Search

Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR2-null mice compared to Cre expressing PAR2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2.

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.

Summary Proteases are enzymes that specifically cleave the peptide backbone of their target proteins. As an important type of irreversible post-translational modification, protein cleavage underlies many key physiological processes. When dysregulated, proteases’ actions are associated with numerous diseases. Many proteases are highly specific, cleaving only those target substrates that present certain particular amino acid sequence patterns. Therefore, tools that successfully identify potential target substrates for proteases may also identify previously unknown, physiologically relevant cleavage sites, thus providing insights into biological processes and guiding hypothesis-driven experiments aimed at verifying protease–substrate interaction. In this work, we present PROSPERous, a tool for rapid in silico prediction of protease-specific cleavage sites in substrate sequences. Our tool is based on logistic regression models and uses different scoring functions and their pairwise combinations to subsequently predict potential cleavage sites. PROSPERous represents a state-of-the-art tool that enables fast, accurate and high-throughput prediction of substrate cleavage sites for 90 proteases. Availability and implementation http://prosperous.erc.monash.edu/ Supplementary information Supplementary data are available at Bioinformatics online.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis., Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine–containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.

SummaryPlasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.

Proteases have central roles in "life and death" processes due to their important ability to catalytically hydrolyze protein substrates, usually altering the function and/or activity of the target in the process. Knowledge of the substrate specificity of a protease should, in theory, dramatically improve the ability to predict target protein substrates. However, experimental identification and characterization of protease substrates is often difficult and time-consuming. Thus solving the "substrate identification" problem is fundamental to both understanding protease biology and the development of therapeutics that target specific protease-regulated pathways. In this context, bioinformatic prediction of protease substrates may provide useful and experimentally testable information about novel potential cleavage sites in candidate substrates. In this article, we provide an overview of recent advances in developing bioinformatic approaches for predicting protease substrate cleavage sites and identifying novel putative substrates. We discuss the advantages and drawbacks of the current methods and detail how more accurate models can be built by deriving multiple sequence and structural features of substrates. We also provide some suggestions about how future studies might further improve the accuracy of protease substrate specificity prediction.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1β, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

Thrombin (EC 3.4.4.13) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin.

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

Background and Objective: Porphyromonas gingivalis, an anaerobic bacterium associated with adult periodontal disease, employs a number of pathogenic mechanisms, including protease/adhesin complexes (gingipains), fimbriae and hemagglutinins, to maintain attachment within colonized hosts. Here we examined the binding of gingipains and whole, live P. gingivalis cells to immobilized extracellular matrix proteins in the presence of soluble forms of the same proteins, to investigate whether this may constitute a colonization mechanism in the oral environment. Material and Methods: Binding of purified gingipain molecules and whole bacterial cells to immobilized matrix proteins was examined in the presence and absence of soluble competitors using enzyme-linked immunosorbent assays. Results: Purified gingipains or whole, live bacteria preferentially bound immobilized forms of matrix proteins, even in the presence of soluble forms of the same proteins. Fimbriae appeared to be redundant for adhesion to immobilized proteins in the presence of the gingipains, indicating that the protease/adhesins and hemagglutinins may be more important for adhesion under these conditions. Conclusion: The data presented here provide evidence for a model of adhesion for P. gingivalis within the fluid environment of the oral cavity, where preferential binding of matrix-located proteins over soluble forms facilitates colonization of the host.

Proteases play vital roles in a range of biological processes, such as cell cycle, cell growth and differentiation, apoptosis, haemostasis and signalling. Fundamental to our knowledge of protease action is an understanding of how the active site operates; this has been examined through extensive studies of the substrate specificity of the enzymes. Kinetic and structural analyses have shown that the binding of a particular substrate residue at a protease subsite can have either a positive or negative influence on the binding of particular residues at other subsites. This phenomenon has been termed subsite cooperativity and has been observed in a wide range of proteases, often between non-adjacent subsites. This review aims to highlight studies where subsite cooperativity has been observed, experimental techniques used in the past and potential methods that can be employed to comprehensively examine this phenomenon. Further understanding of how the protease active site recognises and chooses its substrates for cleavage will have a significant impact on the development of pharmaceuticals that target these enzymes.

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.

Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1β, granulocyte-macrophage colony stimulatory factor and interferon-γ were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1β and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages.

Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.

Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site.

Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 A, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A beta-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90 degrees to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 +/- 0.9 x 10(6) m(-1) s(-)1) and cathepsin G (ka: 7.9 +/- 0.9 x 10(5) m(-1) s(-)1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4-P6' region of the RCL, the distal hinge, and the loop between strands 4B and 5B.

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia).

Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field.

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2′-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.

PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption.Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated.Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR.RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells.These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Factor Xa is a central protease in the coagulation cascade and the target for many anticoagulant compounds currently under development. The preferences of the enzyme for substrates incorporating residues N-terminal to the cleavage site (P1, P2, etc.) have been elucidated, but little is known of its preferences for residues C-terminal to the cleavage site (P1′, P2′, etc.). The preferences of bovine factor Xa for substrate residues in the P1′, P2′ and P3′ positions were mapped using fluorescence-quenched substrates. Bovine factor Xa, often used as a model for factor Xa, was most selective for the P2′ position, less selective at the P1′ position and almost completely non-selective at the P3′ position. It appears that while the prime side subsites of factor Xa impose some selectivity towards substrates, the influence of these sites on factor Xa cleavage specificity is relatively low in comparison to related enzymes such as thrombin.

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine-specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (Kd=1.31±0.26×10−7 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule.

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.

The C1s protease of the classical complement pathway propagates the initial activation of this pathway of the system by cleaving and thereby activating the C4 and C2 complement components. This facilitates the formation of the classical pathway C3 convertase (C4bC2a). C1s has a Lys residue located at position 628 (192 in chymotrypsin numbering) of the SP domain that has the potential to partially occlude the S2–S2′ positions of the active site. The 192 residue of serine proteases generally plays an important role in interactions with substrates. We therefore investigated the role of Lys628 (192) in interactions with C4 by altering the Lys residue to either a Gln (found in many other serine proteases) or an Ala residue. The mutant enzymes had altered specificity profiles for a combinatorial peptide substrate library, suggesting that this residue does influence the active site specificity of the protease. Generally, the K628Q mutant had greater activity than wild type enzyme against peptide substrates, while the K628A residue had lowered activity, although this was not always the case. Against peptide substrates containing physiological substrate sequences, the K628Q mutant once again had generally higher activity, but the activity of the wild type and mutant enzymes against a C4 P4–P4′ substrate were similar. Interestingly, alteration of the K628 residue in C1s had a marked effect on the cleavage of C4, reducing cleavage efficiency for both mutants about fivefold. This indicates that this residue plays a different role in cleaving protein versus peptide substrates and that the Lys residue found in the wild type enzyme plays an important role in interacting with the C4 substrate. Understanding the basis of the interaction between C1s and its physiological substrates is likely to lead to insights that can be used to design efficient inhibitors of the enzyme for use in treating diseases caused by inflammation as result of over-activity of the classical complement pathway.

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease,Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases fromP. gingivalismight mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.

Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.

Protease cascades and their inhibitors are a common feature of many biological regulatory systems, and the various components of such cascades have been subjected to a long and concerted evolution. We present here evidence that in the coagulation cascade, the sequence of the protease-binding reactive-site loop of antithrombin has evolved such that the majority of its residues has been acquired not for the efficient inhibition of its target proteases, thrombin and factor Xa, but to avoid the inhibition of activated protein C (APC). We substituted residues of the reactive-site loop of antithrombin into alpha(1)-antitrypsin and tested the chimeras against thrombin, factor Xa, and APC. With respect to factor Xa and thrombin, the difference in association rate between the fastest and the slowest inhibitors was 5.5- and 88-fold, respectively. However, with respect to APC the difference was 12,500-fold. While most of the variation in the inhibition rates of thrombin could be accounted for by P2 Gly-to-Pro substitutions, for APC almost every residue had an effect on inhibition. In 22 of 25 direct comparisons of antitrypsin residues with antithrombin residues, either singly or in blocs, the antithrombin residues caused a decrease in the rate of inhibition of APC. The antithrombin residue Asn393, at position P'3, emerged as particularly important for avoiding the inhibition of APC, however, its 190-fold effect was seen only when in conjunction with antithrombin P7 to P'2 residues. Cooperative effects among residues of the reactive-site loop thus emerged as critical for restricting the activity of this sequence against APC.

