Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Macrocyclic inhibitors for the serine protease plasmin

Macrocyclic inhibitors for the serine protease plasmin were synthesized and evaluated. The inhibitors were constructed starting from a cyclohexanone core. This core was linked to either the C- or N-terminus of a peptide so that the inhibitors were designed to interact with the non-primed or primed binding sites of the protease. Macrocycles were prepared by connecting the side chain of Tyr or Trp, via a short linker, to one end of the peptide. The activities of the macrocyclic inhibitors, while modest, were up to 10-fold more potent than a related non-cyclic analog.     

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.     

Novel Peptides Inhibit Zika NS2B-NS3 Serine Protease and Virus Replication in Human Hepatic Cell Line

Zika virus (ZIKV) is a Flavivirus associated with several neurological complications. Currently, there are no vaccines or cures available and an efficient antiviral treatment is urgently needed to combat ZIKV infection. Herein, we targeted ZIKV NS2B-NS3 serine protease with short peptides to inhibit ZIKV replication in human hepatic cell line (WRL-68). The short peptide inhibitors were designed using Hyperchem 8.0.10 software. Docking energy and binding configuration were calculated using HADDOCK webserver. ZIKV NS2B-NS3 protease was produced as a recombinant single peptide in Escherichia coli and the protease activity was examined by measuring the cleavage of a fluorescent substrate in the presence of the peptides or aprotinin as a standard protease inhibitor. Computational analysis revealed that the short peptides, AYA2 and AYA9, exhibited lower docking energy to ZIKV protease than aprotinin. Both peptides also possessed lower half maximal inhibitory concentration (IC₅₀), 30.9 and 22.1 µM respectively, against ZIKV protease activity when compared to aprotinin (35.4 µM). Interestingly, AYA2 and AYA9 exhibited minimal cytotoxic effects in WRL-68 cells and showed considerable inhibition against ZIKV replication in vitro at half maximal effective concentration (EC₅₀) of 40.73?±?2.3 µM and 34.65?±?1.8 µM respectively. Fusion of these two peptides to MAP30 peptide substantially reduced the IC₅₀ of ZIKV protease inhibition to 1.1 µM and inhibited ZIKV replication at EC₅₀ of 0.5157?±?0.03 µM. In sum, we reported novel peptides that effectively inhibited ZIKV replication in vitro. This study represents a cost-effective strategy of developing peptide inhibitors by shortening the peptides and producing them in recombinant form.

     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

Protease Inhibitors Cause Necrotic Cell Death in Chlamydomonas reinhardtii by Inducing the Generation of Reactive Oxygen Species

Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N‐tosyl‐lysine‐chloromethyl‐ketone (TLCK) and N‐tosyl‐phenylalanine‐chloromethyl‐ketone (TPCK), which affect the trypsine‐ and chymotrypsine‐like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Propeptide-Mediated Specific Inhibition of a Recombinant Serine Protease from Indian Malaria Vector, Anopheles culicifacies

Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles c ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its three deletion mutants and leupeptin (a general serine protease inhibitor) were tested in vitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

SjAPI,the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Background

Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.

Principal Findings

Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.

Conclusions/Significance

To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.     

Neutralization of Burkholderia pseudomallei Protease by Fabs Generated through Phage Display

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.     

丹参SmPI1和SmPI2基因克隆及胁迫表达研究

该研究从药用植物丹参中克隆了SmPI1和SmPI2蛋白酶抑制剂基因,采用生物信息学和实时荧光定量PCR方法对其序列及表达模式进行了分析。序列分析结果表明,丹参SmPI1和SmPI2基因分别含有一个长度为222bp和216bp的开放阅读框,编码73和71个氨基酸;2个编码蛋白都无跨膜结构域和信号肽,预测都定位于细胞质中;SmPI1和SmPI2蛋白与川桑、可可、苜蓿等植物的蛋白酶抑制剂基因相似性较高,分别为58%、52%、53%和54%、56%、51%。实时荧光定量PCR结果表明,SmPI1和SmPI2基因受茉莉酸甲酯(MeJA)和甘蓝黑腐病黄单胞菌(Xanthomonas campestris pv.Campestris,XC)显著诱导,说明丹参SmPI1和SmPI2基因可强烈响应这两类胁迫,可能参与丹参中两类胁迫分子途径相关的抗性反应。     

