Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.     

Proteinases and proteinase inhibitors as modulators of animal cell growth.

1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.     

The basic difference in catalyses by serine and cysteine proteinases resides in charge stabilization in the transition state

Besides the mechanistic similarities, in particular acylenzyme formation, kinetic investigations and X-ray diffraction studies have revealed some differences between the mechanisms of serine and cysteine proteinases: general base-catalysis in acylation, catalytic contribution by oxyanion binding, and a negatively charged catalytic triad in serine proteinases, but not in cysteine proteinases. In this paper we point out that all these differences are related and connected with the mode of stabilization of the zwitterionic species developing in the transition state of the reactions. In the case of serine proteinases this charge separation requires facilitation by the oxyanion binding and the negative charge of the catalytic triad. On the other hand cysteine proteinases do not require such contributions as they are capable of stabilizing the ion-pair even in the ground state of the reaction. Therefore, cysteine proteinases, in contrast to serine proteinases, may be regarded as "activated" enzymes.     

Proteinases of Streptomyces fradiae. II. Specificity.

It has been shown that extracellular proteinases synthesized by a keratinolytic strain of S. fradiae are characterized by diversified activity in the decomposition of both proteins and synthetic substrates. Among the six proteinases isolated, apart from the ones dominating and having relatively low specificity, there are two enzymes characterized by narrow catalytic abilities--extremely similar to those of trypsin. These proteinases intensively degraded all the trypsin substrates studied, but they were inactive or showed slight activity toward others. They were also highly sensitive to such specific inhibitors of trypsin as TLCK, SBTI and TIO.     

Proteinase inhibitory activity inPenicillium urticae

Intracellular proteinase activity in cell extracts ofPenicillium urticae increased twofold during storage. Three proteinase fractions showed different sensitivities to an inhibitory component which was present in the original crude mycelial extract. The inhibitory component proved to be nondialyzable and heat stable, but lost activity during storage of the cell extracts, probably as a result of proteolytic digestion. Following electrophoresis of the two most stable proteinase fractions, 18 individual proteinases were separated. All but 2 of these enzymes were shown to have undergone activation during the storage period.     

Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.     

Appearance of Endoproteolytic Enzymes during the Germination of Barley

Barley endoproteolytic enzymes are important to germination because they hydrolyze endosperm storage proteins to provide precursors for new protein synthesis. We recently developed an electrophoretic method utilizing gel-incorporated protein substrates to study the endoproteinases of 4-d-germinated barley (Hordeum vulgare L. cv Morex) grain. This work extends those findings to determine the temporal pattern of the appearance of the endoproteinases during germination, the sensitivities of the proteinases to class-specific proteinase inhibitors, and where, in germinating caryopses, the proteinases reside. Six endoproteinase activity bands (representing a minimum of seven enzymes) were present in 5-d-germinated barley grain extracts subjected to electrophoresis in nondenaturing gels at pH 8.8. The activities of two of the enzyme bands (“neutral” proteinases) increased as the pH was increased from 3.8 to 6.5. The activities of the remaining four (“acidic”) bands diminished abruptly as the pH increased above 4.7. Two proteinase bands hydrolyzed gelatin but not edestin, four of the proteinases hydrolyzed both gelatin and edestin at nearly the same rates, and one enzyme degraded only edestin. One neutral endoproteinase was sensitive to diisopropyl fluorophosphate inhibition, and the other was not inhibited by any of inhibitors tested. Four of acidic enzymes were cysteine proteinases [inhibited by trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and N-ethylmaleimide]; the other was an aspartic acid endoproteinase (sensitive to pepstatin). Only the aspartic proteinase was detected in either ungerminated or steeped barley grain. During the germination (malting) process, the aspartic endoproteinase activity decreased until the second day of germination and then increased until germination day 5. The first endoproteinase(s) induced during germination was a neutral enzyme that showed activity on the 1st day of the germination phase after steeping. Most of the endoproteinases became active on the 2nd or 3rd germination day, but one cysteine proteinase was not detected until the 5th day. Acid cysteine proteinases were present in the aleurone, scutellum, and endosperm tissues but not in shoots and roots. The aleurone layer and endosperm contained almost exclusively band B1 neutral proteinases, whereas the scutellum, shoots, and roots contained both B1 and B2 bands. This work shows that germinating barley contains a complex set of proteinases whose expression is temporally and spatially controlled. But, at the same time, it also shows that this electrophoretic method for separating and studying individual enzymes of this complex will allow us to more readily characterize and purify them.     

