Erythrocyte glutathione transferase: a potential new biomarker in chronic kidney diseases which correlates with plasma homocysteine.

The erythrocyte glutathione S-transferase (e-GST) is a member of a superfamily of inducible enzymes involved in cell detoxification that shows an increased expression in chronic kidney disease (CKD) patients. We propose a new automated analysis procedure for e-GST activity that has been validated in 72 CKD patients and 62 maintenance hemodialysis patients (MHD). Regression analysis was carried out to assess association between e-GST activity data, main clinical variables, and plasma homocysteine (Hcy), a modified sulfur amino acid known as potential risk factor for cardiovascular disease that is increased above normal levels in more than 90% of the uremic patients. An increased e-GST activity was confirmed in MHD patients (N=62; 10.2±0.4 U/gHb) compared with healthy subjects (N=80; 5.8±0.4 U/gHb), and as an original finding, a significant increase of e-GST activity was observed in pre-dialysis CKD patients with a positive correlation with disease severity weighted according to the four stages of "Kidney Disease Outcomes Quality Initiative" classification (7.4±0.5, 8±1, 9.5±0.6, 12±1 U/gHb, respectively). No correlation was found between e-GST activity and hemoglobin, transferrin, blood iron and the markers of systemic inflammation and renal function such as alpha-1 acid glycoprotein and high-sensitive C-Reactive Protein, beta-2 microglobulin and the index of malnutrition-inflammation PINI, while a significant correlation was observed for the first time between plasma Hcy and e-GST activity (r2=0.64, P<0.0001) in MHD patients. Hcy, however, was not identified as an inhibitor of e-GST enzyme. The results in this study suggest the potential for automated e-GST analysis as a valuable tool to further explore phase II-related uremic toxicity in CKD and MHD patients.

Trypanothione efficiently intercepts nitric oxide as a harmless iron complex in trypanosomatid parasites.

Trypanosomatids are protozoan organisms that cause serious diseases, including African sleeping sickness, Chagas' disease, and leishmaniasis, affecting about 30 million people in the world. These parasites contain the unusual dithiol trypanothione [T(SH)(2)] instead of glutathione (GSH) as the main intracellular reductant, and they have replaced the otherwise ubiquitous GSH/glutathione reductase redox couple with a T(SH)(2)/trypanothione reductase (TR) system. The reason for the existence of T(SH)(2) in parasitic organisms has remained an enigma. Here, we show that T(SH)(2) is able to intercept nitric oxide and labile iron and form a dinitrosyl-iron complex with at least 600 times higher affinity than GSH. Accumulation of the paramagnetic dinitrosyl-trypanothionyl iron complex in vivo was observed in Trypanosoma brucei and Leishmania infantum exposed to nitric oxide. While the analogous dinitrosyl-diglutathionyl iron complex formed in mammalian cells is a potent irreversible inhibitor of glutathione reductase (IC(50)=4 microM), the T(SH)(2) complex does not inactivate TR even at millimolar levels. The peculiar capacity of T(SH)(2) to sequester NO and iron in a harmless stable complex could explain the predominance of this thiol in parasites regularly exposed to NO.-Bocedi, A., Dawood, K. F., Fabrini, R., Federici, G., Gradoni, L., Pedersen, J. Z., Ricci, G. Trypanothione efficiently intercepts nitric oxide as a harmless iron complex in trypanosomatid parasites.

The extended catalysis of glutathione transferase.

Glutathione transferase reaches 0.5-0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S]≪[E]. As glutathione transferase lowers the pK(a) of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate-enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual "inverted" kinetics whereby the classical trends of v versus E and v versus S are reversed.

Tetramerization and cooperativity in Plasmodium falciparum glutathione S-transferase are mediated by atypic loop 113-119.

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.

Electrostatic association of glutathione transferase to the nuclear membrane. Evidence of an enzyme defense barrier at the nuclear envelope.

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.

Glutathione transferases sequester toxic dinitrosyl-iron complexes in cells. A protection mechanism against excess nitric oxide.

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.