R995); a new salt bridge is formed with K324 on the F3 domain. Binding to β plus the F2.F3 contact with the membrane can also influence the orientation of the β-helix, again helping promote separation of the TM and cytoplasmic domains.
Structural Studies of Intact IntegrinsAlthough crystallography of ectodomains and NMR of TM domains have provided detailed information about integrin structure, other techniques have been applied to intact integrins in attempts to distinguish among current models, such as “switchblade” and “deadbolt.” In principle this should be relatively easy because the switchblade model predicts a near doubling in molecular height on activation (C), whereas the deadbolt model predicts a modest change. In general, however, the various studies have not been in good agreement, possibly because of multiple integrin conformations in solution.Cryoelectron tomography of ice-embedded specimens was used to obtain three-dimensional images of full-length αIIbβ3 incorporated into small liposomes. No significant height change was observed between the inactive state, and the active state induced by Mn. In a FRET study, Mn activation caused an increase of 5 nm in the separation between membrane and ligand binding site, consistent with a conformational change to the upright configuration.
However, FRET between fluorescently labeled Fab fragments and the β3 β-I domain indicated only small changes on platelet activation. A small angle neutron scattering (SANS) study of intact αIIbβ3 in Ca 2+/detergent solutions found “arched” and “handgun” forms although a Mn activated form was not studied. Analytical ultracentrifugation and EM were used to investigate αIIbβ3; measurement of frictional changes under different conditions suggested considerable plasticity in the structure.EM studies of ectodomains are also not entirely consistent although, on balance, they favor the switchblade model. Single-particle reconstructions of the negatively stained αvβ3 ectodomain bound to a fibronectin (FN) fragment suggested that the bent conformation can bind its physiological ligand. EM of negatively stained αvβ3 ectodomain with a cleavable clasp engineered into the carboxyl terminus showed a majority of molecules in the bent conformation when inactive, and a majority in the upright conformation when active.
Negative stain EM of a shorter ectodomain construct of α5β1 bound to FN showed similar results. The recent x-ray structure papers of intact ectodomains contain EM results consistent with the switchblade model (; ).A recent study of integrin activation using EM and other studies gives useful insight. Membrane nanodiscs were synthesized with a single lipid-embedded integrin. The majority (∼90%) of the class-averaged integrin nanodisc EM images in the absence of ligand and the talin head domain had a compact structure with a height of 11±1 nm; i.e., corresponding to the bent conformation in C. In the presence of talin, ∼25% of unliganded integrins had an extended structure with a height of 19±1 nm. In contrast, at least 40% of the fibrin-bound integrins were extended.
This study provides evidence that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3 without applied force or clustering.There is evidence that mechanical force is important for regulating integrin adhesiveness (; ). Talin contains both integrin and actin binding sites , and therefore the cytoskeleton could exert a lateral force on the β subunit. Steered molecular dynamics was applied to a complete ectodomain to mimic effects of cell generated tension.
Evidence was found that lateral force could be transmitted through the β leg to the hybrid domain and promote the active form. LIGAND BINDINGHistorically, the pairing of integrins and their ligands has been uncovered either by ligand affinity chromatography or through the use of subunit-specific monoclonal antibodies (mAbs) to block ligand-mediated cell adhesion. In most cases, protein-protein binding assays have confirmed the associations established by these biochemical or cell biological approaches. A characteristic feature of most integrin receptors is their ability to bind a wide variety of ligands.
Conversely, many extracellular matrix (ECM) and cell surface adhesion proteins bind to multiple integrin receptors (;; ). One molecular explanation for this complexity is the evolutionary selection of common acidic peptide motifs in ECM proteins that mediate integrin binding via coordination to a divalent cation-containing binding pocket.Integrin-ligand combinations can be clustered into four main classes, based on the nature of the molecular interaction. All five αV integrins, two β1 integrins (α5, α8), and αIIbβ3 recognize ligands containing an RGD tripeptide active site.
Crystal structures of αVβ3 and αIIbβ3 complexed with RGD ligands have been reported and they reveal an identical atomic basis for this interaction (; ). RGD binds at an interface between the α and β subunits, with the basic residue fitting into a cleft in a β-propeller module in the α subunit, and the acidic residue coordinating a cation bound in the β-I-domain. RGD-binding integrins bind to a large number of ECM and soluble vascular ligands, but with different affinities that presumably reflect the preciseness of the fit of the ligand RGD conformation with the specific α,β active site pockets. Although RGD is an essential element of the ligand binding process, macromolecular ligands can contain other binding sites, the best characterized of which is a synergy sequence in fibronectin that also binds the α5 β-propeller (, ).α4β1, α4β7, α9β1, the four members of the β2 subfamily, and αEβ7 recognize related sequences in their ligands. Α4β1, α4β7, and α9β1 bind to an acidic motif, termed “LDV,” that is functionally related to RGD. Fibronectin contains the prototype LDV ligand in its type III connecting segment region, but other ligands (such as VCAM-1 and MAdCAM-1) employ related sequences.
