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PubMed2006

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Amino Acid Sequence↗
PubMed2006

Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1.

OBJECTIVE: Thrombin is generated in significant quantities during cardiopulmonary bypass and mediates adverse events, such as platelet aggregation and proinflammatory responses, through activation of the high-affinity thrombin receptor protease-activated receptor 1, which is expressed on platelets and endothelium. Thus antagonism of protease-activated receptor 1 might have broad therapeutic significance. Aprotinin, used clinically to reduce transfusion requirements and the inflammatory response to bypass, has been shown to inhibit protease-activated receptor 1 on platelets in vitro and in vivo. Here we have examined whether aprotinin inhibits endothelial protease-activated receptor 1 activation and resulting proinflammatory responses induced by thrombin. METHODS: Protease-activated receptor 1 expression and function were examined in cultured human umbilical vein endothelial cells after treatment with alpha-thrombin at 0.02 to 0.15 U/mL in the presence or absence of aprotinin (200-1600 kallikrein inhibitory units/mL). Protease-activated receptor 1 activation was assessed by using an antibody, SPAN-12, which detects only the unactivated receptor, and thrombin-mediated calcium fluxes. Other thrombin-dependent inflammatory pathways investigated were phosphorylation of the p42/44 mitogen-activated protein kinase, upregulation of the early growth response 1 transcription factor, and production of the proinflammatory cytokine interleukin 6. RESULTS: Pretreatment of cultured endothelial cells with aprotinin significantly spared protease-activated receptor 1 receptor cleavage (P < .0001) and abrogated calcium fluxes caused by thrombin. Aprotinin inhibited intracellular signaling through p42/44 mitogen-activated protein kinase (P < .05) and early growth response 1 transcription factor (P < .05), as well as interleukin 6 secretion caused by thrombin (P < .005). CONCLUSIONS: This study demonstrates that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular basis to help explain the anti-inflammatory properties of aprotinin reported clinically.

Aprotinin↗
PubMed2005

Differential expression of protease M/neurosin in oligodendrocytes and their progenitors in an animal model of multiple sclerosis.

To determine the possible involvement of protease M/neurosin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model of multiple sclerosis (MS). In situ hybridization, immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) demonstrated that EAE caused an increase in the expression of protease M/neurosin mRNA and its protein product throughout the white and gray matter surrounding demyelinating lesions. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing protease M/neurosin mRNA within control spinal cord showed immunoreactivity for CNPase or NG2, cell-specific markers for oligodendrocytes and their progenitors, respectively. In the spinal cord from mice with EAE, the expression of protease M/neurosin mRNA in CNPase-positive cells appeared to be increased while double-labeled cells positive for protease M/neurosin mRNA and NG2 were rarely found in areas associated with demyelinating lesions. Although a prominent accumulation of inflammatory cells including T-cells was observed in the vicinity of demyelinated lesions, these cells were not associated with protease M/neurosin mRNA expression. The levels of protease M/neurosin mRNA expression were unchanged in the spleen and even decreased in the thymus during the course of EAE. These observations suggest that the differential expression of protease M/neurosin in mature oligodendrocytes and their progenitors is involved in the pathogenesis of MS and EAE.

Animals↗
PubMed2004

Polyprotein processing: cis and trans proteolytic activities of Sesbania mosaic virus serine protease.

Sesbania mosaic virus (SeMV) polyprotein was shown to undergo proteolytic processing when expressed in E. coli. Mutational analysis of the proposed catalytic triad residues (H181, D216, and S284) present in the N-terminal serine protease domain of the polyprotein showed that the protease was indeed responsible for this processing. Analysis of the cleavage site mutants confirmed the cleavage between protease-viral protein genome linked (VPg) and VPg-RNA-dependent RNA polymerase (RdRP) at E(325)-T(326) and E(402)-T(403) sites, respectively. An additional suboptimal cleavage at E(498)-S(499) site was also identified which resulted in the further processing of RdRP to 10- and 52-kDa proteins. Thus, the protease has both E-T and E-S specificities. The polyprotein has a domain arrangement of protease-VPg-p10-RdRP, which is cleaved by the protease. The purified serine protease was also active in trans and cleaved the polyprotein at the same specific sites. These results demonstrate that the serine protease domain is responsible for the processing of SeMV polyprotein both in cis and in trans.

Escherichia coli↗
PubMed2005

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket.

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.

Amino Acid Substitution↗
PubMed1998

Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes.

