Differential gene expression of 3D primary human airway cultures exposed to cigarette smoke and electronic nicotine delivery system (ENDS) preparations.

BACKGROUND: Acute exposure to cigarette smoke alters gene expression in several biological pathways such as apoptosis, immune response, tumorigenesis and stress response, among others. However, the effects of electronic nicotine delivery systems (ENDS) on early changes in gene expression is relatively unknown. The objective of this study was to evaluate the early toxicogenomic changes using a fully-differentiated primary normal human bronchial epithelial (NHBE) culture model after an acute exposure to cigarette and ENDS preparations. RESULTS: RNA sequencing and pathway enrichment analysis identified time and dose dependent changes in gene expression and several canonical pathways when exposed to cigarette preparations compared to vehicle control, including oxidative stress, xenobiotic metabolism, SPINK1 general cancer pathways and mucociliary clearance. No changes were observed with ENDS preparations containing up to 28 µg/mL nicotine. Full model hierarchical clustering revealed that ENDS preparations were similar to vehicle control. CONCLUSION: This study revealed that while an acute exposure to cigarette preparations significantly and differentially regulated many genes and canonical pathways, ENDS preparations containing the same concentration of nicotine had very little effect on gene expression in fully-differentiated primary NHBE cultures.

Pancreatic secretory trypsin inhibitor reduces multi-organ injury caused by gut ischemia/reperfusion in mice.

Intestinal ischemia/reperfusion (I/R) injury occurs during transplantation, mesenteric arterial occlusion, trauma and shock, causing systemic inflammation, multiple organ dysfunction and high mortality. Pancreatic secretory trypsin inhibitor (PSTI), a serine protease inhibitor expressed in gut mucosa may function as a mucosal protective/repair peptide. We examined whether PSTI affected mesenteric I/R-induced injury. Hypoxia/normoxia (H/N) caused 50% drop in cell viability of AGS, RIE1 and Caco-2 cells but PSTI (10 µg/ml) given prior- or during-hypoxic period improved survival by 50% (p<0.01). Similarly, Caco-2 monolayers exposed to H/N had 300% increase in transepithelial permeability, PSTI truncated this by 50% (p<0.01). Mice underwent mesenteric I/R by clamping jejunum, causing severe mucosal injury, increased apoptotic markers and 3-fold increases in plasma IL-6, IL1ß, TNFα, and tissue lipid peroxidation (MDA) and inflammatory infiltration (MPO) levels. Lungs showed similar significant injury and inflammatory infiltrate markers. Smaller increases in MDA and MPO were seen in kidney & liver. PSTI (20 mg/kg) reduced all injury markers by 50-80% (p<0.01). In vitro and in vivo studies showed PSTI reduced pro-apoptotic Caspase 3, 9 and Baxα levels, normalised Bcl2 and caused additional increases in HIF1α, VEGF and Hsp70 above rises caused by I/R alone (all p<0.01). PSTI also prevented reduction of tight junction molecules ZO1 and Claudin1 (all p<0.01) but did not affect increased ICAM-1 caused by I/R in gut or lung. PSTI may be a useful clinical target to prevent I/R injury.

Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties.

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

Autocrine induction of invasion and metastasis by tumor-associated trypsin inhibitor in human colon cancer cells.

From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.

Characterization and comparative 3D modeling of CmPI-II, a novel 'non-classical' Kazal-type inhibitor from the marine snail Cenchritis muricatus (Mollusca).

