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PubMed1986

Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex.

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.

Binding Sites↗
PubMed2000

New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also determined on the fraction.

Bacillus subtilis↗
PubMed2002

Parameters for the recovery of proteases from surimi wash water.

Proteases are important bioactive compounds that have many applications in food processing. In this study, laboratory scale experiments were performed to establish conditions for recovery of a heat stable, acid protease from Pacific whiting (Merluccius productus) surimi process water. Reduction of proteinaceous solids and recovery of protease activity was maximized when process water was pre-treated with acid (pH 4) followed by heat (60 degrees C). In addition, acid plus heat treatment of process water appeared to improve membrane flux and concentration of protease activity (10-fold) was achieved in half as much time as treatments using acidification but not heat. Neither purification nor concentration of protease was effective using either 300 and 1000 kDa ultrafiltration or 0.3 microm microfiltration membranes. However, concentration of protease using 50 kDa ultrafiltration membranes was successful in recovering about 80% of original protease activity. Results provide conditions for further investigations into pilot plant recovery of protease from surimi process water.

Endopeptidases↗
PubMed1996

Bcl-2 regulates activation of apoptotic proteases in a cell-free system.

BACKGROUND: Apoptosis plays an important role in the normal development and homeostasis of metazoans. Aberrations in this process have been implicated in several major human diseases, but its molecular mechanism is poorly understood. In animals as diverse as humans and nematodes, the Bcl-2 oncoprotein prevents or delays apoptosis, whereas proteases of the interleukin-1beta-converting enzyme (ICE) family are required, suggesting that they are components of a conserved mechanism controlling the onset of apoptosis. RESULTS: A cell-free system produced from Xenopus laevis eggs reproduces nuclear events characteristic of apoptosis after a lag phase. We have used this system to define the temporal sequence of biochemical events and to examine the relationship between Bcl-2 and apoptotic proteases. Bcl-2 prevents apoptotic chromatin condensation and DNA cleavage, but only when added prior to the activation of a protease which has characteristics similar to the Ced-3 sub-family of ICE-like proteases and which cleaves poly(ADP-ribose) polymerase (PARP). Bcl-2 attenuates activation of this protease, an effect that does not require de novo protein synthesis or the presence of intact nuclei. The Ced-3-related protease CPP-32 is cleaved during the late stages of apoptosis in this system and after PARP cleavage. Generation of CPP-32-cleaving activity is inhibited by Bcl-2. CONCLUSIONS: These experiments provide direct biochemical evidence that Bcl-2 protects against apoptosis, at least in part, by regulating the activation of a series of apoptotic proteases that cleave PARP and other substrates. This cell-free system provides a useful biochemical model for analyzing the molecular mechanism controlling the activation of these proteases.

Animals↗
PubMedDate not supplied

Purification and characterization of a protease degrading 30 kDa yolk proteins of the silkworm, Bombyx mori.

The second major yolk proteins, 30 kDa proteins (30kPs) of the silkworm, Bombyx mori, which have been provided during oogenesis, are kept continuously unused during embryogenesis and are utilized just before larval hatching. The crude extracts of newly hatched larvae cleaved 30kPs in an in vitro incubation system. A protease was highly purified from newly hatched larvae using ammonium sulfate precipitation, gel filtration and ionic exchange chromatography, and non-denaturing-polyacrylamide gel electrophoresis (ND-PAGE). The protease shared the NH2-terminal amino acid sequence conserved in many serine proteases, and the apparent molecular mass was estimated to be approximately 600 kDa by gel filtration column chromatography. The enzymatic activity was strongly inhibited by elastatinal and diisopropyl fluorophosphate (DFP), indicating that this protease is an elastase-like serine protease. The protease selectively hydrolysed 30kP-1 and 30kP-4 between Ser6 and Ala7, but could not attack other 30kPs such as 30kP-2, 30kP-3 and 30kP-5. Consequently, the protease characterized in the present study is a unique protease which may be specialized for the selective degradation of yolk proteins in silkworm eggs.

Amino Acid Sequence↗
PubMed2003

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease.

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.

BACKGROUND: Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility. RESULTS: We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry. CONCLUSIONS: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.

Affinity Labels↗
PubMed2001

Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets.

BACKGROUND: Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts. RESULTS: Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle. CONCLUSIONS: Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

Animals↗
PubMed1999

Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01.

An organic solvent-stable protease (PST-01 protease) in a culture broth of organic solvent-tolerant Pseudomonas aeruginosa PST-01 was purified by successive hydrophobic interaction chromatography using Butyl-Toyopearl gels. The purified enzyme was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. PST-01 protease had a molecular mass of 38 kDa. The optimum temperature and pH for casein hydrolysis were 55 degrees C and 8.5, respectively. PST-01 protease was stable at pH 8-12 and below 50 degrees C and was determined to be a metalloprotease which was inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon. PST-01 protease inhibited by EDTA was reactivated completely by the addition of zinc or cobalt ions. The stability of PST-01 protease in solutions containing water-soluble organic solvents or alcohols was higher than that in the absence of organic solvent. Furthermore, in general, PST-01 protease was more stable than commercially available proteases, namely, subtilisin Carlsberg, thermolysin, and alpha-chymotrypsin, in the presence of water-soluble organic solvents or alcohols.