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.

Amino Acid Sequence↗
PubMed2001

Promoted expression of mast cell-specific proteases in IgE-dependent passive cutaneous anaphylaxis responses.

BACKGROUND: Various factors can influence the protease expression phenotype of mast cells. METHODS: In an effort to understand the potential role of the mast cell proteases in the IgE-dependent passive cutaneous anaphylaxis (PCA) responses of murine tissues, we studied the changes of proteases expression. The expressions of proteases were examined by Northern blotting and immunohistochemistry. RESULTS: Promoted expression phenotypes of mouse mast cell protease (mMCP)-4, and rat mast cell protease I were accompanied by initiation of anti-dinitrophenyl (DNP) IgE-induced PCA responses, suggesting that the induction of these proteases expression are associated with IgE-mediated anaphylaxis responses. Elevated level of the L-histidine decarboxylase (HDC) mRNA expression was also observed in the PCA tissues and the activated mast cells, compared with that of the corresponding control tissue and cells, due to the activation of mast cells. CONCLUSIONS: Promoted protease expression phenotype appears to be linked with the induction of HDC expression.

Animals↗
PubMed2000

Unraveling the role of proteases in cancer.

Investigators have been studying the expression and activity of proteases in the final steps of tumor progression, invasion and metastasis, for the past 30 years. Recent studies, however, indicate that proteases are involved earlier in progression, e.g., in tumor growth both at the primary and metastatic sites. Extracellular proteases may co-operatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. In this review, we use cathepsin B as an example to examine the involvement of proteases in tumor progression and metastasis. We discuss the effect of interactions among tumor cells, stromal cells, and the extracellular matrix on the regulation of protease expression. Further elucidation of the role of proteases in cancer will allow us to design more effective inhibitors and novel protease-based drugs for clinical use.

Endopeptidases↗
PubMed2001

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease.

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.

Calcium↗
PubMed2003

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera).

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.

Animals↗
PubMed1983

The SOS regulatory system: control of its state by the level of RecA protease.

Our current understanding of the SOS regulatory system suggests that it can exist in two extreme states: in the repressed state, LexA protein is active, and it represses a particular set of genes called SOS genes. In the induced state, which results from various impairments to DNA replication, LexA repressor is cleaved by the specific protease activity of the RecA protein; in consequence, the SOS genes are derepressed and they express various functions that are believed to aid cell survival in induced cells. Since high levels of RecA protease activity turn on this system, it seems plausible that the level of protease activity will also control the transitions between the two states of the system. In order to assess the in vivo level of protease activity, antibody techniques were used to study the stability of LexA repressor during various phases of the SOS regulatory cycle. Repressor was reasonably stable in the repressed state, but it was degraded within a few minutes after an inducing treatment. Cleavage depended upon the RecA protease activity and resulted in the same products as seen in vitro. Cleavage preceded, and did not depend upon, derepression of any SOS gene. During the transition to the repressed state, LexA repressor became increasingly stable with time, suggesting that as DNA damage was repaired the level of protease declined. This decline depended upon derepression of the regulatory system, consistent with the belief that an inducing signal, resulting from DNA damage, reversibly activates the RecA protease and is removed by the action of one or more SOS functions. At low levels of DNA damage, a subinduced state was observed in which repressor level was reduced by a low level of cleavage. These data indicate that the level of RecA protease activity controls the state of the system and the transitions between its two states.

Bacterial Proteins↗
PubMed2002

Proteases involved in long-term potentiation.

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.

Animals↗
PubMed1998

Distinct interleukin-1beta-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells.

Interleukin-1beta converting enzyme (ICE) family proteases (caspases) are known to be implicated as important effectors of apoptotic pathways. The purpose of this study was to elucidate the role of ICE family proteases in apoptosis of mouse cells derived from the terminal proximal tubule (S3) treated with cisplatin, an anti-tumor drug, or staurosporine, a protein kinase C inhibitor. For this purpose, we measured the activities of ICE family proteases and examined the effects of tetrapeptide and viral ICE family protease inhibitors on the activities of ICE family proteases in and the degree of apoptosis of S3 cells treated with cisplatin and staurosporine. RT-PCR analysis revealed that S3 cells as well as mouse kidney express mRNA for ICE and CPP32, an ICE family protease. Results of enzymatic analysis, determination the degree of DNA fragmentation and cytotoxicity test suggest that CPP32 mediates cisplatin-induced apoptosis of S3 cells, whereas ICE family proteases other than CPP32 mediate staurosporine-induced apoptosis of S3 cells. In conclusion, distinct ICE family proteases mediate apoptosis of mouse proximal tubule cells depending on the stimuli to which the cells are exposed.

