Cysteine Protease-specific Inhibitor Research Articles

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Characterization of proteases activities in Ficus carica cultivars.

In this study, we characterized protease activities of 23 Ficus carica cultivars. Extracts of fruit, branch, and leaf of Masui Dauphine, one of the most representative F. carica cultivars in Japan, exhibited gelatin-hydrolyzing activity, both in the absence and presence of a cysteine protease-specific inhibitor, E-64, suggesting that not only ficin (classified as cysteine protease) but also collagenase (classified as serine protease) were involved in the digestion of gelatin. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-l-Lys-l-Pro-l-Leu-Gly-l-Leu-[N3 -(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2 , all branch extracts of 23 F. carica cultivars exhibited the activity both in the absence and presence of cysteine protease-specific inhibitor E-64, indicating that they contain ficin and collagenase. During digestion of acid-solubilized type I collagen by the branch extract of Masui Dauphine at 40-55 °C, collagen was completely digested in the absence of E-64, while it was partially digested in the presence of the inhibitor, indicating that the manner of digestion differed between ficin and collagenase contained in the extract. These results suggest that F. carica is attractive for industrial use to digest collagen. PRACTICAL APPLICATION: The industrial use of F. carica might be enhanced by efficiently utilizing these proteases and/or selecting the appropriate F. carica cultivar. Collagen is one of the targets to which our results might be applied. It is widely accepted today that collagen and its digestion products could be useful as functional food. F. carica is a potential candidate for use in not only complete but also partial digestion of collagen.

Characterization of Female Reproductive Proteases in a Butterfly from Functional and Evolutionary Perspectives.

Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae, the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.

Identification and characteristics of a cathepsin L-like cysteine protease from Clonorchis sinensis.

Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recombinant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited considerable activity at temperatures ranging from 28 to 42°C, with the highest activity observed at 42°C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH4.0-8.0), with an optimal pH of 5.5. The Km and Vmax of the recombinant CsCPL-m towards Z-Phe-Arg-AMC were determined to be 5.71 × 10-6M and 0.6μM/min, respectively, at 37°C and pH5.5. The recombinant CsCPL-m could degrade BSA and gelatine, but could not degrade human hemoglobin and human immunoglobulin G. These results implied that CsCPL might participate in the catabolism of host proteins for nutrition during the parasitic life cycle of C. sinensis; thus, CsCPL could be used as a potential vaccine antigen and drug target against C. sinensis infection.

Host-synthesized cysteine protease-specific inhibitor disrupts Cuscuta campestris parasitism in tomato

Dodder (Cuscuta campestris) is one of the most important pests of tomato causing severe losses in yield. Cuscutain is a pre-pro-protein produced by dodder that has a cysteine proteinase function essential for normal development of the haustoria and parasitism, which involves the secretion and activation of cuscutain cysteine protease in the host plant tissue. The propeptide subunit of this enzyme has an inhibitory function and restricts the enzymatic activity of cuscutain. Here, we transformed the inhibitory propeptide segment of this enzyme into tomato and examined the tomato resistance to C. campestris. We demonstrate the expression of inhibitory propeptide in transgenic plants and find that it effectively interrupted cuscutain enzyme activity and haustoria development at the endophytic stage. Mature haustoria infecting transgenic hosts showed defects in searching hyphae development and these structures were not elongate, and in most cases no functional haustoria were formed due to inhibitor expression in the transgenic plants after prehaustoria contact. Dodder grown on transgenic lines showed an overall reduction in vigor and fecundity due to defective attachment of haustoria. The increased growth of dodder-challenged transgenic plants relative to controls, demonstrates the efficacy of cysteine protease inhibition in parasite plant control.