Journal Article↗
PubMed1980

Interaction of chymotrypsinogens with alpha 1-protease inhibitor.

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.

Animals↗
PubMed2002

ADAM-9 is an insulin-like growth factor binding protein-5 protease produced and secreted by human osteoblasts.

IGF binding protein-5 (BP-5) is an important bone formation regulator. Therefore, elucidation of the identity of IGF binding protein-5 (BP-5) protease produced by osteoblasts is important for our understanding of the molecular pathways that control the action of BP-5. In this regard, BP-5 protease purified by various chromatographic steps from a conditioned medium of U2 human osteosarcoma cells migrated as a single major band, which comigrated with the protease activity in native PAGE and yielded multiple bands in SDS-PAGE under reducing conditions. N-Terminal sequencing of these bands revealed that three of the bands yielded amino acid sequences that were identical to that of alpha2 macroglobulin (alpha2M). Although alpha2M was produced by human osteoblasts (OBs), it was not found to be a BP-5 protease. Because alpha2M had been shown to complex with ADAM proteases and because ADAM-12 was found to cleave BP-3 and BP-5, we evaluated if one of the members of ADAM family was the BP-5 protease. On the basis of the findings that (1) purified preparations of BP-5 protease from U2 cell CM contained ADAM-9, (2) ADAM-9 is produced and secreted in high abundance by various human OB cell types, (3) purified ADAM-9 cleaved BP-5 effectively while it did not cleave other IGFBPs or did so with less potency, and (4) purified ADAM-9 bound to alpha2M, we conclude that ADAM-9 is a BP-5 protease produced by human OBs.

ADAM Proteins↗
PubMedDate not supplied

Appearance of protease activities coincides with p10 and polyhedrin-driven protein production in the baculovirus expression system: effects on yield.

A study of proteolysis effects on recombinant protein yield was completed using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. Activities of protease and beta-galactosidase (beta-gal), a marker heterologous protein, were assayed at various multiplicities of infection (MOI = 1, 5, and 20) on a time course postinfection. Also, several protein-substrate gel electrophoresis assays were run using gelatin, beta-gal, and bovine serum albumin (BSA) in the gel matrix, to determine the protein specificity of the proteases. The most abundant protease activity (cysteine), found at 49 kDa, degraded all three substrates, pre- and post-infection. Two other protease activities (40 and 36 kDa) appeared on polyacrylamide gel electrophoresis (PAGE) gels after 72 hpi (hours postinfection). In addition, the culture with the highest MOI had the highest beta-gal activity until 72 hpi, when the activity dramatically decreased coincidentally with a 2.5-fold increase in protease activity. This result and the electrophoresis evidence that the protease is specific to beta-gal, indicate that there is a negative correlation between protease activity and recombinant protein yield. These results guide efforts to control product-degrading proteolysis in insect cell-baculovirus expression systems by harvest timing and the addition of protease inhibitors.

Animals↗
PubMedDate not supplied

Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases.

Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.

B-Lymphocytes↗
PubMedDate not supplied

Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer.

Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases.

Amino Acid Sequence↗
PubMed2003

Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds.

During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 microm) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Proteases augment the effects of lipopolysaccharide in rat gingiva.

OBJECTIVES: Lipopolysaccharide (LPS) and proteases have been implicated as important factors in the initiation and progression of human periodontal diseases. A single application of LPS or proteases is insufficient to induce periodontal pocket formation or periodontitis. The aim of the present study was to assess the combined effect of lipopolysaccharide and proteases on rat periodontal tissues, and create a periodontal disease model. MATERIALS AND METHODS: Forty male Wistar rats were divided into four groups: combination group (treated with both LPS and proteases solutions); LPS group; proteases group; and control. Each solution was introduced daily into the palatal gingival sulcus of maxillary molars for 8 weeks. The tissues were evaluated histometrically and immunohistochemically. RESULTS: In the LPS group, elongation of rete ridge, apical migration of junctional epithelium (JE), increased numbers of B cells in connective tissue, and resorption of alveolar bone were observed. In the proteases group, the increase in the number of infiltrating polymorphonuclear leukocytes and blood vessels in the connective tissue was greater than that of the LPS group. CONCLUSIONS: The effects of LPS on periodontal tissues differed from those of proteases. The addition of proteases augmented and increased the effects of LPS, which were apical migration, intraepithelial cleavage of JE, and increased B cell density. The lesions in the combination group resembled established lesions of human periodontitis, with the exception of the low density of plasma cells.

Alveolar Bone Loss↗
PubMed2003

Engineering of a macromolecular scaffold to develop specific protease inhibitors.

The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications. Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface. Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface. The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal). Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (K(i)*) of 11 pM, whereas K(i)* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher. The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa.

Amino Acid Sequence↗
PubMed2005

The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells.

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.

Boron Compounds↗