Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.
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PMID:The role of the low-density lipoprotein receptor-related protein (LRP) in the plasma clearance and liver uptake of recombinant single-chain urokinase-type plasminogen activator in rats. 913 48

We immobilized urokinase (UK) by covalent attachment to activated Sepharose 6B-CL through multi-point amine coupling and evaluated its performance in cleaving a fusion protein, which consisted of recombinant human growth hormone (hGH) and a fragment of glutathione S-transferase that was linked by a tetrapeptide of a UK-specific recognition sequence. Packing densities of aldehyde groups on the activated agarose surface could be controlled in a gel range of 7-60 micromol/ml aldehyde by the amount of glycidol used. The immobilization yield was nearly 100% at pH 10.5, and the specific activity of the immobilized UK was equivalent to about 80% of soluble UK under the assay conditions. The immobilized UK showed an improvement in pH and thermal stability, probably due to the structural rigidity imparted by multi-point linkages to the matrix. The cleavage rate by the immobilized UK was lower than that of the soluble enzyme but the side reaction of cryptic cleavage was significantly decreased, which might suggest that the enzyme's specificity was altered by the immobilization. Cleavage yield in the column packed with immobilized UK was dependent on the feed rate, and the yield was approx. 80% of that of the soluble UK. The monomeric hGH could be obtained by selectively precipitating the uncleaved fusion protein and the GST fragments at an acidic pH.
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PMID:Enzymic cleavage of fusion protein using immobilized urokinase covalently conjugated to glyoxyl-agarose. 1263 Sep 3

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.
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PMID:Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1). 1549 75

To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1-, pXO2-). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong to the M4 and M6 families, respectively. Both enzymes digested various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins, with some differences in specificity. In addition, InhA accelerated urokinase-mediated plasminogen activation, suggesting that InhA acts as a modulator of plasmin in the host inflammatory system. Relevant to epithelial barrier function, Npr599 and InhA significantly enhanced syndecan-1 shedding from cultured normal murine mammary gland cells without affecting their viability through stimulation of the host cell ectodomain shedding mechanism. In addition, Npr599 and InhA directly cleaved recombinant syndecan-1 fused to glutathione S-transferase. Mass spectrometric analysis suggested that the cleavage sites of Npr599 and InhA are the Asp(39)-Asp(40) and Gly(48)-Thr(49) bonds, respectively. We propose that Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology through direct degradation of host tissues, increases in barrier permeability, and/or modulation of host defenses.
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PMID:Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors. 1692 47

We tested the hypothesis that cell invasiveness and tumorigenesis are driven by hypomethylation of genes involved in tumor progression. Highly invasive human prostate cancer cells PC-3 were treated with either the methyl donor S-adenosylmethionine (SAM) or methyl DNA-binding domain protein 2 antisense oligonucleotide (MBD2-AS). Both treatments resulted in a dose- and time-dependent inhibition of key genes, such as urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor expression to decrease tumor cell invasion in vitro. No change in the levels of expression of genes already known to be methylated in late-stage prostate cancer cells, such as glutathione S-transferase P1 and androgen receptor, was seen. Inoculation of PC-3 cells pretreated with SAM and MBD2-AS into the flank of male BALB/c nu/nu mice resulted in the development of tumors of significantly smaller volume compared with animals inoculated with PC-3 cells treated with vehicle alone or MBD2 scrambled oligonucleotide. Immunohistochemical analysis of tumors showed the ability of SAM and MBD2-AS to significantly decrease tumoral uPA and MMP-2 expression along with levels of angiogenesis and survival pathway signaling molecules. Bisulfite sequencing analysis of tumoral genomic DNA showed that inhibition of both uPA and MMP-2 expression was due to methylation of their 5' regulatory region. These studies support the hypothesis that DNA hypomethylation controls the activation of multiple tumor-promoting genes and provide valuable insight into developing novel therapeutic strategies against this common disease, which target the demethylation machinery.
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PMID:Alteration of the methylation status of tumor-promoting genes decreases prostate cancer cell invasiveness and tumorigenesis in vitro and in vivo. 1698 64

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas where it promotes invasion and delays tumor growth, both in vitro and in vivo. SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Additionally in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in an extracellular matrix-specific and concentration-dependent manner. Because the signaling aspect of this migration is neither well understood nor characterized, we overexpressed SPARC in both the minimally-invasive U87 cell line and in the most aggressive invasive cell line, SNB19. We first performed RT-PCR analysis and observed an upregulation of uPA and its receptor, uPAR. We also observed increased expression levels of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). Western blot analysis confirmed these results, and the enzymatic activity of the metalloproteinases and uPA was further supported by zymography. Downstream of the uPA-uPAR interaction, upregulation of PI3-K occurred in cells overexpressing SPARC. Using GST-TRBD, we showed the upregulation of active GTP-bound RhoA, but neither Rac1 nor Cdc42 were activated. The inhibition of uPA and uPAR downregulated PI3-K activity and cell migration, as shown by matrigel invasion assay. A dorsal skin-fold chamber model revealed the high angiogenic activity of SPARC, though the proliferation of SPARC overexpressing cells was unaffected. Our results show that the small GTPase RhoA was a critical mediator of invasion or migration in the uPA-uPAR/PI3-K signaling pathway.
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PMID:SPARC-induced migration of glioblastoma cell lines via uPA-uPAR signaling and activation of small GTPase RhoA. 1708 72

The hamster buccal pouch (HBP) carcinogenesis model is one of the most well characterized animal systems for analyzing the development of oral squamous cell carcinoma (OSCC), a common malignancy worldwide. HBP carcinomas that closely mimic human OSCC are useful in understanding the molecular mechanisms of neoplastic transformation. The present study is a comparative evaluation of markers of carcinogen activation, oxidative stress, cell proliferation, apoptosis, invasion, and angiogenesis in human and hamster OSCCs. Enhanced expression of CYP1A1 and CYP1B1 isoforms in both human and hamster oral tumours was associated with significantly increased expression of 8-hydroxy 2-deoxyguanosine (8-OHdG) indicating oxidative DNA damage. Analysis of markers of cell survival and proliferation revealed increased expression of PCNA, GST-P, and NF-kappaB with downregulation of p21, p53 and IkappaB in both human and hamster OSCCs. In addition, both human and hamster oral carcinomas displayed invasive, and angiogenic properties as revealed by dysregulated cytokeratin expression, downregulation of RECK, and increased expression of uPA, MMP-2 and-9, HIF-1alpha, and VEGF. The results reveal aberrant expression of multiple molecules in key signaling pathways in both human OSCCs and HBP carcinomas rendering the HBP model as an important tool for monitoring oral oncogenesis.
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PMID:Of humans and hamsters: a comparative evaluation of carcinogen activation, DNA damage, cell proliferation, apoptosis, invasion, and angiogenesis in oral cancer patients and hamster buccal pouch carcinomas. 1925 Aug 57

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.
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PMID:Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications. 1963 10

We sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.
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PMID:Evaluation of molecular markers in a rat model of mammary carcinogenesis. 1972 28

Hepatitis B spliced protein (HBSP) is involved in the pathogenicity and/or persistence of hepatitis B virus (HBV). Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma (HCC). However, whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown. This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility. Conversely, small interfering RNA (siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion. By glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and a mammalian two-hybrid assay, HBSP was found to directly interact with cathepsin B (CTSB). Similar to HBSP knockdown, knocking down CTSB also reduced cell migration and invasion. Furthermore, the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), and overexpression of HBSP significantly enhanced tumor-induced vascularization of endothelial cells. In contrast, knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis. Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase (MAPK) and Akt signaling pathway, as evidenced by increases in phosphorylation of p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt. Taken together, these findings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes, increased tumor-induced angiogenesis, and activation of the MAPK/Akt signaling, thereby leading to the aggressiveness of hepatoma cells.
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PMID:Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion. 2303 14


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