Thrombin-like serine protease,antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA₂, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.     

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K_I of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver–Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C′ in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.     

The defensive role of latex in plants: detrimental effects on insects

The defensive role of the latex of Calotropis procera has recently been reported. In this study, latex proteins involved in detrimental effects on insects were evaluated on another important crop pest. The latex was fractionated to obtain its major protein fraction, which was then used to evaluate its insecticidal properties against Callosobruchus maculatus (Coleoptera: Bruchidae) in artificial bioassays. Laticifer proteins (LP) were investigated to characterize their action in such an activity. LP was highly insecticidal at doses as low as 0.1% (W/W). This effect was slightly augmented in F₁ generation reared in artificial seeds containing LP at similar proportions of F₀, but was fully reversed when F₁ developed in LP-free seeds. The insecticidal proteins were not retained in a chitin column, and did not lose their insecticidal activity, even after heat treatment or pronase digestion. However, these samples inhibited papain (EC 3.4.22.2) activity and gut proteases of C. maculatus larvae, and a reverse zymogram showed the presence of protein bands resistant to papain digestion. These activities were not observed in unheated LP as they were probably masked by abundant endogenous cysteine protease (EC 3.4.22.16) activity present in unheated LP. LP was resistant to proteolysis when assayed with C. maculatus gut extract. However, gut proteins of C. maculatus were digested when incubated with LP. These observations and the deleterious effects of LP upon C. maculatus, reinforce the hypothesis that laticifer fluids are involved in plant defense against insects and indicate C. procera latex to be a source of promising insecticidal proteins. The inhibitor of proteolysis present in the latex seems to be resistant to heat and proteolysis and is certainly involved in the detrimental effects observed.     

Laticifer proteins play a defensive role against hemibiotrophic and necrotrophic phytopathogens

Proteins from latex of Calotropis procera (CpLP), Plumeria rubra (PrLP), Carica candamarcensis (P1G10) and Euphorbia tirucalli (EtLP) were tested for antifungal activity against phytopathogens. CpLP and P1G10 inhibited each fungi analyzed. PrLP and EtLP did not exert inhibition. CpLP and P1G10 exhibited preferential inhibitory activity towards R. solani (IC₅₀ = 20.7 and 25.3 μg/ml, respectively). The inhibitory activity was lost after heat treatment or proteolysis, providing evidence for the involvement of proteins in the inhibitory effect. Treatment of CpLP or P1G10 with Dithiothreitol improved both, the endogenous proteolytic activity and the antifungal properties. Conversely, pre-treatment of CpLP or P1G10 with iodoacetamide drastically reduced endogenous proteolytic activities and partially abrogated antifungal activity. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. The purified cysteine proteinase CMS2MS2 from Carica candamarcensis latex or papain (E.C. 3.4.22.2), a cysteine proteinase from latex of Carica papaya L., but not trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine proteases, replicated the results obtained with CpLP or P1G10, thus restricting the antifungal property to latex plant cysteine proteinases. CpLP, CMS2MS2 and papain induced production of reactive oxygen species in spores of F. solani, suggesting that inhibition could be linked to oxidative stress. Proteome analysis of CpLP by 2-D electrophoresis and MALDI-TOF-TOF confirmed the existence of various pathogenic-related proteins such as chitinases, peroxidases and osmotins. The results support that laticifer proteins are part of plant defense repertoire against phytopathogenic fungi.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27–30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds.     

Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex

Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.     

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

Co‐expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.     

Protease inhibitors from Streptomyces violascens

Summary Two protease inhibitors from the culture fluid of Streptomyces violascens U 10600 have been purified with a method including freeze-drying, methanol extraction, dialysis, and ultrafiltration. By gel filtration on Sephadex G-15 a separation in two active inhibitors, one of trypsin and one of chymotrypsin, was made.The inhibitors were stable at 100°C, pH 5, for 20 min and at 24°C between pH 1.8 to 9.7. Both inhibitors were dialysable. They had no bacteriostatic or fungistatic effects. The trypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, Entomophthora coronata, and to some extent Gibberella fujikuroi, but not chymotrypsin, kallikrein, ficin, or pepsin. The chymotrypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, and Gibberella fujikuroi, but not trypsin, kallikrein, ficin, pepsin, or protease from Entomophthora coronata.     

Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.     