Structures of this integrin subfamily are lacking, but it is highly likely that LDV peptides bind similarly to RGD at the junction between the α and β subunits. The β2 family employs a similar mode of ligand binding, but the major interaction takes place via an inserted I-domain in the α subunit. Despite this fundamental mechanistic difference, the characterized sites within ligands that bind β2 integrins are structurally homologous to the LDV motif. The major difference is that β1/β7 ligands employ an aspartate residue for cation coordination, whereas β2 integrins use glutamate.Four α subunits containing an α-I-domain (α1, α2, α10, and α11) combine with β1, and form a distinct laminin/collagen-binding subfamily. A crystal structure of a complex between the α2 I-domain and a triple-helical collagenous peptide has revealed the structural basis of the interaction, with a critical glutamate within a collagenous GFOGER motif providing the key cation-coordinating residue. The mechanism of laminin binding is less well understood, although a recent study has suggested that the extreme carboxyl terminus of the γ chain and an undefined site within α subunit laminin G domains together constitute an integrin-binding site.
Three β1 integrins (α3, α6, and α7), plus α6β4, are highly selective laminin receptors. Analysis of laminin fragments indicates that these receptors and the α-I-domain-containing β1 integrins bind to different regions of the ligands. In neither case has the active site been narrowed down to a particular sequence or residue. ACTIVATIONFor the interaction of integrins with their ligands to be meaningful for cellular function, the binding event must be able to regulate signal transduction. However, adhesion is highly dynamic, with cells continuously sampling their pericellular environment, and responding by rapidly changing their position and state of differentiation, and therefore a highly responsive receptor activation mechanism is required. As integrins lack enzymatic activity, signaling is instead induced by the assembly of signaling complexes on the cytoplasmic face of the plasma membrane.
Formation of these complexes is achieved in two ways; first, by receptor clustering, which increases the avidity of molecular interactions thereby increasing the on-rate of binding of effector molecules, and second, by induction of conformational changes in receptors that creates or exposes effector binding sites. Current evidence suggests that conformational regulation is the primary mode of affinity regulation of integrins. In turn, this demands a mechanism for conveying conformational changes between the cytoplasmic tails and the ligand-binding head domain over a relatively large distance (∼20 nm). Evidence for Conformational RegulationGross conformational changes in integrins have been monitored by a variety of techniques, and for almost all of these studies, αIIbβ3 has served as a prototype. Treatment with RGD peptides elicited alterations in sedimentation coefficient and Stokes radius , and receptor activation on platelets triggered changes in intramolecular FRET and cross-linking. MAbs have proven particularly useful probes of integrin function.
Early studies reported activation-dependent changes in mAb binding to αIIbβ3 that were attributed to conformational changes (; ), and these were followed by the identification of a subset of anti-αIIbβ3 mAbs, the epitopes for which were induced in response to ligand binding (, ). The acronym LIBS was coined to describe these epitopes as ligand-induced binding sites.
In most cases that have been examined, activating mAbs appear to function by increasing the affinity of ligand binding. Most LIBS mAbs have epitopes that are regulated by divalent cations, and because cations also regulate ligand binding, it appears that many cation-responsive, activating mAbs recognize naturally occurring conformers of integrins.
These mAbs may therefore displace a conformational equilibrium in favor of these forms that leads to an increase in the proportion of high affinity integrin. Some other activating mAb epitopes are unaffected by either ligand or cation binding and here the most likely mechanism of action is through inducing an activated conformation in the integrin.The location of LIBS epitopes has contributed significantly to our understanding of the process of receptor activation. The overwhelming majority of activating mAbs recognize the β subunit, and their epitopes are distributed throughout the polypeptide (; ). This is suggestive of a large-scale alteration in the conformation of the whole integrin during activation. The regions recognized include the β-I-domain, the extreme amino terminus of the β subunit in the PSI domain, the hybrid domain, the β-subunit knee region, and distal EGF-like repeats. A few activating anti-α subunit mAbs have been reported, the epitopes for which are found in the β-propeller, the heavy-light chain border and close to the transmembrane domain, suggestive of conformational changes in these regions of the molecule (; ). How Are Conformational Changes Coupled?As discussed above, the various structural studies in the last 10 years have greatly stimulated functional analyses.
The relevance of the observed bend in the legs of the integrin dimer has been a highly contentious issue. It has been proposed that integrins are always bent, but several lines of evidence indicate that bent integrins are inactive, and extended integrins are primed. In the original crystal structure of αVβ3, the integrin was bent at an angle of 135°. Locking integrins in this state through disulphide bond engineering abolishes ligand binding by cell surface-expressed receptors.
