Biomedical subjects

Robert N Pike

Publications and source records attributed to Robert N Pike.

34 records · Page 2Linked to original sources

PubMed2003

The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions.

A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.

Amino Acid Sequence↗

PubMed2003

Characterization of the specificity of arginine-specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes.

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine-specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA(cat), the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA(cat) or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aalpha-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bbeta-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene.

Adhesins, Bacterial↗

PubMed2003

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

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.

Amino Acid Sequence↗

PubMed2003

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability.

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.

Amino Acid Substitution↗

PubMed2003

Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis.

Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.

Amino Acid Sequence↗

PubMed2003

The evolution of enzyme specificity in Fasciola spp.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last approximately 135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

Amino Acid Sequence↗

PubMed2003

The 1.5 A crystal structure of a prokaryote serpin: controlling conformational change in a heated environment.

Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C. The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment.

Actinomycetales↗

PubMed2003

Determination of the P1', P2' and P3' subsite-specificity of factor Xa.

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.

Animals↗

PubMed2003

Inhibition of osteoblast apoptosis by thrombin.

The multifunctional serine protease thrombin has been shown to be a specific agonist for a variety of functional responses of cells including osteoblasts. The current study was conducted to determine if thrombin was capable of inhibiting apoptosis in osteoblasts, and if so, to examine the mechanism by which this occurred. Thrombin (20-100 nM) significantly inhibited apoptosis in serum-starved cultures of the human osteoblast-like Saos-2 cell line and cultures of primary osteoblasts isolated from mouse calvariae, as well as dexamethasone-treated primary mouse osteoblasts. Inhibition of serum deprivation-induced apoptosis was shown to require thrombin's specific proteolytic activity. Primary mouse osteoblasts were found to express two functional thrombin receptors, PAR-1 and PAR-4. Thrombin inhibited serum deprivation-induced apoptosis in osteoblasts isolated from PAR-1 null mice to the same degree as in osteoblasts isolated from wild-type mice. Treatment of serum-deprived osteoblasts, isolated from either PAR-1 null or wild-type mice, with a PAR-4-activating peptide failed to significantly inhibit apoptosis compared to the relevant control. Medium conditioned by thrombin-treated osteoblasts, in which thrombin had been inactivated, was able to inhibit serum deprivation-induced osteoblast apoptosis almost as well as thrombin itself. Blocking protein synthesis, by cycloheximide pretreatment of the conditioning cells, prevented this action. The ability of known osteoblast survival factors, such as transforming growth factor beta1, fibroblast growth factor-2, insulin-like growth factor-II, and interleukin-6, to inhibit serum deprivation-induced osteoblast apoptosis was also tested. None of these factors was able to inhibit serum deprivation-induced osteoblast apoptosis to the same extent as thrombin. The results presented here demonstrate that thrombin treatment of osteoblasts inhibits apoptosis induced either by dexamethasone or by serum deprivation. Furthermore, it does so independently of the known thrombin receptors by bringing about the synthesis and/or secretion of an unknown survival factor or factors, which then act in an autocrine fashion to inhibit apoptosis.

Animals↗

PubMed2003

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection.

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

Amino Acid Sequence↗

PubMed2002

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases.

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.

Amino Acid Sequence↗

PubMed2002

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis.

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 (K(d)=1.31+/-0.26x10(-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.

Adhesins, Bacterial↗

PubMed2002

Molecular determinants of the mechanism underlying acceleration of the interaction between antithrombin and factor Xa by heparin pentasaccharide.

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.

Amino Acid Sequence↗

PubMed2002

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

Amino Acid Sequence↗

PubMed2002

Serpins: finely balanced conformational traps.

Serine protease inhibitors (serpins) play very important roles in the maintenance of various physiologically important systems. As knowledge of the workings of proteins of this family grows, new understanding is gained of the mechanisms by which they inhibit target proteases, using conformational changes for which the structure of serpins is uniquely adapted. This finely balanced system is utilized to healthy benefit in the control of serpin function by modulators, arguably the most striking examples of which occur in the control of proteolytic cascades, such as the coagulation system. Serpins also play very important intracellular roles: one example is the protection of immune cells from their own cytotoxic proteases. The finely balanced serpin mechanism also means that it is prone to disastrous consequences if mutations should occur in vital positions in the serpin structure. Many examples of disease-associated mutations have been shown, which has the dual effect of highlighting how important these molecules are in the maintenance of health and the fine balance that must be maintained in order to preserve their active, inhibitory conformation.

Amino Acid Motifs↗

PubMed2002

Serpins in prokaryotes.

Members of the serpin (serine proteinase inhibitor) superfamily have been identified in higher multicellular eukaryotes (plants and animals) and viruses but not in bacteria, archaea, or fungi. Thus, the ancestral serpin and the origin of the serpin inhibitory mechanism remain obscure. In this study we characterize 12 serpin-like sequences in the genomes of prokaryotic organisms, extending this protein family to all major branches of life. Notably, these organisms live in dramatically different environments and some are evolutionarily distantly related. A sequence-based analysis suggests that all 12 serpins are inhibitory. Despite considerable sequence divergence between the proteins, in four of the 12 sequences the region of the serpin that determines proteinase specificity is highly conserved, indicating that these inhibitors are likely to share a common target. Inhibitory serpins are typically prone to polymerization upon heating; thus, the existence of serpins in the moderate thermophilic bacterium Thermobifida fusca, the thermophilic bacterium Thermoanaerobacter tengcongensis, and the hyperthermophilic archaeon Pyrobaculum aerophilum is of particular interest. Using molecular modeling, we predict the means by which heat stability in the latter protein may be achieved without compromising inhibitory activity.

Amino Acid Sequence↗