The complete amino acid sequence obtained by electrospray ionization tandem mass spectrometry of the proteinase inhibitor CmPI-II isolated from Cenchritis muricatus is described. CmPI-II is a 5480-Da protein with three disulfide bridges that inhibits human neutrophil elastase (HNE) (K(i) 2.6+/-0.2 nM), trypsin (K(i) 1.1+/-0.9 nM), and other serine proteinases such as subtilisin A (K(i) 30.8+/-1.2 nM) and pancreatic elastase (K(i) 145.0+/-4.4 nM); chymotrypsin, pancreatic and plasma kallikreins, thrombin and papain are not inhibited. CmPI-II shares homology with the Kazal-type domain and may define a new group of 'non-classical' Kazal inhibitors according to its Cys(I)-Cys(V) disulfide bridge position. The 3D model of CmPI-II exhibits similar secondary structure characteristics to Kazal-type inhibitors and concurs with circular dichroism experiments. A 3D model of the CmPI-II/HNE complex provides a structural framework for the interpretation of its experimentally determined K(i) value. The model shows both similar and different contacts at the primary binding sites in comparison with the structure of turkey ovomucoid third domain (OMTKY3)/HNE used as template. Additional contacts calculated at the protease-inhibitor interface could also contribute to the association energy of the complex. This inhibitor represents an exception in terms of specificity owing to its ability to strongly inhibit elastases and trypsin.

Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue.

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.

A Second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts with the apoplastic pathogenesis-related protease P69B of tomato.

The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response. Plant pathogenic oomycetes, such as the potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) pathogen Phytophthora infestans, secrete a diverse family of serine protease inhibitors of the Kazal family. Among these, the two-domain EPI1 protein was shown to inhibit and interact with the pathogenesis-related protein P69B subtilase of tomato and was implicated in counter-defense. Here, we describe and functionally characterize a second extracellular protease inhibitor, EPI10, from P. infestans. EPI10 contains three Kazal-like domains, one of which was predicted to be an efficient inhibitor of subtilisin A by an additivity-based sequence to reactivity algorithm (Laskowski algorithm). The epi10 gene was up-regulated during infection of tomato, suggesting a potential role during pathogenesis. Recombinant EPI10 specifically inhibited subtilisin A among the major serine proteases, and inhibited and interacted with P69B subtilase of tomato. The finding that P. infestans evolved two distinct and structurally divergent protease inhibitors to target the same plant protease suggests that inhibition of P69B could be an important infection mechanism for this pathogen.

Inhibition of membrane-type serine protease 1/matriptase by natural and synthetic protease inhibitors.

Membrane-type serine protease 1 (MT-SP1), identical to matriptase, is a recently identified type II transmembrane serine protease. MT-SP1/matriptase is of considerable interest for the development, homeostasis, and cancer invasion and metastasis of epithelial tissues. The administration of inhibitors for MT-SP1/matriptase may be effective to suppress the development of tumors where the enzyme may be involved. In the present study, we produced a secreted form of recombinant MT-SP1/matriptase (ekMT-SP1s) that can be activated by enterokinase in vitro and investigated the inhibitory ability of various protease inhibitors toward the recombinant enzyme. The enterokinase-treated ekMT-SP1s (active ekMT-SP1s) cleaved various peptidyl-4-methylcoumaryl-7-amide (MCA) substrates with arginine (or lysine) residue at position P1, and the best substrate was t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. The specificity for the synthetic and natural substrates of the active ekMT-SP1s was in good agreement with that of the natural enzyme. Endogenous protease inhibitors tested, except for antithrombin III, showed no or little inhibition on the cleavage of Boc-Gln-Ala-Arg-MCA by the active ekMT-SP1s. Aprotinin showed strong inhibitory activity toward the cleavage. Food-derived inhibitors, such as soybean trypsin inhibitor, Bowman-Birk inhibitor, and lima bean trypsin inhibitor inhibited it, while chicken ovomucoid did not. Synthetic inhibitors tested inhibited it, and among them, the inhibitory effect of FOY-305 was strongest. The present findings provide important information for the suppression of cancer invasion and metastasis for which MT-SP1/matriptase is responsible.

Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen.

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.

MMP-9 activation by tumor trypsin-2 enhances in vivo invasion of human tongue carcinoma cells.

