Elastase-like Protease Research Articles

Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters.

The loss of smell (anosmia) related to SARS-CoV-2 infection is one of the most common symptoms of COVID-19. Olfaction starts in the olfactory epithelium mainly composed of olfactory sensory neurons surrounded by supporting cells called sustentacular cells. It is now clear that the loss of smell is related to the massive infection by SARS-CoV-2 of the sustentacular cells in the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamsters infected with an early circulating SARS-CoV-2 strain harboring the D614G mutation in the spike protein; we show here that rather than being related to a first wave of apoptosis as proposed in previous studies, the innate immune cells play a major role in the destruction of the olfactory epithelium. We observed that while apoptosis remains at a low level in the damaged area of the infected epithelium, the latter is invaded by Iba1+ cells, neutrophils and macrophages. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we could reduce the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease in SARS-CoV-2 infection levels in the olfactory epithelium. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus in the early phase of the SARS-CoV-2 infection.

Targeting Necrosis: Elastase-like Protease Inhibitors Curtail Necrotic Cell Death Both In Vitro and in Three In Vivo Disease Models.

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity in vitro and in vivo in animal models of traumatic brain injury, acute myocardial infarction, and drug-induced liver toxicity. Consequently, this study may pave the way for the development of novel therapies for the treatment, inhibition, or prevention of a large number of hitherto untreatable diseases.

A Novel Broad-Spectrum Elastase-Like Serine Protease From the Predatory Bacterium Bdellovibrio bacteriovorus Facilitates Elucidation of Site-Specific IgA Glycosylation Pattern.

The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus: BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0–9.0 but was drastically reduced in the presence of MnCl2 and completely inhibited by ZnCl2. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn459). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail.IMPORTANCEAntibodies are well-established as key components of the immune system, and the importance of antibody glycosylation is steadily gaining recognition. Modifications of antibodies by glycosylation creates a vast repertoire of antibody glycovariants with distinctive and diverse functions in the immune system. Most of the available information regarding antibody glycosylation is based on studies with IgG, which have contributed greatly to the advance of therapeutic antibody treatments. However, much is still unknown regarding the importance of glycosylation and the Fc-structure for the remaining antibody classes. Such research has proven to be technically challenging and demonstrates a need for novel tools to facilitate such investigations. Here we have identified and characterized a novel protease from B. bacteriovorus, facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn459 N-glycan. This further highlights the potential of B. bacteriovorus as a source to identify potential novel biotechnological tools.

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: Implications for coronary artery stent

BackgroundBiodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. MethodsMicroparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. ResultsWe demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation. ConclusionsThis study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.

Combined effects of three crystalline toxins from Bacillus thuringiensis with seven proteinase inhibitors on beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae)

Spodoptera exigua is a highly polyphagous pest and causes extensive damage to many field and truck crops. Since the larvae are not or only sublethally affected by Cry1Ac, the fused Cry1Ac/Cry1Ab, or the stacked Cry1Ac+CpTI transgenic cottons that are widely planted in China, S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in 1997 in China. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against, and for expanding control spectrum for insect pests. In the present paper, we first found that S. exigua larval midgut fluids could remarkably degrade activated Cry1Ca, and slightly hydrolyze Cry1Ac and Cry1Ab. Subsequently, we investigated interactions between the 3 Cry toxins and 7 proteinase inhibitors, i.e., phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-α-tosyl-l-phenylalanine chloromethyl ketone, N-α-tosyl-l-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA), by monitoring larval growth and mortality rate. A 6-day dietary exposure of the newly molted 2nd instars to either the inhibitors (3 test concentrations) or the toxins (Cry1Ac, 31.30ng/cm2; Cry1Ab, 3.2ng/cm2; Cry1Ca, 0.6ng/cm2) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight. In general, the synergisms were more obvious at higher inhibitor concentrations. Regarding larval mortality, the inhibitors except EDTA at the highest test concentration showed significant synergism to both Cry1Ab (0.2μg/cm2) and Cry1Ca (0.04μg/cm2). As the inhibitor concentration reduced, however, the synergistic effects decreased. These results indicated that trypsin-, chymotrypsin-, and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the 3 activated Cry toxins, and protection of Bt Cry toxins from proteinase degradation in the midgut by inhibitors may greatly enhance toxicity against S. exigua larvae.

P5-03-03: Identification of New Substrates for Breast Tumor Specific Low-Molecular-Weight Cyclin E Cyclin-Dependent-Kinase 2.