Animals↗
PubMedDate not supplied

The isolation and purification of a dual specific mast cell-derived protease from parasitised caprine jejunal tissue.

A mast cell granule protease has been isolated and purified from nematode-infected caprine jejunal homogenate by FPLC techniques and termed Goat Mast Cell Protease (GMCP). The purification steps were monitored for proteolytic activity against the synthetic substrate carboxybenzoyl-L-lysine thiobenzyl ester (BLT) and the presence of a homogenous protease preparation in the final sample was shown by SDS-PAGE electrophoresis. This protease was compared with enzymatic activity from isolated mucosal mast cells, which demonstrated the putative mast cell-derived source of the purified enzyme. Rabbit antiserum was raised against the protease and through the use of immunohistochemistry and Western blotting techniques the mast cell origin of the protease was confirmed. NH2-Terminal amino acid sequence analysis demonstrated a high degree of homology between GMCP and other previously isolated mast cell proteases including sheep mast cell protease (SMCP). Substrate analysis showed that GMCP also had an unusual dual chymotrypsin-like and trypsin-like activity similar to SMCP and bovine duodenase.

Abomasum↗
PubMed1993

Enzyme histochemical evidence for the presence of potential blood pressure regulating proteases in cultured villous explants from human first trimester placentae.

The proteases dipeptidyl peptidase IV, angiotensinase A and microsomal alanyl aminopeptidase are present in the human term placenta where they may be involved in the local modulation of placental blood pressure. In order to establish an in vitro model system to study the significance of these proteases in disorders related to pregnancy-induced hypertension, the activity of the proteases was localized histochemically in cultured explants of villi from human first trimester placentae. These studies revealed a similar distribution pattern of the activity of the proteases of cryostat sections of first trimester placental villi and in cultured tissue of the same placentae. Dipeptidyl peptidase IV and angiotensinase A activity were present in cytotrophoblast cells and dipeptidyl peptidase IV activity was found in the syncytiotrophoblast, respectively. Additionally, the activity of the proteases was visualized in various populations of stromal cells. Comparing our results with former studies, the protease activity pattern in first trimester placentae was found to be the same as in term placentae. Despite morphological changes of the tissue after 14 d in culture the localization of the proteases remained unchanged up to 52 d of culture. The results suggest that placental explants may serve as a suitable in vitro model for experimental studies on the role of proteases in pregnancy-induced hypertension.

Aminopeptidases↗
PubMed1998

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae.

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

Amino Acid Sequence↗
PubMed2000

Snake venom proteases affecting hemostasis and thrombosis.

The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.

Amino Acid Sequence↗
PubMed1997

Human immunodeficiency virus protease inhibitors. From drug design to clinical studies.

The discovery of human immunodeficiency virus (HIV) protease inhibitors is an example in which pharmacokinetic evaluation was implemented early in the discovery phase to obtain optimal pharmacological and pharmacokinetic properties. Currently, three HIV protease inhibitors, saquinavir, indinavir and ritonavir are clinically available. As a family, these HIV protease inhibitors are characterized pharmacologically by their ability to inhibit the viral protease enzyme. Pharmacokinetically, they are quite different due to their dissimilarity in physicochemical properties. Bioavailability appears to be limited with saquinavir, but not with indinavir and ritonavir. Although all three drugs are metabolized extensively by cytochrome P-450, saquinavir and indinavir are high clearance drugs while ritonavir is a low clearance drug. Despite their significant differences in elimination clearance, all three HIV proteases are given at high oral doses (600-800 mg) either twice or three times daily. These HIV protease inhibitors show superior therapeutic activity and a more favorable safety profile than those reported for the established reverse transcriptase inhibitors. However, the potential for interactions with other drugs metabolized by the CYP 3A4 isoform appears to be considerable. In addition, repeated administration of enzyme inducers results in a substantial decrease of plasma concentrations of protease inhibitors. Therefore, co-administration of drugs, such as rifampicin and rifabutin, must be avoided. HIV protease inhibitors are promising in the treatment of AIDS. Although they are not a cure, they can significantly inhibit that viral replication and improve the quality of life for people who have HIV infection.

Journal Article↗