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new “tethered ligand” N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca 2+ ] i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca + entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca 2+ ] i . Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca 2+ ] i followed by a sustained plateau; [Ca 2+ ] i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.

Background and Objective: Porphyromonas gingivalis is a major aetiological agent in the development of periodontitis, the major clinical hallmark of which is bone resorption. The cysteine proteases (gingipains) produced by P. gingivalis have a critical role in the pathogenesis of the disease, and previous studies on whole bacteria have implicated these enzymes in osteoclastogenesis, a process which serves to upregulate bone resorption. The effects of the gingipains from P. gingivalis on osteoclast differentiation were investigated here to determine whether the enzymes directly contribute to osteoclastogenesis and thus to bone resorption. Material and Methods: The effects of the gingipains on osteoclast differentiation were investigated in primary mouse bone marrow cultures. The cultures harvested from C57BL6/J mice were incubated in the presence of parathyroid hormone, a known osteoclastogenic factor, or active/inactivated forms of three gingipains. Osteoclast differentiation was quantified by counting the number of multinucleated cells positive for tartrate-resistant acid phosphatase, an enzyme marker for these cells. Results: After 10 days of culture, the gingipains, either active or inactive, failed to stimulate osteoclast differentiation in comparison to the parathyroid hormone. Conclusion: The data presented here demonstrate that the gingipains do not induce osteoclast differentiation in this system, indicating that the bacterium uses other mechanisms to induce bone loss.

Objective and Design: Identify and characterize the matrix metalloproteinase responsible for cartilage proteoglycan degradation mediated by a macrophage cell line in a cell culture model that resembles some aspects of rheumatoid pannus.¶Materials or Subjects: Supernatants from the transformed mouse macrophage cell line J774A.1 were used to purify the proteoglycan degrading activity.¶Methods: J774A.1 macrophage culture supernatants were purified by sequential column chromatography and proteins were identified by zymography, western blotting and amino acid sequence analysis. Cartilage degradation was measured using 35S labeled bovine nasal cartilage.¶Results: The cartilage degrading proteolytic activity in the mouse macrophage supernatants proved to be due to two major proteins with approximate molecular masses of 48 kDa and 22 kDa that were identified as macrophage metalloelastase (MME). Incubation of purified MME at 37°C for up to 16 h resulted in the processing of the 48 kDa protein to several novel bands including a previously undescribed protein of ∼25 kDa without accumulation of fully processed 22 kDa protein. A number of proteinases increased the rate of this processing. J774A.1 macrophage metalloelastase degraded cartilage proteoglycan with an efficiency approximately equal to human macrophage metalloelastase (MMP-12) and matrilysin (MMP-7) and twice that of stromelysin-1 (MMP-3).¶Conclusions: These data identify the cartilage proteoglycan degrading metalloproteinase secreted by J774A.1 macrophages in this cell culture model as MME, and describes mechanisms of activation and processing of this enzyme that may play an important role in cartilage degradation.

The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main β-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6°C) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0°C), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband’s plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.© 1998 by The American Society of Hematology.

Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin-like activity which have been implicated as virulence factors in adult-onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis-derived proteinases are important virulence factors in the development of periodontal disease.

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1′, which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1′ in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

Thrombin stimulates expression of interleukin 6 and cyclooxygenase 2 by osteoblasts, both of which enhance osteoblast-mediated osteoclast differentiation by increasing the ratio of receptor activator of nuclear factor κB ligand (RANKL) expression to that of osteoprotegerin (OPG) in osteoblasts. We hypothesised that thrombin would also increase this ratio and thereby stimulate osteoclast differentiation in mixed cultures of osteoblastic cells and osteoclast precursors. In primary mouse osteoblasts, but not in bone marrow stromal cells, thrombin increased the ratio of RANKL to OPG expression. Thrombin inhibited differentiation of osteoclasts, defined as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei, in mouse bone marrow cultures treated with osteoclastogenic hormones; this effect was not mediated by the major thrombin receptor, protease-activated receptor 1, nor did it require thrombin's proteolytic activity. Thrombin also caused a decrease in the number of TRAP-positive cells with fewer than three nuclei. Thrombin (active or inactive) also inhibited osteoclast differentiation and bone resorption, respectively, in cultures of mouse spleen cells and human peripheral blood mononuclear cells induced to undergo osteoclastogenesis by treatment with RANKL and macrophage colony-stimulating factor. Osteoclast differentiation in spleen cells was inhibited when they were exposed to thrombin from days 0 to 3 or 3 to 5 of culture but not days 5 to 7 when most fusion occurred. Thrombin inhibited expression of RANK by spleen cells. These observations indicate that, although thrombin stimulates production of osteoclastogenic factors by osteoblastic cells, it inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors that does not require thrombin's proteolytic activity.