Various human cancer cells express tumor-associated trypsinogen-2 (TAT-2), which can efficiently activate matrix metalloproteinases (MMPs) in vitro. MMP-2 and MMP-9 are particularly associated with the invasive malignant potential of several tumors. To investigate the role of TAT-2 in tumor invasion, we overexpressed TAT-2 in two malignant human squamous cell carcinoma cell lines of tongue and in non-malignant human papilloma virus transformed gingival keratinocytes. The TAT-2 overexpression significantly increased the levels of active MMP-9 in the most malignant cell line. TAT-2-transfected cells intravasated (invaded blood vessels) up to 60% more efficiently than did the control cells in an in vivo chick embryo chorioallantoic membrane invasion model. This increased intravasation was almost completely abolished by a specific tumor-associated trypsin inhibitor (TATI). These results indicate that TAT-2 has a role in the invasive growth of tumors, either alone or in cascade with gelatinases, especially by generating active MMP-9.

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Structural study of the complex between human pepsin and a phosphorus-containing peptidic -transition-state analog.

The refined crystal structure of the complex between human pepsin and a synthetic phosphonate inhibitor, Iva-Val-Val-Leu(P)-(O)Phe-Ala-Ala-OMe [Iva = isovaleryl, Leu(P) = the phosphinic acid analog of L-leucine, (O)Phe = L-3-phenyllactic acid, OMe = methyl ester], is presented. The structure was refined using diffraction data between 30 and 1.96 A resolution to a final R factor ( summation operator| |F(o)| - |F(c)| | / summation operator|F(o)|, where |F(o)| and |F(c)| are the observed and calculated structure-factor amplitudes, respectively) of 20.0%. The interactions of the inhibitor with the enzyme show the locations of the binding sites on the enzyme from S4 to S3'. Modeling of the inhibitor binding to porcine pepsin shows very similar binding sites, except at S4. Comparison of the complex structure with the structures of related inhibitors bound to penicillopepsin helps to rationalize the observed differences in the binding constants. The convergence of reaction mechanisms and geometries in different families of proteinases is also discussed.

Mechanism of porcine pancreatic alpha-amylase inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose.

The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.

Monitor peptide (rat pancreatic secretory trypsin inhibitor) directly stimulates the proliferation of the nontransformed intestinal epithelial cell line, IEC-6.

BACKGROUND/AIMS: The growth-stimulating activity of monitor peptide [rat pancreatic secretory trypsin inhibitor (PSTI)-61] was evaluated in the rat intestinal epithelial cell line IEC-6. In response to food intake, monitor peptide induces cholecystokinin release and the subsequent secretion of pancreatic enzyme into the rat small intestine. METHODS: Monitor peptide was purified from rat bile-pancreatic juice. The proliferation of IEC-6 cells was determined by both counting cell number and [3H]-thymidine incorporation. Ornithine decarboxylase (ODC) activity was assessed by radiometric technique using L-[1-14C]-ornithine. RESULTS: Monitor peptide (10(2)-10(5) ng/ml) induced a significant increase in the incorporation of [3H]-thymidine into IEC-6 cells, and this effect was observed in a dose-dependent manner. A significant increase in the cell number was also observed. An antibody specific for the synthetic NH2-terminal fragment of monitor peptide completely abrogated the growth-stimulating effects of this peptide. In addition, monitor peptide effectively increased the ODC activity of IEC-6 cells. CONCLUSIONS: These results indicate that monitor peptide stimulates the growth of intestinal epithelial cells, and suggest that polyamine metabolism may be involved in this mechanism of growth induction.

Human pancreatic secretory trypsin inhibitor. Distribution, actions and possible role in mucosal integrity and repair.

