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At least 253 records · Page 14Linked to original sources
PubMed2000

Novel functions of human alpha(1)-protease inhibitor after S-nitrosylation: inhibition of cysteine protease and antibacterial activity.

alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)). We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase. S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity. Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione. NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap. Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase. As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated.

Animals↗
PubMed1991

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis.

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca2+ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca2+ ions and gramicidin S; its pH and temperature optima were 9.0 and 37 degrees C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.

Bacillus subtilis↗
PubMed1978

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Amino Acids↗
PubMed2004

Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.

Amino Acid Sequence↗
PubMed1982

Effects of proteolytic enzymes and monovalent ions demonstrate protease-sensitive and protease-insensitive stereospecific binding sites on dopaminergic receptors in rat striatum.

Rat striatal membranes from different subcellular fractions were treated with various proteolytic and other enzymes and the binding of a dopamine agonist ([3H]NPA) and of an antagonist ([3H]haloperidol) was assayed in several conditions. In membranes of striatal microsomal and mitochondrial fractions, stereospecific binding of both [3H]NPA and [3H]haloperidol assayed in a monovalent ion-poor buffer was potently and rapidly inhibited by trypsin and certain related proteases. The enzymes did not affect the binding of the ligands when assayed in a buffer containing monovalent ions (greater than or equal to 40 mM NaCl or KCl or a physiological mixture of electrolytes). The inhibition, seen in the monovalent ion-poor buffer, was dependent on the enzyme concentration. The endoproteases (trypsin, alpha-chymotrypsin, papain, ficin) showed nanomolar IC50-values for inhibition of both [3H]NPA and [3H]haloperidol binding. The inhibition occurred very rapidly at 0 degree and was different from the slow proteolytic inactivation seen by prolonged incubation at 37 degrees. It was demonstrated that monovalent ions did not themselves interfere with the interaction between the proteases and the membranes. The observations provide evidence for two different types of stereospecific dopaminergic binding sites which are differentially exposed for ligand binding depending on the concentration of monovalent ions. There sites are protease-sensitive sites, labelled in monovalent ion-poor media and protease-insensitive sites, labelled in media with higher concentrations of monovalent ions. Both types of binding sites bind dopamine agonists and dopamine antagonists with high affinity, but some differences were noted in the binding properties and the drug binding selectivity of the sites. It is argued that both sites form part of the same dopamine receptor macromolecular complex. The findings corroborate the hypothesis that dopamine receptors are composed of different sub-unit binding sites, but these are not distinct agonist and antagonist specific sites. The mechanism by which the protease-sensitive sites are rapidly inactivated by particular proteases, is probably a complexation between the enzymes and certain essential peptide moieties of the receptor sites involved.

Animals↗
PubMed1992

Regulation of neuronal migration and neuritogenesis by distinct surface proteases. Relative contribution of plasmin and a thrombin-like protease.

The relative contribution of two neuronal surface proteases, plasmin and a protease with thrombin-like specificity, on NB2a/dl neuroblastoma migration and neuritogenesis were examined. Exogenous plasmin induced cell body rounding and increased cell migration, but did not prevent or reverse neurite outgrowth. Inhibition of endogenous plasmin by its specific inhibitor, aprotinin, suppressed migration but did not induce neuritogenesis. Removal or inhibition of the thrombin-like protease by serum deprivation or hirudin addition, respectively, induced neurite outgrowth, as shown in our previous studies, but did not suppress migration. By contrast, trypsin induced simultaneous cell rounding and neurite retraction. These findings indicated that plasmin may regulate cell migration, while the thrombin-like protease may regulate facets of neurite outgrowth. Although unable to induce de novo neuritogenesis, plasmin inhibition potentiated the otherwise transient neurites induced by simultaneous inhibition of the thrombin-like protease. Since cultured neuronal cells migrate primarily in the direction of newly elaborated neurites, this finding is interpreted to indicate that cessation of neuronal migration by plasmin inhibition enhances net neurite outgrowth by inhibition of the putative thrombin-like protease.

Cell Adhesion↗
PubMed1988

The Ca2+-dependent protease inhibitor of rat ventral prostate: properties of the inhibitor and effects of castration on Ca2+-dependent protease and inhibitor activities.

1. The rat ventral prostate contains a heat stable inhibitor of Ca2+-dependent protease. This inhibitor was found to exist in a wide range of molecular weights (approx. 40-270 kDa) in adult rats. 2. However, in rats immediately post puberty (45 days of age) the inhibitor was predominantly of the higher molecular weight forms. 3. The inhibitor was also found in the dorsolateral and anterior (coagulating gland) prostate lobes but was of lower specific activity than in the ventral lobe. 4. Although the activities of the Ca2+-dependent protease and inhibitor decreased per ventral prostate gland after castration, these activities were not different during the first 10 days postcastration when expressed per g wet wt or per unit cytosol protein. 5. With a longer duration of castration, there was a decline in the specific activity (per unit protein) of the protease and an increase in that of the inhibitor. 6. Thus, the activities of the protease and inhibitor change in concert with the amount of cellular cytosol protein during the active period of castration-induced atrophy. 7. However, in long term castrated rats, functions carried out by the Ca2+-dependent protease may be effectively suppressed. 8. These data suggest that the Ca2+-activated protease probably is involved in the regulation of some metabolic processes in the active gland and is not prominent in the castration induced atrophy of the ventral prostate unless it functions through the proteolysis of some select protein(s).

Animals↗
PubMed2005

Suppression of macrophage responses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease inhibitor (SLPI) is independent of its anti-protease function.

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.

Amino Acid Substitution↗
PubMed2006

A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus.

A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K(i) with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.