Abstract Background: Cyclin E overexpression occurs in 25% of breast cancer tumors and is linked to poor prognosis. In tumor cells full length cyclin E (FL-E) is processed by an elastase-like protease into low-molecular weight isoforms (LMW-E) that are biochemically hyperactive. When overexpressed in breast cancer cells, LMW-E induced genomic instability and resistance to p21, p27, and antiestrogens. LMW-E are tumor specific and are associated with the aggressive triple negative breast cancer (TNBC). Transgenic mice overexpressing LMW-E had increased incidence of mammary tumors and distant metastasis when compared to FL-E. Furthermore, when the role of CDK2 in LMW-E mediated mammary tumorigenesis was interrogated by crossing LMW-E transgenic mice with CDK2 knock out mice, we found that the mice are protected against mammary tumor formation. Additionally, treatment of LMW-E transgenic mice with roscovitine (a CDK2 inhibitor) delays mammary tumor formation. These results indicate that kinase inhibition may have therapeutic activity in LMW-E overexpressing tumors as shown in this preclinical model. These results lead us to hypothesize that the biological and biochemical differences between FL-E and LMW-E may be due to the phosphorylation of a distinct set of substrates when complexed with CDK2. Our goal is to identify distinct LMW-E/CDK2 substrates on a proteome-wide scale that could serve as novel therapeutic targets for the treatment of the aggressive LMW-E expressing TNBC. Material and Methods: We used two different approaches to identify LMW-E/CDK2 substrates. 1) We generated an analog sensitive CDK2 kinase, (F80A or F80G)-CDK2 to specifically radiolabel its substrates in cell extracts followed by their identification by mass spectroscopy. 2) We incubated ProtoArrayMicroarrays spotted with 3000 GST-tagged human protein on high density glass slides either with recombinant active FL-E/CDK2 or LMW-E/CDK2. Results: In the first approach, we expressed and purified wild-type CDK2 in complex with FL-E or LMW-E and CDK2 (F80A) and CDK2 (F80G) from insect cells. Although all 3 CDK2 kinases can use ATP to phosphorylate GST-Rb protein, only the F80G mutant can use PE-ATP-γ-S. In the second approach, we incubated protein arrays either with recombinant active FL-E/CDK2 or LMW-E/CDK2 at a concentration of 50 nM. Our first screen identified a total of 122 potential substrates to both FL-E/CDK2 or LMW-E/CDK2 kinase complexes. We only identified 1 protein that is phosphorylated by FL-E/CDK2 significantly more than by LMW-E/CDK2 as compared to the 32 potential substrates specific to LMW-E/CDK2 suggesting that by losing the N-terminal portion, the LMW-E/CDK2 kinase complex is able to specifically interact and phosphorylate novel proteins. Discussion: The identification of new physiological LMW-E/CDK2 substrates will lead to the development of novel targets for therapeutics and the identification of the biological function for the treatment of the aggressive LMW-E expressing TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-03-03.

A new factor in the Aedes aegypti immune response: CLSP2 modulates melanization

Microbial infections in the mosquito Aedes aegypti activate the newly identified CLSP1 and CLSP2 genes, which encode modular proteins composed of elastase-like serine protease and C-type lectin domains. These genes are predominantly regulated by the immune deficiency pathway, but also by the Toll pathway. Silencing of CLSP2, but not CLSP1, results in the activation of prophenoloxidase (PPO), the terminal enzyme in the melanization cascade, suggesting that CLSP2 is a negative modulator of this reaction. Haemolymph PPO activation is normally inhibited in the presence of Plasmodium parasites, but in CLSP2-depleted mosquitoes, the Plasmodium-induced block of melanization is reverted, and these mosquitoes are refractory to the parasite. Thus, CLSP2 is a new component of the mosquito immune response.

Comparison of the nucleotide sequences of elastase-like serine protease genes from Metarhizium anisopliae

Comparison of the nucleotide sequences of elastase-like serine protease genes from Metarhizium anisopliae

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

BackgroundThe serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPα, EspPβ, EspPγ, and EspPδ), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown.Methodology/Principal FindingsThe structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the β-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His127, Asp156 and Ser263 in Domain 1 form the catalytic triad of EspP.Conclusions/SignificanceOur data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPβ and EspPδ.

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage

A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5′ cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27–35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two β-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.

