Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Induction of HLA-DR antigen expression by interferon-gamma (IFN-gamma) is inhibited by trypsin inhibitors and an anti-trypsin monoclonal antibody, but not by chymotrypsin inhibitors, suggesting a requirement for trypsin-like protease (TLP) activity in IFN-gamma-induced HLA-DR expression. Using p-nitroanilide and thioester substrates, TLP activity was demonstrated in cellular extracts of a hybrid epidermal cell line and judged to be essential for HLA-DR expression. TLP activity was inhibited by the trypsin inhibitors soybean trypsin inhibitor, ovomucoid trypsin inhibitor, and tosyl-lysyl-chloromethyl ketone and by an anti-trypsin monoclonal antibody, closely paralleling inhibition of HLA-DR expression by such agents. TLP activity was enhanced by exposure to trypsin-linked agarose, indicating that the protease normally exists in an inactive form, perhaps in an enzyme-inhibitor complex or as an activatable proenzyme. Finding glucocorticoids (GC) to also inhibit IFN-gamma-induced HLA-DR expression and to regulate serine protease, especially urokinase plasminogen activator (uPA), activity raised the possibility of GC regulation of TLP activity. However, TLP activity was found to be constitutively expressed, regulated by neither GC nor IFN-gamma, nor was uPA activity involved in HLA-DR regulation. Trypsin inhibitors and GC also inhibited induction of intracellular 2',5'-oligoadenylate (2-5A) synthetase by IFN-gamma. Thus, TLP activity is required for IFN-gamma induction of HLA-DR and 2-5A synthetase.
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PMID:Induction of HLA-DR by interferon-gamma requires a trypsin-like protease. 177 67

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
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PMID:Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris. 754 63

Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators.
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PMID:Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA. 930 22

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.
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PMID:Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. 943 34

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50- to 65-kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA-dependent, plasmin-mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated-suPAR (B-suPAR) bound in a specific fashion to THP-1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B-suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B-suPAR to THP-1 cells was enhanced four- to sevenfold by 24-h exposure of cells to PMA or by co-incubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B-suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by co-incubation with uPA. B-suPAR biding to PMA-differentiated THP-1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA-differentiated THP-1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B-suPAR. Whereas the binding of suPAR did not measurably affect cell-associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane-associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin-sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand.
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PMID:A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells. 971 60

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.
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PMID:A novel murine tryptase involved in blastocyst hatching and outgrowth. 1142 30

One of the potential therapeutic interventions to hormone-independent breast cancer would be to reactivate the expression of estrogen receptor or progesterone receptor (PR) in the tumor cells so as to render the tumor responsive to the hormones. We have reported previously that progesterone markedly inhibited cell growth and induced remarkable focal adhesions in PR-transfected MDA-MB-231 cells. The aim of this study was to determine the effects of progesterone on the invasive properties and in vivo tumor growth of PR-transfected MDA-MB-231 cells. It was found that progesterone has increased cell resistance to trypsin digestion and increased cell attachment to extracellular matrix proteins, especially laminin and fibronectin. In vitro invasion assays using modified Boyden chambers showed that progesterone increased cell migration through matrix protein-coated membranes. However, Northern blotting analysis demonstrated that progesterone strongly down-regulated (up to 60-fold) the gene expression of urokinase plasminogen activator and increased (up to 5-fold) the expression of tissue-type plasminogen activator in these cells. This pattern of gene regulation suggested an inhibition of cell invasiveness because numerous clinical studies have indicated that low levels of urokinase plasminogen activator and high levels of tissue-type plasminogen activator in breast cancer are associated with favorable prognosis. Furthermore, animal studies showed that progesterone strongly inhibited the tumor formation and growth in Scid mice. After 12 weeks of inoculation, the median weight of tumors in the progesterone-treated group was 25 mg compared with 203 mg in the placebo group (P < 0.001). These results suggest that progesterone may provide effective treatment for estrogen receptor- and PR-negative breast cancer if the PR expression were reactivated. Alternatively, activation of progesterone-mediated molecular pathways in hormone-independent breast cancer may achieve similar therapeutic effects.
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PMID:Effect of progesterone on the invasive properties and tumor growth of progesterone receptor-transfected breast cancer cells MDA-MB-231. 1155 6

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.
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PMID:Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans. 1451 79

The preparation and assessment of biological activity of 6-substituted 2-naphthamidine inhibitors of the serine protease urokinase plasminogen activator (uPA, or urokinase) is described. 2-Naphthamidine was chosen as a starting point based on synthetic considerations and on modeling of substituent vectors. Phenyl amides at the 6-position were found to improve binding; replacement of the amide with other two-atom linkers proved ineffective. The phenyl group itself is situated near the S1' subsite; substitutions off of the phenyl group accessed S1' and other distant binding regions. Three new points of interaction were defined and explored through ring substitution. A solvent-exposed salt bridge with the Asp60A carboxylate was formed using a 4-alkylamino group, improving affinity to K(i) = 40 nM. Inhibitors also accessed two hydrophobic regions. One interaction is characterized by a tight hydrophobic fit made with a small dimple largely defined by His57 and His99; a weaker, less specific interaction involves alkyl groups reaching into the broad prime-side protein binding region near Val41 and the Cys42-Cys58 disulfide, displacing water molecules and leading to small gains in activity. Many inhibitors accessed two of these three regions. Affinities range as low as K(i) = 6 nM, and many compounds had K(i) < 100 nM, while moderate to excellent selectivity was gained versus four of five members of a panel of relevant serine proteases. Also, some selectivity against trypsin was generated via the interaction with Asp60A. X-ray structures of many of these compounds were used to inform our inhibitor design and to increase our understanding of key interactions. In combination with our exploration of 8-substitution patterns, we have identified a number of novel binding interactions for uPA inhibitors.
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PMID:Identification of novel binding interactions in the development of potent, selective 2-naphthamidine inhibitors of urokinase. Synthesis, structural analysis, and SAR of N-phenyl amide 6-substitution. 1471 4

In this letter we report the synthesis and biochemical evaluation of selective, irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator (uPA). A diphenyl phosphonate group was introduced on the substratelike peptide Z-d-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A k(app) value in the 10(3) M(-1) s(-1) range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.
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PMID:Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator. 1511 82


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