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

Ralat, Luis A.; Misquitta, Stephanie A.; Manevich, Yefim; Fisher, Aron B.; Colman, Roberta F.

doi:10.1016/j.abb.2008.02.043

Cited by 49 publications

(62 citation statements)

References 32 publications

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“…This involves heterodimerization with GSH transferase (GST), followed by GSH-mediated reduction and enzyme reactivation (27). GST is thought to serve as a source of activated GSH catalyzing glutathionylation of the oxidized peroxidatic cysteine residue, and GSH is the reductant in this mechanism which regenerates the active 1-Cys peroxiredoxin (28,40). More recently, ascorbate has been proposed to function as a reductant for 1-Cys peroxiredoxins from diverse species (31).…”

Section: Discussionmentioning

confidence: 99%

Antioxidant Activity of the Yeast Mitochondrial One-Cys Peroxiredoxin Is Dependent on Thioredoxin Reductase and Glutathione In Vivo

Greetham

Grant

2009

Molecular and Cellular Biology

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Peroxiredoxins are ubiquitous enzymes which protect cells against oxidative stress. The first step of catalysis is common to all peroxiredoxins and results in oxidation of a conserved peroxidatic cysteine residue to sulfenic acid. This forms an intermolecular disulfide bridge in the case of 2-Cys peroxiredoxins, which is a substrate for the thioredoxin system. 1-Cys Prx's contain a peroxidatic cysteine but do not contain a second conserved cysteine residue, and hence the identity of the in vivo reduction system has been unclear. Here, we show that the yeast mitochondrial 1-Cys Prx1 is reactivated by glutathionylation of the catalytic cysteine residue and subsequent reduction by thioredoxin reductase (Trr2) coupled with glutathione (GSH). This novel mechanism does not require the usual thioredoxin (Trx3) redox partner of Trr2 for antioxidant activity, although in vitro assays show that the Trr2/Trx3 and Trr2/GSH systems exhibit similar capacities for supporting Prx1 catalysis. Our data also indicate that mitochondria are a main target of cadmium-induced oxidative stress and that Prx1 is particularly required to protect against mitochondrial oxidation. This study demonstrates a physiological reaction mechanism for 1-Cys peroxiredoxins and reveals a new role in protection against mitochondrial heavy metal toxicity.All aerobic organisms are exposed to reactive oxygen species (ROS) during the course of normal aerobic metabolism or following exposure to radical-generating compounds. ROS cause wide-ranging damage to macromolecules, which can result in genetic degeneration, physiological dysfunction, and eventual cell death (18,19). Sulfhydryls play a key role in the response to oxidative stress, regulated primarily by the glutathione (GSH)/glutaredoxin and thioredoxin systems (9,19,42,44). These redox systems were originally identified as hydrogen donors for ribonucleotide reductase, but they also act upon metabolic enzymes that form a disulfide as part of their catalytic cycle. They have proposed roles in diverse processes, including protein folding and regulation, reduction of dehydroascorbate, repair of oxidatively damaged proteins, and sulfur metabolism (20,42). Glutaredoxins and thioredoxins are structurally and functionally conserved. Despite this considerable functional overlap, they are differentially regulated. The oxidized disulfide form of thioredoxin is reduced directly by NADPH and thioredoxin reductase, whereas glutaredoxin is reduced by GSH using electrons donated by NADPH via GSH reductase. The two systems are therefore thermodynamically linked, as each uses NADPH as a source of reducing equivalents.During respiration, mitochondria are the primary source of ROS in the cell, and complex III of the respiratory chain is responsible for approximately 80% of ROS production (4, 5). Mitochondrial ROS cause wide-ranging damage to various cellular organelles and tissues and have been implicated in a number of disease processes. Not surprisingly, therefore, mitochondrial thiols are major ROS targets, a fact which...

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Section: Discussionmentioning

confidence: 99%

Antioxidant Activity of the Yeast Mitochondrial One-Cys Peroxiredoxin Is Dependent on Thioredoxin Reductase and Glutathione In Vivo

Greetham

Grant

2009

Molecular and Cellular Biology

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“…GSTpi was shown to form a complex with the inactive form of 1-CysPrx (both in vivo and in vitro), which accounts for reactivation of 1-Cys-Prx. 4,13 Mitogen-activated protein kinases (MAPKs) are eukaryotic enzymes at the core of the MAPK cascade. They transduce signals sent by cell surface receptors to important intracellular targets (both cytosolic and nuclear), controlling cell proliferation, differentiation, inflammation, and apoptosis.…”

Section: Introductionmentioning

confidence: 99%

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

et al. 2011

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Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK 1 ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.

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“…Chaotropic salts have been used to disrupt electrostatic interactions in proteins by shielding charges between subunits (20 -22). Studies conducted in our laboratory showed that the molecular mass of GST is decreased by the addition of potassium bromide, implying that electrostatic interactions are important forces holding the subunits together (23).…”

mentioning

confidence: 99%

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Huang

Misquitta

Blond

et al. 2008

Journal of Biological Chemistry

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Human glutathione transferase (GST ) has been crystallized as a homodimer, with a subunit molecular mass of ϳ23 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 , 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 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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Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Cited by 49 publications

References 32 publications

Antioxidant Activity of the Yeast Mitochondrial One-Cys Peroxiredoxin Is Dependent on Thioredoxin Reductase and Glutathione In Vivo

Antioxidant Activity of the Yeast Mitochondrial One-Cys Peroxiredoxin Is Dependent on Thioredoxin Reductase and Glutathione In Vivo

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

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