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Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of n H are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an approximately 3 to approximately 5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113-119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (GSTs) with extraordinary high affinity (K(D) = 10(-10) m). This complex is formed spontaneously through NO-mediated extraction of iron from ferritin and transferrin, in a reaction that requires only glutathione. In hepatocytes, DNDGIC may reach concentrations of 0.19 mm, apparently entirely bound to Alpha class GSTs, present in the cytosol at a concentration of about 0.3 mm. Surprisingly, about 20% of the dinitrosyl-glutathionyl-iron complex-GST is found to be associated with subcellular components, mainly the nucleus, as demonstrated in the accompanying paper (Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z., and Ricci, G. (2007) J. Biol. Chem. 282, 6372-6379). DNDGIC is a potent irreversible inhibitor of glutathione reductase, but the strong complex-GST interaction ensures full protection of glutathione reductase activity in the cells, and in vitro experiments show that damage to the reductase only occurs when the DNDGIC concentration exceeds the binding capacity of the intracellular GST pool. Because Pi class GSTs may exert a similar role in other cell types, we suggest that specific sequestering of DNDGIC by GSTs is a physiological protective mechanism operating in conditions of excessive levels of nitric oxide.

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoprotein-positive tumors.

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.

Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK.GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a sigma complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the sigma complex was the favored event. Docking studies demonstrated the likely localization of the sigma complex in the GST active sites and provide a structural explanation for its strong stabilization.

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions.

Increasing evidence suggests that iron-mediated oxidative stress might underlie the development of neurodegeneration in Friedreich's ataxia (FRDA), an autosomal recessive ataxia caused by decreased expression of frataxin, a protein implicated in iron metabolism. In this study, we demonstrate that, in fibroblasts of patients with FRDA, the cellular redox equilibrium is shifted toward more protein-bound glutathione. Furthermore, we found that actin is glutathionylated, probably as a result of the accumulation of reactive oxygen species, generated by iron overload in the disease. Indeed, high-pressure liquid chromatography analysis of control fibroblasts in vivo treated with FeSO4 showed a significant increase in the protein-bound/free GSH ratio, and Western blot analysis indicated a relevant rise in glutathionylation. Actin glutathionylation contributes to impaired microfilament organization in FRDA fibroblasts. Rhodamine phalloidin staining revealed a disarray of actin filaments and a reduced signal of F-actin fluorescence. The same hematoxylin/eosin-stained cells showed abnormalities in size and shape. When we treated FRDA fibroblasts with reduced glutathione, we obtained a complete rescue of cytoskeletal abnormalities and cell viability. Thus, we conclude that oxidative stress may induce actin glutathionylation and impairment of cytoskeletal functions in FRDA fibroblasts.

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (KD congruent with 10-10 m), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (KD congruent with 10-9 m). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (KD congruent with 10-7 m), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitroso-glutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione S-transferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1/2 = 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1/2 = 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.

The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.