Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.     

Synthesis of active site-directed organometallic irreversible protease inhibitors.

p-Antimonybenzenesulfonyl fluoride and p-mercurybenzenesulfonyl fluoride irreversibly inhibit chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), and chromosomal protease, and these inhibitors appear to be as active as phenylmethanesulfonyl fluoride. The pretreatment of the proteases interferes with the phosphorylation of the active-site serine by diisopropylfluorophosphate suggesting that the organometallic inhibitors may also interact with the active site serine. The organometallic inhibitors may be used for localization of proteases in different parts of the cell by electron microscopy and p-mercurybenzenesulfonyl fluoride could also be used for isolation of proteases by sulfhydryl affinity chromatography.     

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.     

Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations approximately 10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

Evidence for involvement of serine proteases in the late stages of the natural killer cell lytic reaction

An irreversible inhibitor (L-1-tosylamide-2-phenylethyl-chloromethylketone) and substrate (N-acetyl-L-tyrosineethylester) of the neutral serine protease chymotrypsin were evaluated for their effects on the natural killer cell lytic reaction sequence. During direct cell-mediated cytolysis these inhibitors had no effect on natural killer cell binding to target cells but were able to inhibit the "trigger" mechanism which initiates killing. In addition, they inhibited later calcium-dependent events in the lytic reaction and killer cell-independent lysis. These findings suggest that serine proteases may be required during several stages of natural killer cell lysis, including calcium-dependent programming as well as the actual lethal hit.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations In Vitro

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.     

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.     

Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators

Plant serine protease inhibitors are defense proteins crafted by nature for inhibiting serine proteases. Use of eco-friendly, sustainable and effective protein molecules which could halt or slow down metabolism of nutrients in pest would be a pragmatic approach in insect pest management of crops. The host-pest complexes that we observe in nature are evolutionary dynamic and inter-depend on other defense mechanisms and interactions of other pests or more generally speaking symbionts with the same host. Insects have co-evolved and adapted simultaneously, which makes it necessary to investigate serine protease inhibitors in non-host plants. Such novel serine protease inhibitors are versatile candidates with vast potential to overcome the host inhibitor-insensitive proteases. In a nutshell exploring and crafting plant serine proteinase inhibitors (PIs) for controlling pests effectively must go on. Non-host PI seems to be a better choice for coevolved insensitive proteases. Transgenic plants expressing wound inducible chimaeric PIs may be an outstanding approach to check wide spectrum of gut proteinases and overcome the phenomenon of resistance development. Thus, this article focuses on an entire array of plant serine protease inhibitors that have been explored in the past decade, their mode of action and biological implications as well as applications.     

Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.     

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous β-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of β-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with β-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous β-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Tumor-associated protein SPIK/TATI suppresses serine protease dependent cell apoptosis

Serine protease dependent cell apoptosis (SPDCA) is a recently described caspase independent innate apoptotic pathway. It differs from the traditional caspase dependent apoptotic pathway in that serine proteases, not caspases, are critical to the apoptotic process. The mechanism of SPDCA is still unclear and further investigation is needed to determine any role it may play in maintaining cellular homeostasis and development of disease. The current knowledge about this pathway is limited only to the inhibitory effects of some serine protease inhibitors. Synthetic agents such as pefabloc, AEBSF and TPCK can inhibit this apoptotic process in cultured cells. There is little known, however, about biologically active agents available in the cell which can inhibit SPDCA. Here, we show that over-expression of a cellular protein called serine protease inhibitor Kazal (SPIK/TATI/PSTI) results in a significant decrease in cell susceptibility to SPDCA, suggesting that SPIK is an apoptosis inhibitor suppressing this pathway of apoptosis. Previous work has associated SPIK and cancer development, indicating that this finding will help to open the doorway for further study on the mechanism of SPDCA and the role it may play in cancer development.     

Serine protease inhibitors block N-terminal arginylation of proteins by inhibiting the arginylation of tRNA in rat brains

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10^–5M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

Characterisation of proteases involved in egg hatching of the sheep blowfly, Lucilia cuprina

A number of proteases were identified in the egg shell washings (ESW) collected during the egg hatching of Lucilia cuprina (sheep blowfly). Characterization of these proteases indicated a pH optima in a similar pH range that was optimal for L. cuprina egg hatching. Mechanistic characterization of these proteases indicated that they were predominantly of the serine class. Several protease inhibitors were tested for their ability to inhibit L. cuprina egg hatching in vitro. Egg hatching was significantly (P<0.05) inhibited by PMSF (61%), 1,10-Phenanthroline (42%) and Pepstatin (29%). The inhibition of egg hatching by PMSF showed a strong concentration dependence, with its effects ranging from inhibition at high concentrations to enhancement of egg hatching at low concentrations. Addition of ESW to unhatched eggs, significantly (P<0.05) enhanced their rate of hatching above untreated control eggs. This enhancement of egg hatching was significantly (P<0.05) reversed by the protease inhibitors Elastatinal (40%), 1,10-Phenanthroline (40%) and PMSF (38%). These studies indicate a role for serine and/or metallo-proteases in facilitating L. cuprina egg hatch.