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Trypsin-like Protease (trypsin-like + protease)
Selected AbstractsHuman airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF ,-converting enzyme activity in airway epithelial cellsFEBS JOURNAL, Issue 24 2005Manabu Chokki Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway in the airway epithelial cell line NCI-H292. In this study, the mechanisms by which HAT-induced AR release can occur were investigated. HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2. Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production. However, in contrast to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release. Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor ,-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release. These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism. [source] Antifungal Activity of a Bowman,Birk-type Trypsin Inhibitor from Wheat KernelJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2000G. Chilosi A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC50) values ranging from 111.7 to above 500 ,g/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens. Zusammenfassung Ein Trypsinhemmer aus Weizenkörnern (WTI) zeigte eine starke antifungale Aktivität gegenüber verschiedenen pathogenen Pilzen und hemmte deren trypsinähnliche Aktivität. WTI hemmte in vitro die Sporenkeimung und das Hyphenwachstum der Pathogene, wobei die IC50 -Werte zwischen 111,7 und mehr als 500 ,g/ml lagen. Elektronenmikroskopische Untersuchungen zeigten, dai WTI morphologische Veränderungen bewirkte, die aus einer Hemmung des Hyphenwachstums und einer veränderten Verzweigung bestanden. Eine der untersuchten Pilzarten, Botrytis cinerea, bildete eine trypsinähnliche Protease, die durch den Trypsininhibitor gehemmt wurde. Ebenso wie andere sameneigene Abwehrproteine scheint WTI während der Keimruhe und in den frühen Stadien der Keimung, wenn die Pflanzen gegenüber möglichen bodenbürtigen Pathogenen besonders exponiert sind, ein wichtiger Resistenzfaktor in Weizenkörnern zu sein. [source] Activity-based mass spectrometric characterization of proteases and inhibitors in human salivaPROTEOMICS - CLINICAL APPLICATIONS, Issue 7 2009Xiuli Sun Abstract Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50,75,kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer. [source] Characterization of the trypsin-like protease (Ha-TLP2) constitutively expressed in the integument of the cotton bollworm, Helicoverpa armigera,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Yang Liu Abstract Trypsins belong to the serine endoproteases. They are the most important proteases in insects because of their key roles in food digestion and zymogens activation. But there has been little study of the trypsins in the integuments of insects. In this work, we cloned a trypsin-like protease gene from Helicoverpa armigera and named it trypsin-like protease 2 (Ha-TLP2). Semi-quantitative reverse transcription PCR analysis showed that Ha-TLP2 is constitutively expressed in the integument and can be down-regulated by 20-hydroxyecdysone (20E) and up-regulated by the juvenile hormone (JH) analog methoprene. Immunohistochemistry showed that Ha-TLP2 is located not only in the epidermis, but also in new and old cuticles. Immunoblotting and gelatin-SDS-PAGE revealed that Ha-TLP2 is constitutively expressed with activity in the integument during larval feeding, molting, and metamorphosis. This evidence suggests that Ha-TLP2 is involved in the remodeling of the integument. © 2009 Wiley Periodicals, Inc. [source] Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoataseBIOPOLYMERS, Issue 2 2001Adrian Murza Abstract Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 85,94, 2001 [source] Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like proteaseGENES TO CELLS, Issue 11 2004Jernej Murn Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis-inducing potential and dissected the death-signalling pathways of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin- and trypsin-like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP-ribose) polymerase 1 (PARP-1) and inhibitor of caspase-activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk). These results suggest that inhibition of anti-apoptotic serine proteases governs the onset of the caspase-dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin-like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK-pre-treated WEHI 231 cells undergoing apoptosis triggered either by anti-IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin-like protease. [source] Specificity and reactive loop length requirements for crmA inhibition of serine proteasesPROTEIN SCIENCE, Issue 2 2005Lisa D. Tesch Abstract The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (,102,104 M,1sec,1), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (kdiss ,10,7 sec,1), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (kcat/KM of ,106 M,1 sec,1). N-terminal sequencing confirmed that the P1 Arg,P1,Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1,Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ,10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (105,106 M,1sec,1), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1,Cys,P2,Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites. 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