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)

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.
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PMID:Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems. 760 44

PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity.
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PMID:Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity. 861 3

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.
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PMID:Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. 926 34

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.
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PMID:RNA aptamers specifically interact with the prion protein PrP. 934 39

The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.
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PMID:Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli. 1049 69

We investigated the expression, activation and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38 MAPKs) and extracellular signal-regulated kinases (ERKs), using western blotting and immunohistochemistry, in the brains of hamsters infected with 263K scrapie agent, to clarify the role of these kinases in the pathogenesis of prion disease. The immunoblot analysis demonstrated that activation of JNK, p38 MAPK and ERK in whole brain homogenates was increased in infected animals. Phosphorylation of cAMP/calcium responsive element binding protein (CREB), a downstream transcription factor of active ERK, was significantly increased in scrapie-infected hamsters. The immunohistochemical study showed that active ERK was enhanced in infected hamsters compared with controls. Active ERK immunoreactivity was observed within neurons in the dentate gyrus and in glial fibrillary acidic protein (GFAP)-positive reactive astrocytes of infected animals. The expression level of c-Jun mRNA as well as protein, a substrate of active JNK, was increased in infected animals. A significant increase in JNK activity upon glutathione S-transferase (GST)-c-Jun was observed in infected compared with control animals. Phospho-c-Jun immunoreactivity was observed only in neurons of the thalamus in infected groups. These findings indicated that the JNK pathway was activated in the scrapie-infected group. The chronological activation of MAPKs using immunoblot analysis indicates that the kinases are sequentially activated during the pathophysiology of prion disease. Taken together, these results lend credence to the notion that MAPK pathways are dysregulated in prion disease, and also indicate an active role for this pathway in disease pathogenesis.
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PMID:Activation of mitogen-activated protein kinases in hamster brains infected with 263K scrapie agent. 1613 77

To develop monoclonal antibodies (MAbs) to react with normal prion protein (PrPC) and abnormal isoform of prion protein (PrPSc), PrPSc was isolated from brains of 263 K scrapie-infected hamsters and immunized to PrP knockout mice. We developed two hybridomas, 3F10 and 1C5 (IgG1), of which epitope mappings were screened by using glutathione S-transferase (GST) fusion proteins of recombinant hamster prion protein and suitable peptides. 3F10 showed a high affinity for hamster and mouse PrP and was demonstrated to recognize the residues 137-151. 1C5 recognizes the region 119-130 corresponding to the GXXXG motif, the glycine zipper region, conserved in all mammals. In the immunohistochemical analysis, the positive staining for PrPSc was observed in the extracellular compartment of scrapie-infected brains but not in the normal brains. However, in Western blot, these antibodies recognized both normal and abnormal prion proteins. These results suggested that the developed mouse MAbs are specific to prion protein and can recognize abnormal prion protein more effectively than normal prion protein in immunohistochemistry. Therefore, these antibodies could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties between PrPC and PrPSc.
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PMID:Generation of monoclonal antibody recognized by the GXXXG motif (glycine zipper) of prion protein. 1704 82