Furthermore, when the gross structure of integrins was examined by electron microscopy under conditions in which ligand binding was low, e.g., in Ca 2+-containing buffers or following the introduction of intersubunit covalent bond constraints, predominantly bent structures were observed (; ). In bent integrins, the ligand-binding pocket may be oriented toward the plasma membrane, thereby impeding ligand engagement, but flexibility at the juxtamembrane domain could enable a “breathing” movement for the conversion of bent to extended integrin (; ). In this context, a cryo-EM reconstruction of unstimulated αIIbβ3 indicated a partially extended conformation.
The binding of stimulatory mAbs might then displace a conformational equilibrium, leading to activation. Similarly, breaking the interactions between the α and β cytoplasmic tails appears to lead to a loss of the interactions among the leg regions, disruption of an interface between the head and legs, and a repositioning of the head to point away from the cell surface. Major support for this model comes from studies of soluble recombinant integrins by electron microscopy and from the large number of epitopes of stimulatory mAbs that have now been shown to lie in the knee or leg regions. Exposure of these epitopes is low in the bent state of the integrin (where they are masked) but high in the extended state.The pathway of conformational change from the interior of the cell to the ligand-binding site of the integrin is incompletely understood, but movement of the hybrid domain appears to be a central feature of the conformational changes accompanying unbending. By EM, an acute angle between the hybrid domain and β-I was observed in the bent state, and an obtuse angle in the extended, ligand-occupied state.
In the bent conformation, any movement of the hybrid domain relative to the β-I domain is prevented, and therefore unbending is probably an essential prerequisite to hybrid domain motion. A central role for hybrid domain movement in affinity regulation has been established by a number of approaches.
Activating mAb epitopes in the hybrid domain map close to an interface between the hybrid domain and the α subunit β-propeller. These epitopes would be masked in bent integrins, but would become exposed when the hybrid domain swings away from the propeller. Furthermore, engineering of glycosylation sites between the hybrid domain and the β-I-domain produces a putative wedge that leads to integrin activation.Conformational changes in the head are the key determinant of ligand-binding activity, specifically, the conformation of the β-I domain, which, in turn, is determined by the position of the hybrid domain. Thus, a swing-out of the hybrid domain away from the α-subunit pulls downward on the α7 helix of the β-I domain and favors the upward movement of the α1 helix. The motion of these two helices shifts the β-I domain from a low-affinity into a high-affinity conformation by backbone movements of loops that contain cation-coordinating residues. Mutations that favor a downward shift of the α7 helix (;; ) also result in a high-affinity state.Although current models of integrin function strongly suggest that conversion to a high-affinity receptor requires extension, there is some evidence to suggest that ligand-bound integrin can adopt a bent conformation. Crystallized αVβ3 can bind a cyclic RGD peptide in the bent conformation and electron microscopy images also show bent αVβ3 in complex with a fragment of fibronectin.
In addition, studies that have either used FRET or competition ELISA to measure the distance between a fluorescently tagged ligand peptide and labeled cell membrane or between mAbs directed against the head piece and leg regions of αIIbβ3 on platelets have revealed partial unbending (;, ). Nevertheless, when FRET-FLIM is employed to analyze the conformation of α5β1 in adherent cells, by measuring FRET between a fluorescently labeled Fab bound to α5β1 and fluorescent dye intercalated into the cell membrane, it has been shown that integrins are extended in focal adhesions and bent elsewhere. Integrin Antagonists as Therapeutic AgentsThe short acidic peptides that serve as ligand active sites are essentially pro-drugs, and both RGD and LDV peptides have been converted into small molecules therapeutics. RGD-based, peptidomimetic antagonists of αIIbβ3, such as eptifibatide (from 1998) and tirofiban (from 1998), are now used widely as antithrombotic agents , and LDV-based compounds are in development for treatment of asthma and multiple sclerosis.
In addition, mAbs that block integrin function and cell adhesion have been developed as therapeutic agents. These agents were originally assumed to compete with ligands for receptor binding, but this now appears not to be the case, with many anti-integrin mAbs having been shown to function via allosteric mechanisms.
Current evidence suggests that mAbs inhibit ligand binding either by stabilizing the unoccupied state of the receptor or by preventing a conformational change necessary for ligand occupancy. In turn, the allosteric inhibition of ligand binding by anti-functional anti-integrin mAbs implies that it may be feasible to synthesize small molecule inhibitors that function in the same way.
Such inhibitors could have advantages over competitive inhibitors in that a partial inhibition of function may be obtained and therefore adhesion may be more easily controlled, and they may not possess the agonistic properties of ligand mimetics, and may therefore not suffer from mechanism-related side-effects. Small molecule allosteric inhibitors that bind to α-I-domains have now been reported.
