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1.
Redox Biol ; 6: 326-333, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26335398

ABSTRACT

2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.


Subject(s)
Gene Expression Regulation, Fungal , Molecular Chaperones/chemistry , Peroxidases/chemistry , Recombinant Fusion Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Sulfinic Acids/chemistry , Chromatography, Gel , Hydrogen Peroxide/pharmacology , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Peroxidases/genetics , Peroxidases/metabolism , Plasmids/chemistry , Plasmids/metabolism , Protein Multimerization , Protein Structure, Quaternary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sulfinic Acids/metabolism
2.
Physiol Genomics ; 37(2): 140-6, 2009 Apr 10.
Article in English | MEDLINE | ID: mdl-19141541

ABSTRACT

The tet-inducible system has been widely used to achieve conditional gene expression in genetically modified mice. To alleviate the frequent difficulties associated with recovery of relevant transgenic founders, we tested whether a controlled strategy of transgenesis would support reliable cell-specific, doxycycline (Dox)-controlled transgene expression in vivo. Taking advantage of the potent hypoxanthine-aminopterin-thymidine selection strategy and an embryonic stem (ES) cell line supporting efficient germ-line transmission, we used hypoxanthine phosphoribosyltransferase (HPRT) targeting to insert a single copy tet-inducible construct designed to allow both glucocorticoid receptor (GR) and beta-galactosidase (beta-Gal) expression. Conditional, Dox-dependent GR and beta-Gal expression was evidenced in targeted ES cells. Breeding ES-derived single copy transgenic mice with mice bearing appropriate tet transactivators resulted in beta-Gal expression both qualitatively and quantitatively similar to that observed in mice with random integration of the same construct. Interestingly, GR expression in mice was dependent on transgene orientation in the HPRT locus while embryonic stem cell expression was not. Thus, a conditional construct inserted in single copy and in predetermined orientation at the HPRT locus demonstrated a Dox-dependent gene expression phenotype in adult mice suggesting that controlled insertion of tet-inducible constructs at the HPRT locus can provide an efficient alternative strategy to reproducibly generate animal models with tetracycline-induced transgene expression.


Subject(s)
Embryonic Stem Cells/metabolism , Hypoxanthine Phosphoribosyltransferase/genetics , Receptors, Glucocorticoid/metabolism , Tetracycline/pharmacology , Animals , Bacterial Proteins/genetics , Blotting, Western , Carrier Proteins/genetics , Cell Line , Doxycycline/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Female , Gene Expression Regulation/drug effects , Gene Knock-In Techniques , Genetic Vectors/genetics , Humans , Lac Operon/genetics , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Myocardium/metabolism , Receptors, Glucocorticoid/genetics , Staining and Labeling , Transfection , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
3.
Pharmacogenetics ; 11(6): 521-35, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11505222

ABSTRACT

The von Hippel-Lindau (VHL) tumour suppressor gene is commonly mutated in renal cell carcinoma of clear cell type (CCRCC). We investigated the possible relationship between VHL mutations in sporadic CCRCC and polymorphism of genes encoding enzymes involved in carcinogen metabolism: two cytochrome P450 monooxygenases (CYP1A1 and CYP2D6), one NAD[P]H:quinone oxidoreductase (NQO1), three glutathione S-transferases (GSTM1, GSTT1 and GSTP1) and two arylamine N-acetyltransferases (NAT1 and NAT2). We analysed DNA from tumour and nontumoural kidney tissue from 195 CCRCC patients. Single VHL mutations were identified in 88 patients and double mutations were present in two patients. Nine of 18 transversions were GC to TA, four were AT to TA, four were GC to CG and one was AT to CG. Ten of 19 transitions were GC to AT and nine were AT to GC. We also identified 53 frameshifts and two GC to AT at CpG. An excess of transversions was observed in a subset of patients with active GSTT1 [GSTT1 (+) genotype] and probably defective NAT1 (NAT1 S/R variant genotype). All 18 transversions were in GSTT1 (+) patients, whereas only 76% of transitions (P = 0.05) and 81% of the other mutations (P = 0.06) occurred in this genotype. We found that 28% of the transversions were in the NAT1 S/R genotype versus 12% of the transitions (P = 0.40) and 4% of the other mutations (P = 0.01). This suggests that pharmacogenetic polymorphisms may be associated with the type of acquired VHL mutation, which may modulate CCRCC development.


Subject(s)
Acetyltransferases/genetics , Arylamine N-Acetyltransferase , Carcinoma, Renal Cell/genetics , Genes, Tumor Suppressor , Glutathione Transferase/genetics , Ligases , Mutation , Proteins/genetics , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Adult , Aged , Chromosome Aberrations , Female , Frameshift Mutation , Gene Frequency , Genotype , Humans , Isoenzymes , Loss of Heterozygosity , Male , Middle Aged , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded Conformational , Von Hippel-Lindau Tumor Suppressor Protein , Xenobiotics/metabolism
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