Targeting the cell cycle for prognosis and therapy of breast cancer

Cyclin E is a G1-cyclin that plays a key role in the G1 to S transition of the cell cycle. Cyclin E is processed in tumor cells by an elastase-like protease into low-molecular-weight (LMW) isoforms that are biochemically hyperactive. The LMW isoforms of cyclin E are unique to cancer cells. In breast cancer, such alteration of cyclin E is a very strong predictor of poor patient outcome. Alterations in the binding properties of these LMW isoforms to CDK2 and the CDK inhibitors (CKIs), p21 and p27, result in their functional hyperactivity. The LMW forms of cyclin E are several-fold more effective at binding to CDK2. Additionally, compared with the full-length cyclin E–CDK2 complexes, the LMW cyclin E–CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the CKIs to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity. When overexpressed in breast cancer cells, the LMW forms of cyclin E, but not the full-length form, result in their hyperactivity due to increased affinity for cdk2 and resistance to inhibition by the CDK inhibitors p21 and p27, result in resistance to the growth inhibiting effects of anti-estrogens, and result in chromosomal instability. Finally, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. To assess the oncogenic role of cyclin E-LMW as compared with full-length cyclin E, we examined the consequences of overexpressing these isoforms in the mammary glands of transgenic mice using the MMTV promoter. Four constructs were generated: MMTV-M46A coding for the full-length cyclin E (EL1), MMTV-EL1/EL4 coding for EL1 and the isoform translated at methionine 46 (EL4), and MMTV-T1 and MMTV-T2 coding for the isoforms generated by elastase cleavage at the first site (EL2 + EL3) and at the second site (EL5 and EL6), respectively. For each construct at least two transgenic lines were established. Transgene expression was demonstrated by RT-PCR, northern blotting and western blotting. Overexpression of cyclin E was seen in more than 90% of ductal and lobular cells of the mammary glands for each independent line. Mammary-specific LMW cyclin E overexpression induced extensive abnormalities at 2 months, including perturbed architecture, polyploidy, anysocytosis and apoptosis. Whole-mount preparations of mammary glands at different development stages showed that overexpression of EL1/EL4 and cyclin E-T1 induced growth delay, while at 6 months of age an increased proportion of cells in the S phase was found (25.6 ± 5.6% for EL1/EL4, 9.0 ± 2.7% for T1 compared with 3.9 ± 1.9% for non-transgenic animals). We observed a 34% (13/38) incidence of mammary adenocarcinomas in the EL1/EL4 transgenic lines with a mean latency of 18.3 months, and observed a 20% (5/25) incidence in the T1 transgenic lines with a mean latency of 17.1 months. The tumor incidence rate of the other transgenic lines, M46A and T2, are still unknown due to the young age of the mice (all under 7 months of age) and the long latency of cyclin E-mediated tumor generation. Thirty percent (4/13) of the EL1/EL4 and 40% (2/5) of the T1 tumor-bearing animals developed lung metastasis. The tumors induced by the EL1/EL4 and T1 transgenes were mainly solid adenocarcinomas with very little differential to glandular for EL1/EL4 and mostly glandular for T1. Since p53 alterations are common in human breast carcinomas, we bred a T1 line with p53+/- mice. The T1 × p53+/- cross generated tumors that are much more malignant than the T1 tumors; the incidence increased to 100%, with a much shorter latency of 11 months. Biochemical analysis of the tumors revealed that 64% (9/14) retained cyclin E expression and that, on average, the cyclin E-overexpressing tumors had threefold higher cyclin E kinase activity than the non-cyclin E-expressing tumors. Taken together, these data indicate that tumor progression in cyclin E transgenic mice follow sequential steps of dysplasia, mammary intraepithelial neoplasia and invasive/metastatic tumors. Collectively, the biochemical and biological differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. The transgenic mouse model system can serve as a useful system in which to study the mechanisms responsible for LMW cyclin E-induced genetic instability and may help identify those factors that promote tumor progression and metastasis. The properties of the LMW forms of cyclin E suggest that they are not just surrogate markers of poor outcome, but that they are bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.

Activity of gut proteinases from Cry1Ab‐selected colonies of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae)

Susceptibility to the Cry1Ab protoxin and toxin from Bacillus thuringiensis (Berliner) and activity of gut proteinases were assessed in both susceptible and Cry1Ab-selected colonies of European corn borer, Ostrinia nubilalis (Hubner). Resistance in two different selected colonies was at least 6- and 15-fold for the Cry1Ab protoxin and 108- and 484-fold for the Cry1Ab toxin. Activities of trypsin-like, chymotrypsin-like and elastase-like proteinases were variable among the colonies tested and not indicative of a major contribution to Cry1Ab resistance. Activation of the 130-kDa Cry1Ab protoxin occurred rapidly in all colonies, with no apparent differences among colonies. In addition, there were no apparent changes in activated Cry1Ab processing, indicating that proteolytic degradation was not associated with resistance. These results suggest that mechanisms other than proteolytic activation of protoxin and toxin degradation, such as target site modification may be involved in the resistance to B thuringiensis Cry1Ab in these O nubilalis colonies.

