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Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

ABSTRACT: Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M(w) of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M(w) of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K(m) value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.

SUBMITTER: Ralat LA

PROVIDER: S-EPMC4939725 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

Ralat Luis A LA Misquitta Stephanie A SA Manevich Yefim Y Fisher Aron B AB Colman Roberta F RF

Archives of biochemistry and biophysics 20080310 1

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M(w) of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between ...[more]

PMID: 18358825

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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.

Project description:IntroductionPlasma biomarkers may be useful in diagnosing acute cerebral infarction requiring urgent reperfusion, but their performance remains to be confirmed. If confirmed, these molecules could be used to develop rapid and reliable decentralised measurement methods, making it possible to initiate reperfusion therapy before hospital admission. The FLAG-1 large prospective study will constitute a plasma bank to assess the diagnostic performance of two biomarkers: glutathione S-transferase-π and peroxiredoxin 1. These molecules are involved in the oxidative stress response and could identify cerebral infarction within a therapeutic window of less than 4.5 hours following the onset of symptoms. Secondary objectives include assessing performance of these biomarkers within 3-hour and 6-hour windows; identifying additional biomarkers diagnosing cerebral infarction and significant criteria guiding therapeutic decisions: ischaemic features of stroke, presence of diffusion/fluid-attenuated inversion recovery mismatch, volume of cerebral infarction and penumbra on cerebral MRI.Methods and analysisThe exploratory, prospective, multicentre FLAG-1 Study will include 945 patients with acute stroke symptoms (onset ≤12 hours, National Institute of Health Stroke Scale score ≥3). Each patient's 25 mL blood sample will be associated with cerebral MRI data. Two patient groups will be defined based on the time of blood collection (before and after 4.5 hours following onset). Receiver operating characteristic analysis will determine the diagnostic performance of each biomarker, alone or in combination, for the identification of cerebral infarction <4.5 hours.Ethics and disseminationThe protocol has been approved by an independent ethics committee. Biological samples are retained in line with best practices and procedures, in accordance with French legislation. Anonymised data and cerebral imaging records are stored using electronic case report forms and a secure server, respectively, registered with the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés (CNIL)). Results will be disseminated through scientific meetings and publication in peer-reviewed medical journals.Trial registration numberClinicalTrials.gov Registry (NCT03364296).

Dataset Information

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

Publications

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

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