Inhibition of streptococcal pyrogenic exotoxin B using allicin from garlic

Streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) and inactivation of SpeB results in the significantly decreased virulence of the bacterium. The protein is secreted as an inactive zymogen of 40 KDa (SpeBz) and undergoes proteolytic truncation to result in a 28 KDa mature active protease (SpeBm). In this study the effect of allicin on the proteolytic activity of SpeBm was evaluated using azocasein assay. Allicin neutralized the SpeBm proteolytic activity in a concentration dependent manner (IC50 = 15.71 ± 0.45 μg/ml). The loss of activity was completely reversed by subsequent treatment with a reducing agent, dithiothreitol (DTT; 10 mM final concentration), suggesting that allicin likely inhibits the SpeBm by forming a disulfide linkage with an active thiol group in its active site. This mechanism of action was further confirmed with the fact that DTT did not reverse the SpeBm activity in the presence of E−64, a cysteine protease-specific inhibitor, which works specially by forming a thioether linkage with free sulfhydryl groups in enzymes active site. The MIC of allicin against GAS was found to be 32 μg/ml. Exposure of GAS culture to allicin (25 μg/ml) inhibited maturation of SpeBz to the SpeBm.In conclusion, the results of this study suggest that allicin inhibits the maturation of SpeBz and proteolytic activity of SpeBm and could be a potential therapeutic agent for the treatment of GAS infections.

Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins

Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni(2+)-affinity chromatography. Purified protein was evaluated by SDS-PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km=12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60°C, thermal stability T50 value was 44°C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS-PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.

Partial Characterization of Protease from the Leaves of Jatropha curcas

The crucial roles of proteases in food and other industries stimulate research to find additional sources of the enzyme especially from non-conventional sources. In this study, protease was partially characterized from Jatropha curcas leaves.The enzyme had pH and temperature optima of 4.0 and 45oC respectively. A decline in residual activity of the enzyme was observed at above 45oC and the activation energy (Ea) from Arrhenius plot was 0.57kJ/mol. The enzyme showed specificity in the order; casein>hemoglobin> albumin> ovalbumin. Initial velocity studies for the determination of kinetic parameters revealed a KM and Vmax of 0.48 mg/ml and 0.014µmol/min respectively with a computed index of physiological efficiency (Kcat) of 0.029 min-1. Furthermore, Dixon-Webb’s plot identified ionizable groups at the active site with pKa1 and pKa2 of 5.0 and 5.3 respectively as well as an enthalpy of ionization of 0.047 kcal/mol implicating aspartate as an important amino acid at the active site of the enzyme. The protease was highly sensitive to cysteine protease specific inhibitor iodoacetate while 1,10phenanthroline and ethylenediaminetetraacetate slightly inhibited the enzyme. Data from this study suggest that J. curcas protease possesses closely similar properties to other known industrial proteases.

An enzyme activity as a potential biomarker for Alzheimer's disease

Background: Six different N-terminal amyloid precursor protein (APP) fragments, with molecular weight ∼12 kDa, have previously been identified in human cerebrospinal fluid (CSF). In a pilot study, both the sum of their concentrations, measured by western blot, and the relative abundance pattern, measured by mass spectrometry, were different in Alzheimer's disease (AD) patients compared to healthy controls. To test if these differences were also reflected in protease activities that possibly give rise to the ∼12 kDa fragments an enzymatic assay was developed and the activity in CSF was investigated for its potential as a biomarker for AD. Methods: The substrate in the protease activity assay was a custom made fluorochrome/quencher labeled peptide that covers the cleavage sites in APP (APP118-APP127) corresponding to the C-termini of the six ∼12 kDa APP fragments. The activity was measured in CSF from 55 AD patients and 17 controls. Results: There was a significant increase in the protease activity in CSF from AD patients compared to the controls (p = 0.001). This is in line with previous results which indicate that the sum of the ∼12 kDa fragments are elevated in AD. Results from inhibition studies strongly suggests that the enzyme responsible for the cleavage of the substrate is an aspartic protease since a sub nM IC50 value was recorded for Pepstatin A while no inhibition was observed for the cysteine protease specific inhibitor E64 at concentrations up to100 nM. Conclusions: There exists an enzymatic activity in CSF capable of cleaving a peptide substrate that spans a portion, close to the N-terminal, of APP. In a pilot study the activity is increased in AD patients compared to controls suggesting that it can be used as a biomarker. (Less)

Effect of aging on functional brain connectivity: Association with apolipoprotein E genotype