Interplay between antibacterial effectors: a macrophage antimicrobial peptide impairs intracellular Salmonella replication.

Antimicrobial peptides have established an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated. Macrophage expression of the murine cathelicidin-related antimicrobial peptide (CRAMP) was increased after infection by the intracellular pathogen Salmonella typhimurium, and this increase required reactive oxygen intermediates. By using CRAMP-deficient mice or synthetic CRAMP peptide, we found that CRAMP impaired Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria. This impaired bacterial cell division also depended on intracellular elastase-like serine protease activity, which can proteolytically activate cathelicidins. Macrophage serine protease activity induced filamentation and enhanced the activity of CRAMP in vitro. A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment. Although cationic peptides have been hypothesized to have activity against pathogens within macrophages, this work provides experimental evidence that the antimicrobial arsenal of macrophages includes cathelicidins. These results show that intracellular reactive oxygen intermediates and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen, and they highlight the cooperativity between macrophage antibacterial effectors.

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.

Involvement of a serine protease, but not of neutrophil elastase, in tumor necrosis factor-induced lethal hepatitis and induction of platelet-activating factor

Background/Aims : Tumor necrosis factor (TNF) plays an essential role in several types of acute and chronic hepatitis. Our aims in the present study were to elucidate the mechanism by which TNF leads to acute lethal hepatitis, thereby focusing on the role of serine proteases and platelet-activating factor (PAF). Methods : All experiments were performed in a model of acute hepatitis, induced by TNF in combination with d-(+)-galactosamine (GalN). Neutrophil elastase (NE)-deficient mice, generated by gene targeting were used in the studies. Results : We found that a serine protease plays an essential mediating role in the in vivo TNF effect as α 1-antitrypsin ( α 1-AT), soybean trypsin inhibitor (STI) and turkey trypsin inhibitor (TTI), confer complete protection. α 1-AT and TTI, but not STI, reduce PAF blood levels, induced by TNF/GalN, which is compatible with an elastase-like serine protease involvement in PAF synthesis. In our search for relevant serine proteases we believed that NE was an excellent candidate protease. However, we found that TNF/GalN-induced lethality is not attenuated in mice deficient in NE. Conclusions : The data suggest that TNF-induced lethal hepatitis is accompanied by increases in circulating PAF and plasma clotting time, and mediated by a serine protease, but not by NE.

Cleavage of Human IgE Mediated by Schistosoma mansoni

Background: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. Methods: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcε receptors. Results: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cε2/Cε3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcεRII. Conclusions: An elastase-like protease in S. mansoni is able to render IgE non-functional.

Some characteristics of the proteinase—Inhibitor systems in adaptive response to strenuous exercise

In rats, adaptation to strenuous exercise was accompanied by phasic changes in the activities of the kallikrein-kinin system, elastase-like proteinases, and proteinase inhibitors, and total antioxidant activity in the serum, myocardium, liver, and cerebral cortex. After 30-min physical exercises, activity of the kallikrein-kinin system decreased in the serum and increased in tissue with parallel activation of elastase-like proteinases in the myocardium and cerebral cortex. After 3-h exercises the activity of the kallikrein-kinin system showed some indications of exhaustion, especially in the myocardium and cerebral cortex. Activities of elastase-like proteinases tended to normal due to activation of α1-proteinase inhibitor and normalization of total antioxidant activity.

Spruce budworm elastase precipitates Bacillus thuringiensis δ-endotoxin by specifically recognizing the C-terminal region

A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain δ-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75) 1 Abbreviations used: BAPNA, Nα-benzoyl-DL-arginine p-nitroanilide; BBMV, brush border membrane vesicles; CAPS, 3-(cyclohexylamino)-propanesulfonic acid; CBB, Coomassie brilliant blue R250; Cf, Choristoneura fumiferana; DFP, diisopropyl flurophosphate; DIP, diisopropyl phosphate; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; OSU, Ohio State University; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TOAD, toxin overlay agarose diffusion; TPP-75, 75-kDa toxin precipitating protein. 1 resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody–antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of δ-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.