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PubMed2000

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.

Amino Acid Sequence↗
PubMed2001

Secretory leukocyte protease inhibitor in ovarian endometriomas following GnRH agonist therapy.

OBJECTIVE: To determine whether expression of secretory leukocyte protease inhibitor is affected in tissue and peritoneal fluid of women with ovarian endometriomas treated with GnRH analogues. METHODS: In 32 women with endometriomas (17 untreated and 15 treated with GnRH analogue) and 21 with ovarian cystadenomas, we examined the expression of secretory leukocyte protease inhibitor messenger RNA (mRNA) by Northern blot analysis; protein distribution was measured immunohistochemically. Concentrations of secretory leukocyte protease inhibitor in peritoneal fluid were measured by enzyme-linked immunosorbent assay. Expression of secretory leukocyte protease inhibitor in endometrioma explants in vitro was also studied with and without the GnRH analogue treatment. RESULTS: Secretory leukocyte protease inhibitor mRNA expression was identified only in untreated endometriomas. In the GnRH agonist-treated endometriomas, the semiquantitative H-score for secretory leukocyte protease inhibitor immunostaining was significantly lower than that for untreated endometriomas (P <.001). The peritoneal fluid of the GnRH agonist-treated women also contained significantly lower concentrations of secretory leukocyte protease inhibitor (median 76 ng/mL, interquartile range 51-131 ng/mL; P <.001) than untreated women (124 ng/mL, 70-186 ng/mL). Secretory leukocyte protease inhibitor in endometrioma explants in vitro was significantly inhibited by the GnRH analogue (P <.05). CONCLUSION: Expression of secretory leukocyte protease inhibitor in tissue and peritoneal fluid of women with ovarian endometriomas was decreased by GnRH agonist treatment.

Adult↗
PubMed2000

Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes*

A protease-producing microorganism was isolated in northern Taiwan and identified as a strain of Bacillus subtilis. B. subtilis Y-108 thus isolated can be used for deproteinization of crustacean wastes in the preparation of chitin. For deproteinization tests, liquid phase fermentation of untreated shrimp shell, crab shell, and lobster shell wastes with this microbe showed protein removal of 88, 67, and 83%, respectively. In contrast, the protein removal of the acid treated wastes was 76, 62, 56%, respectively. The optimized conditions for protease production was found when the culture was shaken at 30 degrees C for 3 days in 100 ml of medium (phosphate buffer adjusted to pH 6.0) containing 7% shrimp and crab shell powder (SCSP), 0.1% K(2)HPO(4), 0.05% MgSO(4), 1.0% arabinose, 1.5% NaNO(3), and 1.5% CaCl(2). Under such conditions, the protease of B. subtilis Y-108 attained the highest activity. It was as high as 20.2 U/ml. The protease was purified in a three-step procedure involving ammonium sulfate precipitation, DEAE-Sepharose CL-6B ionic exchange chromatography, and Sephacryl S-200 gel permeation chromatography. The enzyme was shown to have a relative molecular weight of 44 kDa by SDS polyacrylamide gel electrophoresis. The protease was most active at pH 8.0 and 50 degrees C with casein as substrate. The protease was activated by Mn(+2), Fe(+2), Zn(+2), Mg(+2), Co(+2), but inhibited completely by Hg(+2). The protease was also inhibited by metal-chelating agent such as EDTA, sulfhydryl reagents as beta-mercaptoethanol, and by cysteine hydrochloride, histidine, glycerol. The EDTA was the most effective inhibitor that caused complete inhibition of protease. It was concluded that this enzyme is a metal-chelator-sensitive neutral protease.

Journal Article↗
PubMed2000

Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes.

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.

Journal Article↗
PubMed2000

An immunoassay for detection of heat-stable proteases from thermoduric psychrotrophic Bacillus spp. of dairy origin.