These molecules appear to stabilize the low affinity conformation of the α-I-domain by blocking downward movement of the terminal α7-helix and thereby preventing rearrangements at the ligand-binding pocket necessary for high affinity ligand binding. CONCLUSIONThere has been remarkable progress in our understanding of integrin structure and function in the last 10 years. The basis of much previous work on conformation, which was performed with conformationally sensitive antibodies, and ligand binding, which was largely based on mutational analyses, can now be modeled at atomic resolution. A unifying biophysical model of integrin function, which incorporates features such as catch bonds, extreme flexibility at the knees and the on- and off-rates of ligand and effector binding is therefore within reach. The process of integrin activation from inside the cell is also now quite well understood at a structural level.
However, a number of major questions remain unresolved. These include outside-in signaling, which is much less well understood compared with inside-out signaling and it is unclear how similar or different the two processes are. We still do not know how an integrin allows a cell to interpret the binding of different ligands, and therefore how microenvironmental sensing is achieved at a molecular level. Looking further ahead, the process of “inactivation,” where integrins return to their resting state, is not understood, and we are just starting to develop approaches to measure force transduction at adhesion sites.
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1, 2, 2. and 1. 1Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA. 2Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USAPeptidylarginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs) via PAD4-mediated histone citrullination.
Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors.
A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis. The Peptidyl Arginine Deiminase FamilyThe 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 ).
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. Five 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.PADI 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. PAD4Of 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.PAD4 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.PAD4 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.
Biochemical Activation of PAD4While 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 K d 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 (; ).The 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 10 2- to 10 3-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.In 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. PAD Mechanism and InhibitionGiven 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.Briefly, 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 ). 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 ). 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 ). 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 ), 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. (A) PAD4 in complex with Histone H4 1–5 (SGRGK). PAD4 active site residue side chains are colored gray (D350, H471, D473, C645A) and residues that are involved in binding the H4 1–5 backbone and S1 are colored yellow (Q346, W347, R347). N-terminally acetylated H4 1–5 is shown in cyan with Arginine 3 bound in the PAD4 active site.
Polar residue interactions between PAD4 and H4 1–5 are indicated by dashed lines. The mutation of the active site cysteine residue (C645) to an alanine (C645A) was necessary to achieve substrate binding in the crystal structure as described in. Figure was created from the structure filed under PDB code 2dey. (B) Proposed catalytic mechanism for PAD4. (C) Haloacetamidine-based inhibitors targeting the PAD isozymes.
The Function of PAD4 in NeutrophilsNeutrophils 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.In 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.Histone 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.Neutrophil 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. Stimulation of PAD4 ActivityA 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 H 2O 2 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 catalogs the variety of stimuli reported to stimulate NET formation. Although 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. PAD4 Activity and ROSThe 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 ( O 2.
−) is generated by the NADPH complex (Nox) and by the electron transport chain in mitochondria. O 2. − is then converted to H 2O 2 spontaneously or by the enzyme superoxide dismutase (SOD; ). H 2O 2 acts directly on target cells and is converted to additional effectors by enzymes such as myeloperoxidase (MPO). Interestingly, the addition of H 2O 2 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.The 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 H 2O 2 or exogenous glucose oxidase, which generates H 2O 2 , indicating that the ROS production facilitated by Nox2 is necessary for NETs. Catalase removes intracellular H 2O 2 by reduction to water and oxygen , and catalase inhibition increases intracellular H 2O 2 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 H 2O 2 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 ). The 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 O 2. − , 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 H 2O 2 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 H 2O 2 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 H 2O 2. These studies indicate a model in which NADPH oxidase activity generates O 2. −, which then dismutates to H 2O 2 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. PAD4 and AutophagyLike 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.
ConclusionNeutrophil 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.PAD4 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 ), 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. Conflict of Interest StatementThe 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. AcknowledgmentsThe work in Dr. Mowen’s laboratory was supported grants GM085117 and AI067460 from the National Institutes of Health (Kerri A.
The work in Dr. Thompson’s laboratory was supported by grants GM079357 and CA151304 from the National Institutes of Health as well as an American College of Rheumatology Research Foundation Within Our Reach Grant. We apologize to investigators whose important contributions were not included due to space limitations. This is manuscript #21941 from The Scripps Research Institute. Reviewed by:, Indiana University School of Medicine, USA, Istituto Clinico Humanitas, Italy, Rega Institute, K. Leuven, BelgiumCopyright: © 2012 Rohrbach, Slade, Thompson and Mowen. This is an open-access article distributed under the terms of the, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Correspondence: Paul R.
Thompson, Department of Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA. E-mail: [email protected]; Kerri A. Mowen, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: [email protected].
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