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS., Author Summary The type 2 secretion system (T2SS) is a sophisticated, multi-component molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. In Vibrio cholerae, for example, the T2SS mediates the secretion of cholera toxin. We find that there are three distinct forms of T2SS, based on the sequence characteristics of the secretin. A targeting paradigm, developed for the Klebsiella-type secretin PulD, could not previously be applied to the T2SS in Vibrio cholerae and many other bacterial species whose genomes encode no homolog of the crucial targeting factor PulS (also called OutS, EtpO or GspS). Using bioinformatics we find, remarkably, that these bacteria have instead evolved a structurally distinct protein to serve in place of PulS. We crystallized and solved the structure of this distinct factor, AspS, measured its activity in novel assays for T2SS assembly, and show that the protein is essential for the function of the Vibrio-type T2SS. A structural homolog of AspS found here in Pseudomonas suggests widespread use of the pilotin-secretin targeting paradigm for T2SS assembly.

Ragweed (Ambrosia artemisiifolia), the major cause of late summer hay fever (allergic rhinitis) in the United States and Canada, is clinically the most important source of the seasonal aeroallergens. A novel endopeptidase was extracted from the pollen of this plant and purified by a series of column chromatographic steps. It has a molecular mass of 82 kDa according to gel filtration and SDS-polyacrylamide gel electrophoresis and a pH optimum near 9.0, and its activity is unaffected by chelating or reducing agents. A 17-amino acid amino-terminal sequence of this protein showed no similarity with any other proteases. The enzyme was inhibited by diisopropyl fluorophosphate, a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone, a chymotrypsin-like proteinase inhibitor. Various synthetic substrates were efficiently cleaved with a strong preference for Phe in the P1 and P3 position and Pro in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. In addition to synthetic substrates, the neuropeptides, vasoactive intestinal peptide and substance P, which are required for normalized lung functions, were also rapidly hydrolyzed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor, which occurred through cleavage within the reactive site loop. These results indicate that the purified enzyme is a novel endopeptidase, which may be involved in both the degradation of neuropeptides and the inactivation of protective proteinase inhibitors during pollen-initiated allergic reactions.

African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and alpha2-macroglobulin effectively inhibited trypanopain-Tb, with the Ki values for cystatin C and low-molecular-mass kininogen (approximately 10(-11) M) predicting, that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or (a2-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats displayed no trypanopain-Tb-like activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.

Periodontitis is an excellent model of local tissue destruction due to the uncontrolled action of host and microbial proteinases. Although host enzymes are responsible for direct degradation of connective tissue, proteinases from the periodontopathogenic bacteriumPorphyromonas gingivalis are able to activate the kallikrein/kinin cascade, disrupt coagulation, and activate complement-mediated chemotaxis. Since the hallmarks of adult onset of periodontitis include infection by this pathogen, bleeding, increased crevicular flow, and neutrophil accumulation, investigations of this disease at a biochemical level indicate a major role for bacterial proteinases in infections byP. gingivalis.

The scabies mite (Sarcoptes scabiei) is a parasitic mite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. The scabies mite produces 33 proteins that are closely related to the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed Scabies Mite Inactivated Protease Paralogues (SMIPPs). The precise function of SMIPPs remains unclear. However, it has been suggested that these proteins may function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. The high-resolution crystal structures of two members of the SMIPP family have been solved and provide insight into how SMIPPs have lost the ability to bind substrates in a classical ‘canonical’ fashion, and instead have evolved alternative functions in the lifecycle of the Scabies mite.

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.