Microinjected glutathione S-transferase Yb subunits translocate to the cell nucleus.
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ABSTRACT:
SUBMITTER: Bennett CF
PROVIDER: S-EPMC1148376 | biostudies-other | 1987 Oct
REPOSITORIES: biostudies-other
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Project description:production of glutathione s-transferase from Lactobacillus plantarum Ku720558
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Project description:Human glutathione transferase pi (GST pi) has been crystallized as a homodimer, with a subunit molecular mass of approximately 23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST pi, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K(m) for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K(m)(GSH), whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V(max). The GST pi retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.
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Project description:Glutathione transferase (formerly GST) catalyzes the inactivation of various electrophile-producing anticancer agents via conjugation to the tripeptide glutathione. Moreover, several data link the overexpression of some GSTs, in particular GSTP1-1, to both natural and acquired resistance to various structurally unrelated anticancer drugs. Tumor overexpression of these proteins has provided a rationale for the search of GST inhibitors and GST activated cytotoxic prodrugs. In the present review we discuss the current structural and pharmacological knowledge of GST-activated cytotoxic compounds.
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Project description:Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1',3 and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1', 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has a C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal-tetrapeptide-deleted form was not observed for rat liver GST 1.
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Project description:Considering the pleiotropic roles of glutathione transferase (GST) omega class members in redox homeostasis, we hypothesized that polymorphisms in GSTO1 and GSTO2 might contribute to prostate cancer (PC) development and progression. Therefore, we performed a comprehensive analysis of GSTO1 and GSTO2 SNPs' role in susceptibility to PC, as well as whether they might serve as prognostic biomarkers independently or in conjunction with other common GST polymorphisms (GSTM1, GSTT1, and GSTP1). Genotyping was performed in 237 PC cases and 236 age-matched controls by multiplex PCR for deletion of GST polymorphisms and quantitative PCR for SNPs. The results of this study, for the first time, demonstrated that homozygous carriers of both GSTO1*A/A and GSTO2*G/G variant genotypes are at increased risk of PC. This was further confirmed by haplotype analysis, which showed that H2 comprising both GSTO1*A and GSTO2*G variant alleles represented a high-risk combination. However, the prognostic relevance of polymorphisms in GST omega genes was not found in our cohort of PC patients. Analysis of the role of other investigated GST polymorphisms (GSTM1, GSTT1, and GSTP1) in terms of PC prognosis has shown shorter survival in carriers of GSTP1*T/T (rs1138272) genotype than in those carrying at least one referent allele. In addition, the presence of GSTP1*T/T genotype independently predicted a four-fold higher risk of overall mortality among PC patients. This study demonstrated a significant prognostic role of GST polymorphism in PC.
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Project description:Fusion protein tags are widely used to capture and track proteins in research and industrial bioreactor processes. Quantifying fusion-tagged proteins normally requires several purification steps coupled with classical protein assays. Here, we developed a broadly applicable nanosensor platform that quantifies glutathione-S-transferase (GST) fusion proteins in real-time. We synthesized a glutathione-DNA-carbon nanotube system to investigate glutathione-GST interactions via semiconducting single-walled carbon nanotube (SWCNT) photoluminescence. We found that SWCNT fluorescence wavelength and intensity modulation occurred specifically in response to GST and GST-fusions. The sensor response was dependent on SWCNT structure, wherein mod(n - m, 3) = 1 nanotube wavelength and intensity responses correlated with nanotube diameter distinctly from mod(n - m, 3) = 2 SWCNT responses. We also found broad functionality of this sensor to diverse GST-tagged proteins. This work comprises the first label-free optical sensor for GST and has implications for the assessment of protein expression in situ, including in imaging and industrial bioreactor settings.
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Project description:The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.
| S-EPMC1152740 | biostudies-other
Project description:BackgroundGlutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.ResultsThe immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death.The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors.The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes.ConclusionTherefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in vitro doxorubicin treatment significantly reduces the expression of GCLC and GSS genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer.
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