Storage and mobilization as antagonistic functional constraints on seed storage globulin evolution

When seeds germinate nearly all the proteins are degraded in senescing storage tissue cells. All these proteins act as amino acid reserves which are mobilized to nourish the seedling. Nevertheless, the major amount of the seeds' protein reserve consists of a few enzymatically inactive, abundant, genuine storage proteins. In their metabolism the conflicting processes of biosynthesis, protein turnover and breakdown, are temporally separated. No degradation of correctly formed storage proteins was observed at the time of synthesis and accumulation during seed maturation. Breakdown takes place after a (long) period of rest when seeds germinate and seedlings start growing. At that time genuine storage proteins are no longer synthesized. Genuine storage proteins have evolved structural features permitting controlled temporal patterns of protection and proteolysis. The acquisition of inserted sequence stretches as sites accessible to limited proteolysis played a key role in the evolution of this control system and happened in coevolution of genuine storage proteins with specific proteinases. This can be deduced from the results of current research on the mechanisms of limited and unlimited proteolysis of storage globulins and on storage globulin evolution. The evolved system of controlled structure-function interplay between storage globulins and proteinases is part of a syndrome that, in addition, comprises differential compartmentation and gene expression of storage proteins and proteinases for controlling the total spatial and temporal patterns of globulin storage and mobilization in maturing and germinating seeds.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.     

Biosynthesis of lysosomal proteinases in health and disease

Proteolytic maturation of lysosomal proteinases is initiated after receptor-mediated targeting to prelysosomal compartments, while terminal processing occurs upon delivery to lysosomes. These late processing events are impaired in patients suffering from inherited lysosomal disorders, such as sialic acid storage disease and mucolipidosis II (I-cell disease). Lysosomes in the affected cells display marked changes in their physiological and morphological properties, with features reminiscent of prelysosomal compartments. This indicates that the absence of mature lysosomes interferes with the final processing steps during the biosynthesis of lysosomal proteinases. Thus, impaired proteinase maturation reflects an incompetent lysosomal apparatus and as such can be seen as a hallmark of lysosomal storage diseases.     

ATP-dependent proteinases in bacteria

Cytoplasmic proteolysis is an indispensable process for proper function of a cell. Degradation of many intracellular proteins is initiated by ATP-dependent proteinases, which are involved in the regulation of the level of proteins with short half-lives. In addition, they remove many damaged and abnormal proteins and thus play also an important role during stress. ATP-dependent proteinases are large multi-subunit assemblies composed of proteolytic core domains and ATPase-containing regulatory domains on a single polypeptide chain or on distinct subunits, which can act as molecular chaperones. This review briefly summarizes the data about four main groups of these proteinases in bacteria (i.e. Lon, Clp family, HsIUV and FtsH) and characrizes their structure, mechanism of action and properties.     

Characterization of neutral proteinases from human spleen]

Several proteinases hydrolyzing histone and caseine in neutral media were obtained by Sephadex G-100 fractionation of water and salt (1 M KCl) extracts of human spleen. The level of the activity of proteinases in the extracts was very low as a result of the presence of an inhibitor. Neutral proteinases were found in two protein fractions. The "high-molecular-weight-" proteinases were inhibited by DFP and therefore they were attributed to a group of serine proteinases. The "low-molecular-weight" fraction contained neutral SH-dependent proteinase(s) and DFP-inhibited enzymes. In this fraction, the kininogenase activity and the hydrolysis of Boc-1-ananine p-nitrophenyl ester, N-benzoyl-L-tryosine ethyl ester and N-benzoyl-DL-arginine p-nitroanilide were observed.     

Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.     

Detection of a proteinase inhibitor in Aspergillus nidulans

The activity of proteinases in mycelial extracts of Aspergillus nidulans increased during storage. The rate of activation increased with temperature. Three separate proteinase activities, differing in their electrophoretic mobilities on polyacrylamide gels, were readily detected at pH 6.5. Inhibitory activity, effective against all three proteinase activities, was also detected in fractions prepared from fresh mycelial extracts. The inhibitory factor(s) were heat-stable and non-dialysable. The inhibitory activity was lost during storage of mycelial extracts. It is proposed that the inhibitory factor(s) are digested by the proteinases during storage.     

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

The potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.     

Detection of cathepsin B, plasminogen activators and plasminogen activator inhibitor in human non-small lung cancer cell lines

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.     

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.