Background: Six different N-terminal amyloid precursor protein (APP) fragments, with molecular weight w12 kDa, have previously been identified in human cerebrospinal fluid (CSF). In a pilot study, both the sum of their concentrations, measured by western blot, and the relative abundance pattern, measured by mass spectrometry, were different in Alzheimer’s disease (AD) patients compared to healthy controls. To test if these differences were also reflected in protease activities that possibly give rise to the w12 kDa fragments an enzymatic assay was developed and the activity in CSF was investigated for its potential as a biomarker for AD. Methods: The substrate in the protease activity assay was a custom made fluorochrome/quencher labeled peptide that covers the cleavage sites in APP (APP118-APP127) corresponding to the C-termini of the six w12 kDa APP fragments. The activity was measured in CSF from 55 AD patients and 17 controls. Results: There was a significant increase in the protease activity in CSF from AD patients compared to the controls (p 1⁄4 0.001). This is in line with previous results which indicate that the sum of the w12 kDa fragments are elevated in AD. Results from inhibition studies strongly suggests that the enzyme responsible for the cleavage of the substrate is an aspartic protease since a sub nM IC50 value was recorded for Pepstatin A while no inhibition was observed for the cysteine protease specific inhibitor E64 at concentrations up to100 nM. Conclusions: There exists an enzymatic activity in CSF capable of cleaving a peptide substrate that spans a portion, close to the N-terminal, of APP. In a pilot study the activity is increased in AD patients compared to controls suggesting that it can be used as a biomarker.

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

Crucial Commitment of Proteolytic Activity of a Purified Recombinant Major House Dust Mite Allergen Der p1 to Sensitization toward IgE and IgG Responses

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-gamma. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

Partial characterization of a cysteine protease from Panonychus citri

A cysteine protease activity of Panonychus citri was detected and partially characterized. Proteolytic activity of the enzyme was enhanced by the addition of reducing agent, dithiothreitol (DTT), and also it was absolutely inhibited by cysteine protease specific inhibitors, such as trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E.64) and iodoacetic acid (IAA). The other specific inhibitors of serine- and metallo-proteases could not inhibit the enzyme activity. This result showed for the first time cysteine protease activity of P. citri. Further studies on the biological roles of the enzyme are required.

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.

Cysteine Protease Inhibitors Produced by the Industrial Koji Mold, Aspergillus oryzae O-1018.

Aspergillus oryzae O-1018 (FERM P-15834) separated from industrial koji for brewing sake was found to produce five papain-inhibitory compounds in the culture supernatant. The five isolated inhibitors were named CPI-1 to CPI-5, and their structures were elucidated by spectroscopic analyses and chemical degradation. We determined the structures of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N- [(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl] amino]butane, 5-amino-1-[[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]amino]pentane and N (8)- [N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu-cyl]spermidine, respectively. We also confirmed by a degradation experiment that CPI-1 consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine and spermidine. Although CPI-4 was identified as kojistatin A,(1)) the other CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition potency of CPIs against cysteine proteases was as high as or higher than that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times more inhibitory than E-64 toward cathepsin L.

Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo

Schistosome parasites utilize hemoglobin as a major protein source for their metabolism. Degradation of hemoglobin has been hypothesized to be mediated by both cysteine and aspartyl proteases secreted into the lumen of the parasite intestine. We now show that two distinct types of irreversible cysteine protease-specific inhibitors both arrest schistosome hemoglobin degradation in vitro. Arrest of hemoglobin degradation is followed by death of developing schistosomula 1 week later. Schistosome infected mice treated by a dose of 2 mg inhibitor per day for 1 week early in infection, and 2 weeks at the time of egg production, showed a significant reduction in worm burden, hepatomegaly, and the number of eggs produced per female worm. Histopathology showed a minimal immune response to those eggs which were produced, consistent with a delay in egg production relative to untreated infections. By tagging the inhibitor with biotin, specific cysteine protease targets were identified in extracts of schistosome worms.

The epithelial reaction of mouse lens in traumatic cataract models

Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni2+-affinity chromatography. Purified protein was evaluated by SDS–PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km = 12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60 °C, thermal stability T50 value was 44 °C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12 h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS–PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2 h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.