A RAS-encoded protein in Dictyostelium discoideum is acylated and membrane-associated

Dictyostelium dfscoideum synthesizes a 23000 M_r protein, p23^dd-ras, closely related to the mammalian oncogene-encoded protein p21^ras. To investigate the subcellular localization of P23^dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23^dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23^dd-ras were recovered in the cytosol fraction, suggesting that intact p23^dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un-labelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23^dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23^dd-ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23^dd-ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23^dd-ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21^ras in mammalian cells.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .     

An enzymatic method for the determination of hemoglobinA<Subscript>1C</Subscript>

Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A_1C. To develop an enzymatic method for the measurement of HbA_1C, we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA_1C to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA_1C and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA_1C.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

Synthesis and proteasome inhibition of N‐allyl vinyl ester‐based peptides

Inhibition of the proteasome, the multicatalytic protease complex responsible for the turnover of many cellular proteins, represents an attractive target in the development of new drug therapies, proteasome inhibitors being potentially useful tools for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Based on our previous development of a new class of inhibitors bearing a C‐terminal VE cluster able to interact with catalytic threonine, we report herein the synthesis and activity of new VE‐based peptide analogs bearing an additional allyl pharmacophore unit at the C‐ or N‐terminal position of the pseudotripeptide sequence. In the new series, the structural modification carried out to the prototype determine a decrease of proteasome inhibition. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inactivation of neurohormone D by Malpighian tubules in an insect,Periplaneta americana

Summary The heart rate accelerating peptide neurohormone D is rapidly inactivated by intact Malpighian tubules of cockroaches and also by homogenates of them. The peptide is removed from a solution by an active uptake mechanism. Within the tubule cells one or a set of soluble proteinases with a molecular mass around 45000 Da hydrolyze the neuropeptide. The inhibition of the reaction by synthetic protease inhibitors and chelating agents characterizes the enzyme(s) as metalloendopeptidase with serine or cysteine at the active site. This seems to be the first evidence that a peptidase comparable to the neutral metalloendopeptidase of mammalian kidney microvilli exists in insect Malpighian tubules and could play an important role in the hydrolysis of neuropeptides.Abbreviations EDTA ethylenediamine tetraacetic acid - AEBSF 4--aminoethylbenzenesulfonylfluoride - PMSF pnenylmethanesulfonyl fluoride - TLCK tosyl-lysine chloromethyl ketone - TPCK tosyl-phenylalanyl chloromethyl ketone - CMB chloromercuribenzoic acid - DNP-Ala N-dinitrophenyl-alanine - TFA trifluoroacetic acid     

EprS,an autotransporter protein of Pseudomonas aeruginosa,possessing serine protease activity induces inflammatory responses through protease‐activated receptors

PA3535 (EprS), an autotransporter (AT) protein of Pseudomonas aeruginosa, is predicted to contain a serine protease motif. The eprS encodes a 104.5 kDa protein with a 30‐amino‐acid‐long signal peptide, a 51.2 kDa amino‐terminal secreted passenger domain and a 50.1 kDa carboxyl‐terminal outer membrane channel formed translocator. Although the majority of AT proteins have been reported to be virulence factors, little is known about the functions of EprS in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of recombinant EprS secreted by Escherichia coli. The proteolytic activity of EprS was markedly decreased by changing Ser to Ala at position 308 or by serine protease inhibitors. EprS preferred to cleave substrates that terminated with arginine or lysine residues. Thus, these results indicate that EprS, a serine protease, displays the substrate specificity, cleaving after basic residues. We demonstrated that EprS activates NF‐κB‐driven promoters through protease‐activated receptor (PAR)‐1, ‐2 or ‐4 and induces IL‐8 production through PAR‐2 in a human bronchiole epithelial cell line. Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of PAR‐1, ‐2 and ‐4 at a specific site with exposure oftheir tethered ligands. Collectively, these results suggest that EprS activates host inflammatory responses through PARs.