Raffaele Fabrini

Monomer−Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

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 (KD = 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.

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 (IC50=4 |xM), 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. FASEB J. 24, 1035‐1042 (2010). www.fasebj.org

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.

Erythrocyte glutathione transferase: a new biomarker for hemodialysis adequacy, overcoming the Kt/V(urea) dogma?

Kt/Vurea ratio is commonly used to assess the delivered dose of dialysis in maintenance hemodialysis (MHD) patients. This parameter only reflects the efficacy of dialytic treatments in removing small toxins, but not middle and protein-bound toxins. Erythrocyte glutathione transferase (e-GST), an enzyme devoted to cell depuration against a lot of large and small toxins, is overexpressed in uremic patients. Aim of the present study is to verify whether e-GST may represent a novel biomarker to assess the adequacy of different dialytic techniques complementary to Kt/Vurea parameter. Furthermore, it will be investigated whether e-GST could reflect the ‘average’ adequacy of multiple dialytic sessions and not of a single one treatment as it occurs for Kt/Vurea. One hundred and three MHD patients and 82 healthy subjects were tested. Fourty four patients were treated with standard bicarbonate hemodialysis (HD) and 59 patients were on online hemodiafiltration (HDF). In all MHD patients e-GST activity was 60% higher than in healthy controls. In HDF, e-GST activity was lower than in HD subgroup (8.2±0.4 versus 10.0±0.4 U/gHb, respectively). Single-pool Kt/Vurea and total weekly Kt/Vurea were higher in HDF than in HD, but no correlation was found between e-GST activity and Kt/Vurea data. e-GST, whose level is stable during the erythrocyte life-span, provides information on the long-term depurative efficacy of dialysis treatments.

The Impact of Nitric Oxide Toxicity on the Evolution of the Glutathione Transferase Superfamily: A PROPOSAL FOR AN EVOLUTIONARY DRIVING FORCE*

Background: Why do ancestral GSTs utilize cysteine/serine as catalytic residues, whereas more recently evolved GSTs utilize tyrosine? Results: Only the more recently evolved GSTs display enough affinity to bind and make harmless the toxic DNDGIC (a natural NO carrier). Conclusion: GST evolution could be linked to the defense against NO. Significance: This represents a further piece in the puzzle of evolutive adaptation to NO toxicity. 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 102–104 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.

Erythrocyte glutathione transferase: a novel biomarker to check environmental pollution hazardous for humans

Glutathione transferase (GST) is an enzyme capable of protecting the body from a lot of toxic compounds. Previous studies demonstrated that the erythrocyte GST (e-GST) expression increases as the level of circulating toxins increases. Aim of the present study is to verify if e-GST may represent a biomarker able to signalize an environmental pollution hazardous for humans. The study involved about 500 healthy volunteers living in eight distinct areas at or near the Sacco river valley, a region of the Frosinone district (Lazio-Italy) well known for its environmental pollution. Subjects of six areas displayed increased levels of e-GST ranging from 18% to 44% compared to 400 volunteers living in the Rome hinterland. Higher levels of GSTs are present in the areas where the risk of pollution is higher (areas 7 and 8). Interestingly, women living in the Sacco valley display much higher expression of e-GST than men, possibly due to a greater time exposition to the environmental contamination. Possible oxidative alteration of GST activity has not been observed. In conclusion, e-GST may represent an early and sensitive bio-signal of dangerous pollution for humans.

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.

Inactivation of Human Salivary Glutathione Transferase P1-1 by Hypothiocyanite: A Post-Translational Control System in Search of a Role

Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

Nuclear shield: a multi-enzyme task-force for nucleus protection.

Background In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions. Methodology/Principal Findings Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization. Conclusions/Significance The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes.