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

The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.
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PMID:Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation. 779 59

The immunological relationship between liver flukes and their mammalian hosts is being unravelled by in vivo and in vitro studies. Vaccine studies in cattle and sheep with purified antigens (fatty acid binding protein, FABP; glutathione S-transferase, GST; cathepsin L, CatL; hemoglobin) have shown that high reductions in worm burdens (31-72%) and egg production (69-98%) can be achieved, raising the realistic possibility that immunological control of Fasciola infection is a commercially achievable goal. Combination vaccines may also be feasible since a cocktail of CatL and hemoglobin elicits a significant 72% protection in cattle. Analysis of immune responses to Fasciola during infection in ruminants suggests that chronic infection correlates with a type 2 helper T cell response, implying that type 1 helper T cell responses are down-regulated in fasciolosis. Recent results studying the resistance of Indonesian Thin Tail (ITT) sheep to F. gigantica have shown that this breed exhibits high innate (or rapidly acquired) resistance to infection and acquires a higher level of resistance after a primary challenge. Initial studies suggest that the resistance of ITT sheep to F. gigantica may be determined by a major gene. Merino sheep also acquire resistance to F. gigantica. In contrast, ITT and Merino sheep do not exhibit resistance to F. hepatica. These results suggest that there are fundamental differences between these two species of Fasciola in the biology of their interaction with the sheep immune system. In vitro studies on immune mechanisms of killing of juvenile fluke have shown that juvenile larvae of F. hepatica are susceptible to antibody-dependent killing by activated rat macrophages in vitro which is mediated by nitric oxide. Future studies on the immune effector mechanisms expressed by resistant sheep which control infection by F. gigantica will lead to new knowledge which may allow the design of more effective vaccines for fasciolosis.
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PMID:Immunological approaches for the control of fasciolosis. 939 93

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.
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PMID:Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle. 942 34

To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study [Matsumoto et al. (1997) J. Biochem. 121, 464-476], we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli. The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column. GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA. The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower. The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1.
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PMID:Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro. 953 63

We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.
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PMID:Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells. 963 4

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
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PMID:Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. 1101 86

Strains of mice that differ in voluntary alcohol consumption (VAC) are valuable models for the identification of genes involved in the complex etiology of alcohol effects and alcoholism. These mice offer a novel approach to the identification of strain-specific ethanol responsive (SSER) genes in tissues directly involved in alcohol metabolism and preference. We assessed mRNA from the liver and brain from male mice representing C57BL/6J, BALB/c, A/J, and DBA/2J strains following ethanol treatment (chronic ethanol fed liquid diet for 14 days or acute i.p. injection at two doses; 4 g/kg or 8 g/kg), using an expression array containing 588 genes (Clontech #7741-1). The results have identified NADPH cytochrome P450 oxidoreductase, insulin-like growth factor binding protein-1, glutathione S-transferase Mu 1, and cathepsin L as ethanol responsive genes in the liver. Further, we have established that IkB-alpha and clusterin genes in the brain are ethanol responsive, but only at the lower dose of the ethanol challenge. Although a number of other genes showing subtle (<2X) differences across strains and treatment combinations were reproducible in repeated blots, they were not confirmed by still evolving independent technologies of gene specific mRNA quantitation. The results demonstrate that comparative expression studies are an efficient approach to discover interacting gene networks that underlie the etiology of complex phenotypes including response to alcohols.
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PMID:Examination of ethanol responsive liver and brain specific gene expression, in the mouse strains with variable ethanol preferences, using cDNA expression arrays. 1246 48

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.
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PMID:A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. 1683 18

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.
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PMID:Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host. 1730

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.
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PMID:Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins. 1737 49


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