Amino Acid Sequence↗
PubMed2003

Cockroach proteases increase IL-8 expression in human bronchial epithelial cells via activation of protease-activated receptor (PAR)-2 and extracellular-signal-regulated kinase.

BACKGROUND: We have shown that serine proteases in German cockroach extract increase TNF-alpha-induced expression of IL-8 in human bronchial epithelial cells. The mechanism by which cockroach proteases regulate cytokine expression is unknown; however, protease-activated receptors (PARs) might play a role. OBJECTIVE: We sought to determine the role of PARs and extracellular-signal-regulated kinase (ERK) in cockroach-induced regulation of IL-8 expression. METHODS: 16HBE14o- human bronchial epithelial cells were treated with the specific PAR-1 and PAR-2 agonists, TFRIFD and SLIGKV, respectively. IL-8 transcription was assessed by transiently transfecting cells with a luciferase-tagged IL-8 promoter construct, and in some cases, dominant-negative expression vectors. To block PAR cleavage, antibodies against the cleavage region of PAR-1 and PAR-2 were used. ERK phosphorylation was determined by Western blot. RESULTS: Although both PAR-1 and PAR-2 were endogenously expressed in 16HBE14o- cells, selective activation of PAR-2 but not PAR-1 mimicked the effect of cockroach extract on IL-8 expression. Using a blocking antibody against cleavage of PAR-2 but not PAR-1 attenuated cockroach-extract-induced responses, suggesting that cockroach proteases cleave PAR-2. Treatment of cells with cockroach extract and SLIGKV each increased phosphorylation of ERK. Chemical or genetic inhibition of Ras and mitogen-activated protein kinase/ERK (MEK), upstream activators of ERK, each attenuated cockroach- and PAR-2-induced IL-8 transcription. CONCLUSION: Cockroach proteases and PAR-2 activation synergistically increase TNF-alpha-induced IL-8 transcription via activation of ERK. These data suggest an important role for PAR-2 and ERK activation in the regulation of cytokine expression in airway epithelium in response to cockroach proteases.

Allergens↗
PubMed2002

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR).

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70 degrees C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes.

DNA, Archaeal↗
PubMed1989

Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation.

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.

Amino Acid Sequence↗
PubMed2003

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family.

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.

Amino Acid Sequence↗
PubMedDate not supplied

Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?

Calcium-dependent protease (calcium protease) is apparently involved in a variety of cellular processes. Here we have attempted to clarify the role and regulatory mechanism of calcium protease by analysing its structure. The complete primary structure of calcium protease (relative molecular mass (Mr) 80,000 (80K), 705 amino acids) was deduced from the nucleotide sequence of cloned complementary DNA. The protein contains four distinct domains, and we have observed a marked similarity between the second and fourth domains and the papain-like thiol proteases and calmodulin-like calcium-binding proteins, respectively. This finding suggests that calcium protease arose from the fusion of genes for proteins of completely different function and evolutionary origin. Further, it provides functional insight into cellular regulatory mechanisms mediated by Ca2+ through calcium-binding proteins.

Amino Acid Sequence↗
PubMed1997

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases.

The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1999

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.

Alzheimer Disease↗
PubMed2006

Protease-activated receptors: how proteases signal to cells to cause inflammation and pain.

Certain serine proteases that originate from the circulation (coagulation factors), inflammatory cells (mast cell tryptase, neutrophil granzyme A, and proteinase 3), and epithelial and neuronal tissues (trypsins) can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs on multiple cell types to reveal tethered ligand domains that bind to and activate the cleaved receptors. The proteases that activate PARs are often generated and secreted during injury and inflammation, and PARs orchestrate tissue responses to these insults, including hemostasis, inflammation, nociception, and repair mechanisms. Agonists of PARs, notably PAR2, induce inflammation in many tissues that is characterized by hyperemia, extravasation of plasma proteins, granulocyte infiltration, and alterations in epithelial permeability. These effects are mediated in part by the release of neuropeptides substance P and calcitonin gene-related peptide from sensory nerve fibers in peripheral tissues. Proteases that activate PAR2 also induce the release of neuropeptides from the central projections of these nerves in the dorsal horn of the spinal cord, where they participate in pain transmission. Accumulating evidence from PAR-deficient mice indicates that these mechanisms may contribute to experimental models of disease and raise the possibility that protease inhibitors and PAR antagonists may be useful therapies for a variety of inflammatory and painful conditions.

Animals↗
PubMed1992

A growth-regulated protease activity that is inhibited by the anticarcinogenic Bowman-Birk protease inhibitor.

The Bowman-Birk protease inhibitor (BBI) has been shown to be an effective suppressor of carcinogenesis in vivo and in vitro. To elucidate the mechanism(s) by which BBI suppresses carcinogenesis, we believe it will be necessary to identify and characterize the target enzymes that specifically interact with the BBI. We have shown previously that several cellular proteins in C3H/10T1/2 mouse embryo fibroblast cells specifically bind to a BBI affinity resin. In the current report, we demonstrate that one of these proteins has proteolytic activity as judged by its ability to degrade gelatin. The enzyme has a mass of 45 kDa and subcellular fractionation experiments demonstrate that this enzyme is located in the cytosol. Furthermore, the proteolytic activity was inhibited by diisopropylfluorophosphate but was not affected by EDTA, indicating that this enzyme is a serine protease. Higher levels of protease activity were found in logarithmic-phase C3H/10T1/2 cells compared with nondividing (confluent) cells, suggesting that this protease activity is growth regulated. Similar levels of this activity were present in nontransformed and in radiation-transformed C3H/10T1/2 cells. Treatment of nontransformed C3H/10T1/2 cells with phorbol 12-myristate 13-acetate increased the specific activity of this protease 5- to 10-fold. Our results suggest that this protease is a target enzyme of the BBI in these cells.

Animals↗