Vacuole Cysteine Proteases and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Degradation during Monocarpic Senescence in Cowpea Leaves

Characterisation of proteases degrading ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC: 4.1.1.39) was studied in the cowpea leaf during monocarpic senescence 3 and 9 d after flowering (DAF), representing early and mid pod fill. The stage at 3 DAF coincided with decrease in the metabolic parameters characterising senescence, i.e., contents of total soluble proteins, RuBPCO, and leaf nitrogen. At 9 DAF, there was a decline in total soluble proteins and an appearance of a 48 kDa cysteine protease. Characterisation of the proteases was done using specific inhibitors. Subcellular localisation at 3 DAF was studied by following the degradation of RuBPCO large subunit (LSU) in the vacuole lysates using immunoblot analyses. Cysteine proteases played a predominant role in the degradation of RuBPCO LSU at the crude extract level. At 9 DAF, expression of cysteine protease isoforms was monitored using polyclonal antibodies against papain and two polypeptides of molecular masses 48 and 35 kDa were observed in the vacuole lysates. We confirmed thus the predominance of cysteine proteases in the vacuoles during different stages of pod development in cowpea leaf.     

Kinetically Controlled Peptide Synthesis Mediated by Papain Using the Carbamoylmethyl Ester as an Acyl Donor

A series of dipeptides were synthesized generally in good yields with carbamoylmethyl (Cam) esters as acyl donors in the presence of a cysteine protease, papain, immobilized on Celite. Several segment condensations were also achieved generally in high yields without danger of racemization and formation of the secondary-hydrolysis product. Moreover, partial sequences of some bioactive peptides were prepared through segment condensations, and aimed-at peptides were obtained generally in high yields without the racemization of C-terminal residues of the carboxyl components. Thus, the superiority of the Cam ester in the kinetically controlled peptide synthesis was once again ascertained in couplings mediated by the cysteine protease as in those catalyzed by the serine proteases reported earlier.     

Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias): a phylogenetic test of plant defense escalation

A tremendous diversity of plants exude sticky and toxic latex upon tissue damage, and its production has been widely studied as a defensive adaptation against insect herbivores. Here, we address variation in latex production and its constituent chemical properties (cardenolides and cysteine proteases) in 53 milkweeds [Asclepias spp. (Apocynaceae)], employing a phylogenetic approach to test macroevolutionary hypotheses of defense evolution. Species were highly variable for all three traits, and they showed little evidence for strong phylogenetic conservatism. Latex production and the constituent chemical defenses are thus evolutionarily labile and may evolve rapidly. Nonetheless, in phylogenetically independent analyses, we show that the three traits show some correlations (and thus share a correlated evolutionary history), including a positive correlation between latex exudation and cysteine protease activity. Conversely, latex exudation and cysteine protease activity both showed a trade‐off with cardenolide concentrations in latex. We also tested whether these traits have increased in their phenotypic values as the milkweeds diversified, as predicted by plant defense escalation theory. Alternative methods of testing this prediction gave conflicting results – there was an overall negative correlation between amount of evolutionary change and amount of latex exudation; however, ancestral state reconstructions indicated that most speciation events were associated with increases in latex. We conclude by (i) summarizing the evidence of milkweed latex itself as a multivariate defense including the amount exuded and toxin concentrations within, (ii) assessing the coordinated evolution of latex traits and how this fits with our previous notion of ‘plant defense syndromes’, and finally, (iii) proposing a novel hypothesis that includes an ‘evolving community of herbivores’ that may promote the escalation or decline of particular defensive strategies as plant lineages diversify.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.     

Hybrid protease inhibitors for pest and pathogen control – a functional cost for the fusion partners?

Fusion proteins integrating dual pesticidal functions have been devised over the last 10 years to improve the effectiveness and potential durability of pest-resistant transgenic crops, but little attention has been paid to the impact of the fusion partners on the actual activity of the resulting hybrids. Here we assessed the ability of the rice cysteine protease inhibitor, oryzacystatin I (OCI), to retain its protease inhibitory potency when used as a template to devise hybrid inhibitors with dual activity against papain-like proteases and carboxypeptidase A (CPA). C-terminal variants of OCI were generated by fusing to its C-terminal end: (i) the primary inhibitory site of the small CPA inhibitor potato carboxypeptidase inhibitor (PCI, amino acids 35-39); or (ii) the complete sequence of PCI (a.a. 1-39). The hybrid inhibitors were expressed in E. coli and tested for their inhibitory activity against papain, CPA and digestive cysteine proteases of herbivorous and predatory arthropods. In contrast with the primary inhibitory site of PCI, the entire PCI attached to OCI was as active against CPA as free, purified PCI. The OCI-PCI hybrids also showed activity against papain, but the presence of extra amino acids at the C terminus of OCI negatively altered its inhibitory potency against cysteine proteases. This negative effect, although not preventing dual binding to papain and CPA, was correlated with an increased binding affinity for papain presumably due to non-specific interactions with the PCI domain. These results confirm the potential of OCI and PCI for the design of fusion inhibitors with dual protease inhibitory activity, but also point out the possible functional costs associated with protein domain grafting to recipient pesticidal proteins.     

Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Molecular cloning of a mitogenic proteinase from Carica candamarcensis: its potential use in wound healing

Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.