Pancreatic secretory trypsin inhibitor is a potent protease inhibitor which was originally identified in the pancreas. It has subsequently been shown to be present in mucus-secreting cells throughout the gastrointestinal tract and also in the kidney, lung and breast. Its major roles are likely to be to prevent premature activation of pancreatic proteases and to decrease the rate of mucus digestion by luminal proteases within the stomach and colon. In addition, PSTI increases the proliferation of a variety of cell lines and stimulates cell migration, possibly acting via the EGF receptor. These findings suggest that PSTI may also be involved in both the early and late phases of the healing response following injury. Further studies including the production of transgenic overexpression and knockout models should help elucidate the physiological function of this peptide.

Purification and characterization of a novel Kazal-type serine proteinase inhibitor of neutrophil elastase from sheep lung.

A Kazal-type elastase inhibitor was purified by trichloroacetic acid precipitation of sheep lung lavage fluid followed by chymotrypsin affinity and gel-filtration chromatography of the supernatant. Sheep lung elastase inhibitor (SLEI) is glycosylated. Laser desorption mass spectrometry indicated that SLEI has a molecular mass of 16.8-17.3 kDa. Partial protein sequence of SLEI and of a peptide derived from SLEI showed 31-52% and 51-66% homology at the N-terminus and at the inhibitory site respectively with Kazal-type double-headed proteinase inhibitors (bikazins). SLEI inhibited human leukocyte elastase and porcine pancreatic elastase but not human cathepsin G. It was inactivated by chloramine-T and reactivated when incubated with methionine sulfoxide peptide reductase and dithiothreitol, indicating the presence of a methionine at the active site. The concentration of SLEI in bronchoalveolar lavage fluid (BALF) and lung lymph was 0.28 microM (0.23-0.49); 0.24 microM (0.20-0.31) (median, (range), n = 5), respectively and was undetectable in plasma (< 0.03 microM) suggesting that SLEI is produced in the lung. The median molar ratios of SLEI to alpha1-proteinase inhibitor in BALF and lung lymph were 3.2 to 1 and 0.017 to 1, respectively. These results indicate that SLEI probably makes an important contribution to antielastase defence in epithelial lining liquid.

Stimulatory effect of synthetic luminal cholecystokinin releasing factor (LCRF) fragment (1-35) on pancreatic exocrine secretion in conscious rats.

The effects of a synthetic putative luminal cholecystokinin (CCK) releasing factor (LCRF) fragment (1-35) on pancreatic exocrine secretion were examined in conscious Wistar rats and a mutant strain of rats lacking the CCK-A receptor. Intraduodenal injection of graded doses of the LCRF fragment induced biphasic responses in Wistar rats. The injection of 0.1 microgram of the LCRF fragment produced a maximal response, while 1 microgram produced a lower response. No significant effect was observed in rats lacking the CCK-A receptor. The synthetic LCRF fragment stimulated pancreatic secretion via CCK-A receptors in conscious rats.

Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires.

Given the extent of hepatitis C virus (HCV) infection as a worldwide health problem and the lack of effective treatment, the development of anti-HCV drugs is an important and pressing objective. Previous studies have indicated that proteolytic events mediated by the NS3 protease of HCV are fundamental to the generation of an active viral replication apparatus, as unequivocably demonstrated for flaviviruses. As a result, the NS3 protease has become a major target for discovering anti-HCV drugs. To gain further insight into the biochemical and biophysical properties of the NS3 enzyme binding pocket(s) and to generate biological tools for developing antiviral strategies, we decided to engineer macromolecular ligands of the NS3 protease domain. Phage-displayed repertoires of minibodies ("minimized" antibody-like proteins) and human pancreatic secretory trypsin inhibitor were sampled by using the recombinant NS3 protease domain as a ligate molecule. Two protease inhibitors were identified and characterized biochemically. These inhibitors show marked specificity for the viral protease and potency in the micromolar range but display different mechanisms of inhibition. The implications for prospective development of low-molecular-weight inhibitors of this enzyme are discussed.

Activation of type IV procollagenases by human tumor-associated trypsin-2.

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.