Phagosome as the organelle linking innate and adaptive immunity

The means by which phagocytosis and antimicrobial defense mechanisms are linked have expanded greatly in recent years. It is now clear that the process of phagocytosis does more than just degrade internalized microbes, but also helps coordinate the actions of the innate and adaptive immune system. This review will discuss the means by which Toll-like receptor signaling pathways are coordinated around the processes of phagocytosis, phagosome trafficking and autophagy and how these signaling pathways influence T-cell-mediated immunity. In this regard, we propose that at the subcellular level, phagosomes represent the smallest definable unit that links innate and adaptive immunity.     

93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35–50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.     

Molecular Mechanism That Induces Activation of Sp?tzle,the Ligand for the Drosophila Toll Receptor

The Drosophila Toll receptor is activated by an endogenous cytokine ligand Spätzle. Active ligand is generated in response to positional cues in embryonic dorso-ventral patterning and microbial pathogens in the insect immune response. Spätzle is secreted as a pro-protein and is processed into an active form by the serine endoproteases Easter and Spätzle-processing enzyme during dorso-ventral patterning and infection, respectively. Here, we provide evidence for the molecular mechanism of this activation process. We show that the Spätzle prodomain masks a predominantly hydrophobic region of Spätzle and that proteolysis causes a conformational change that exposes determinants that are critical for binding to the Toll receptor. We also gather that a conserved sequence motif in the prodomain presents features of an amphipathic helix likely to bind a hydrophobic cleft in Spätzle thereby occluding the putative Toll binding region. This mechanism of activation has a striking similarity to that of coagulogen, a clotting factor of the horseshoe crab, an invertebrate that has changed little in 400 million years. Taken together, our findings demonstrate that an ancient passive defense system has been adapted during evolution and converted for use in a critical pathway of innate immune signaling and embryonic morphogenesis.     

Anti-inflammatory Compounds Parthenolide and Bay 11-7082 Are Direct Inhibitors of the Inflammasome

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1β and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-κB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-κB inhibitors revealed that the synthetic IκB kinase-β inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-κB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.     

Structure-function analysis of grass clip serine protease involved in Drosophila Toll pathway activation

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.     

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.     

Crystal Structures of the Toll/Interleukin-1 Receptor (TIR) Domains from the Brucella Protein TcpB and Host Adaptor TIRAP Reveal Mechanisms of Molecular Mimicry

The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys⁸⁹ and Cys¹³⁴. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.     

Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.     

A novel motif in the Crohn's disease susceptibility protein, NOD2, allows TRAF4 to down-regulate innate immune responses

The Crohn's disease and early onset sarcoidosis susceptibility protein, NOD2, coordinates innate immune signaling pathways. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis. In this work, we identify the atypical tumor necrosis factor-associated factor (TRAF) family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-κB activation and directly binds to NOD2 to inhibit NOD2-induced bacterial killing. We find that two consecutive glutamate residues in NOD2 are required for interaction with TRAF4 and inhibition of NOD2 signaling because mutation of these residues abrogated both TRAF4 binding and inhibition of NOD2. This work identifies a novel negative regulator of NOD2 signaling. Additionally, it defines a TRAF4 binding motif within NOD2 involved in termination of innate immune signaling responses.     

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.     

Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability

The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 3₁₀-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.     

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4–P3′ residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 β-sheet B, and the 147-loop directly contacts PAI-1 β-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.     

Molecular battle between host and bacterium: recognition in innate immunity

During infection, our innate immune system is the first line of defense and has evolved to clear invading bacteria immediately. To do so, recognition is the key element. However, how does the innate immune system distinguish self from nonself, and how does it recognize all bacteria (estimated to be far over a million species)? The answer lies in the recognition of evolutionary conserved structures. In this review, we approach this phenomenon from the bacterial perspective. What are the evolutionary conserved structures in bacteria, and what strategies are there in the human innate immune system to sense these structures? We illustrate most examples both at the functional as well as at the molecular level. Furthermore, we highlight how pathogenic bacteria can evade this recognition to survive better in the human host which in turn can result in life‐threatening diseases. Copyright © 2011 John Wiley & Sons, Ltd.     

Ligand specificity determined by differentially arranged common ligand-binding residues in bacterial amino acid chemoreceptors Tsr and Tar

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.     

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.     

Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation

Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.     

Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation. In addition, siRNA to Unc93b1 reduced the secretion of the TLR3 ectodomain (T3ECD) into the cell medium. Furthermore, two human single nucleotide polymorphisms that affected herpesvirus and influenza virus encephalopathy as well as a natural isoform generated by alternative splicing were found to be impaired for T3ECD secretion and decreased the abundance of TLR3 on the cell surface. The locations of the SNP P554S and the deletion in the isoform led to the identification of a loop in the TLR3 ectodomain that is required for secretion and a second whose presence decreased secretion. Finally, a truncated protein containing the N-terminal 10 leucine-rich repeats of T3ECD was sufficient for secretion in an Unc93b1-dependent manner.     

Structural insights into recognition of triple-helical beta-glucans by an insect fungal receptor

The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as β1,3-glucan. β1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. β1,3-Glucan recognition protein (βGRP)/gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured β1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal β1,3-glucan recognition domain of βGRP/GNBP3 (βGRP-N) in complex with the β1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one βGRP-N. The spatial arrangement of the laminarihexaoses bound to βGRP-N is almost identical to that of a β1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of β1,3-glucan.