A homogeneous preparation of a thermostable protease from Bacillus sp. B-17 was used to raise an antiserum in rabbits. IgG of this antiserum was used to study the antigenic relationship of proteases in cell-free extracts of 21 bacilli of milk origin. Based on immunological cross reactivity, the 21 bacilli were divided into 3 serological subgroups. To raise antibodies of broader specificity, protease from Bacillus sp. B-11 (group II) and B-3 (group III) were purified, mixed with purified B-17 protease, and an antiserum was raised against this mixture. IgG of this antiserum was purified (IgG anti-bacilli protease). A sandwich ELISA was standardized using IgG anti-bacilli protease as capture antibody. The assay could detect 1.2 ng ml(-1) of protease in milk or buffer, but the assay failed to detect 4 of 21 bacilli proteases. The results suggest that this assay is useful for the detection of proteases of Bacillus spp. in dairy industry.

Animals↗
PubMed1997

Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families.

Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.

Amino Acid Sequence↗
PubMed1987

Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA.

Although protease E was isolated from human pancreas over 10 years ago [Mallory, P. A., & Travis, J. (1975) Biochemistry 14, 722-729], its amino acid sequence and relationship to the elastases have not been established. We report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. We have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. We also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of rat mast cell protease I (chymase).

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.

Amino Acid Sequence↗
PubMed2001

The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.

Animals↗
PubMed2002

Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown.

Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.

Catalysis↗
PubMed2003

Binding of nonphysiological protein and peptide substrates to proteases: differences between urokinase-type plasminogen activator and trypsin and contributions to the evolution of regulated proteolysis.

Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.

Catalytic Domain↗
PubMed1998

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters.

A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.

Amino Acid Sequence↗
PubMed1997

Bcl-2 acts upstream of the PARP protease and prevents its activation.

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.

Journal Article↗
PubMed2000

Comparative molecular modelling identifies a common putative IgE epitope on cysteine protease allergens of diverse sources.

BACKGROUND: Previous approaches for studying common allergenic epitopes have mainly focused on sequence comparisons, which unfortunately yield little or no information on the shape of the epitope which is the most important determinant of cross-reactivity. OBJECTIVES: The aim of this study was to investigate the structural basis for cross-reactivity between a previously identified immunodominant epitope of the house dust mite allergen Der p 1 (Leu147-Gln160) and the corresponding epitopes on other allergens that are either taxonomically closely related (i.e. cysteine proteases of other mite species) or representing evolutionary conserved structures (i.e. plant, human and parasite cysteine proteases). METHODS: We carried out comparative molecular modelling on a range of cysteine proteases, including those of other mite species (Der f 1 and Eur m 1), human (cathepsins B, K, L, S and O), plants (papain, chymopapain and actinidin) and parasites (cruzain, cathepsin L-like Leishmania protease, Entamoeba ACP1 protease and Schistosoma Q26534, Q11003 and cathepsin L proteases). RESULTS: Our study shows that all the cysteine proteases investigated here display an epitope corresponding to that previously identified on Der p 1, but with varying shapes and degree of accessibility. It appears that the core of the epitope on these homologous cysteine proteases consists of a centrally located conserved Tyr residue flanked on either sides by accessible amino acids. CONCLUSION: Therefore, these cysteine proteases seem to use similar accessible structures, which may form the basis for the rational design of generic epitope-directed treatment strategies for controlling allergic diseases.

Allergens↗
PubMedDate not supplied

[Proteases in helminthic parasites].

Proteases catalyse the cleavage of internal peptide bonds within peptides and proteins. They are classified into four major classes and are involved in a broad range of eukaryotic processes. Proteases have also been found to play a number of critical roles in the virulence of pathogenic agents, particularly of nematode parasites. Parasitic proteases are involved in different aspects of host-parasite interactions. They facilitate the invasion of host tissues and allow nutrition as well as the survival of the parasite in its host. Proteases also participate in the parasite's evasion from the host's immune response. The functional diversity and complexity of these enzymes are described in this review, with a particular focus on the principally identified proteases of four helminths: Schistosoma sp., Fasciola sp., Taenia sp. and Haemonchus sp. Some of these proteases, especially the cysteine proteases secreted by the parasitic trematode Fasciola hepatica, have been successfully tested in experimental immunodiagnosis. Proteases identified in different parasites are currently under study for a use as recombinant vaccines. In this respect, proteases are proposed as major potential targets for immunotherapy and chemotherapy against parasitic diseases.

Animals↗
PubMed1986

Modulation of smooth muscle responses by serine proteases and related enzymes.

Standard isolated smooth muscle tissue preparations were used to screen the musculotropic actions of serine proteases and other substances that are generated during hemostatic activation. Sera obtained from human, rabbit, and guinea pigs produced a dose-dependent contraction of these isolated tissue preparations. These sera also augmented the actions of epinephrine and other agonists on different tissue preparations. Both contractile and relaxant actions of proteases were observed with various serine proteases and their complexes. Thrombins (alpha, beta, and gamma) produced varying degrees of contractile actions of rabbit aortic strips; however, Factor Xa produced a marked relaxation of this preparation. Trypsin and kallikrein did not produce any action on the aortic strip and thrombin, however, produced contraction of the guinea pig ileum. Protease complexes produced varying musculotropic actions on the isolated tissue preparations, which may be related to their composition. The musculotropic actions of serum and thrombin were not blocked by conventional pharmacologic antagonists. However, protease inhibitors, such as hirudin, D-Phe-Pro-Arginal, and other derivatives, produced varying effects of the musculotropic actions of serum and thrombins. These results indicate that thrombin and related proteases generated during the activation of the hemostatic system are capable of producing direct musculotropic actions of various smooth muscles. Various serine proteases and serine protease complexes were found to produce strong hemodynamic effects in a rabbit model for the study of blood plasma. The smooth muscle modulant actions of serine proteases should be taken into account to clarify the vascular pathologic changes in relation to spasmogenic and spasmolytic syndromes observed during hemostasis disorders.

Animals↗
PubMed2000

Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production.

BACKGROUND: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. OBJECTIVE: We sought to assess protease activity in Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus extracts and study the ability of these extracts to cause desquamation and release of proinflammatory cytokines from epithelial cells. METHODS: Protease activities of the fungal extracts were quantified. Changes with respect to cell morphology, cell desquamation, and cytokine production (IL-6 and IL-8) were measured in the absence and presence of the fungal extracts in an airway-derived epithelial cell line (A549) and primary epithelial nasal cells. RESULTS: Fungal proteases differentially induced morphologic changes, cell desquamation, and production of IL-6 and IL-8 in a dose- and time-dependent fashion. Alternaria alternata extracts induced cell shrinking and cell desquamation and strongly enhanced the production of IL-6 and IL-8 at higher concentrations. Aspergillus fumigatus extracts caused cell shrinking, cell desquamation, and production of IL-6 and IL-8, even at low concentrations. The Aspergillus fumigatus-derived extract grown on collagen medium induced a strong dose-dependent decline in cytokine production at higher concentrations. Cladosporium herbarum extracts did not induce morphologic changes or cell desquamation but enhanced IL-6 and IL-8 productions at higher concentrations. The dependence of these effects on intact protease activity was shown by their abrogation by protease inhibitors. CONCLUSION: Proteases present in fungal extracts interact with epithelial cells, leading to morphologic changes, cell desquamation, and induction of proinflammatory cytokines. It is proposed that these fungal proteases may activate epithelial cells through a protease-activated receptor type 2-driven mechanism.

Allergens↗
PubMed1991

Direct selection for sequences encoding proteases of known specificity.

We have developed a simple genetic selection that could be used to isolate eukaryotic cDNAs encoding proteases that cleave within a defined amino acid sequence. The selection was developed by using the transcription factor GAL4 from Saccharomyces cerevisiae as a selectable marker, a cloned protease from tobacco etch virus (TEV), and an 18-amino acid TEV protease target sequence. In yeast, TEV protease cleaves its target even when the target is fused to internal regions of the GAL4 protein. This cleavage separates the DNA binding domain from the transcription activation domain of GAL4, rendering it transcriptionally inactive. The proteolytic cleavage can be detected phenotypically by the inability of cells to metabolize galactose. Cells expressing the TEV protease can also be selected on the suicide substrate 2-deoxygalactose. DNA binding studies show that the TEV protease decreases the activity of the GAL4/target fusion protein. Because another protease target sequence of 55 amino acids can be inserted into GAL4 without any loss of transcriptional activity, this assay offers the opportunity to use high-efficiency cDNA cloning and expression vectors to select coding sequences of other proteases from various species. The assay could also be used to help define both target specificities and functional domains of proteases.

Base Sequence↗