New physiological roles of glautathione transferases

Glutathione transferases (GSTs) are enzymes able to conjugate GSH to a lot of toxic compounds thereby favoring their excretion. Recently, other protective roles of these enzymes have been discovered. In particular, it has been observed that a peculiar and strong interaction exists between some mammalian GSTs and an endogenous carrier of nitric oxide, the dinitrosyl-diglutathionyl iron complex (DNDGIC). This iron complex is a paramagnetic molecule with a characteristic EPR spectrum centered at g = 2.03, that is spontaneously formed when NO enters the cell. This complex is a strong irreversible inhibitor of glutathione reductase. The present work explores the possible role of GSTs like a protection system against DNDGIC. Actually, mammalian GSTs bind DNDGIC with extraordinary affinity (KD = 10-9-10-10 M). When rat hepatocytes are incubated in the presence of GSNO, a natural source of NO, a rapid formation of 0.1 - 0.2 mM intracellular DNDGIC has been observed. This concentration would be lethal for glutathione reductase. However the complex does not appear like a free species but completely bound to GSTs, that are present at the cytosolic level of 0.8 mM. In this form the complex is completely harmless for glutathione reductase. Surprisingly, electron paramagnetic data, reveal that DNGIC-GST is partially associated to subcellular fractions and in particular to nuclei. Our data indicate that about 10% of the cytosolic pool GST 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 and immunofluorescence experiments have been used to detail the electrostatic association in hepatocytes. 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 a potent protection shell for the nucleus and an amazing novelty in cell physiology. A second target of this study is represented by the particular GST isoenzyme expressed by the Plasmodium falciparum (PfGST), the parasite causative of malaria. This enzyme is characterized by a peculiar dimer/tetramer transition that occurs in the absence of GSH and that causes a total loss of its enzymatic activity. Moreover PfGST binds hemin with high affinity and this interaction is finalized to the protection of the parasite against this toxic compound. Binding of hemin is regulated by a cooperative mechanism and does not occur in the tetrameric enzyme. Side directed mutagenesis, steady-state kinetic experiments, fluorescence anisotropy and X-ray crystallography were used to verify the involvement of some protein segment in the tetramerization process and in the cooperative phenomenon. Actually the loop 113-118 represents one the most prominent structural difference between PfGST and other GSTs. Our results demonstrate that truncation, increased rigidity or even a simple point mutation of this loop cause a dramatic change of the tetramerization kinetics that becomes hundred times slower than that observed in the native enzyme. Furthermore all mutants loose the positive cooperativity for hemin binding found in the native structure suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process, that is very fast in the absence of GSH in the native enzyme, is prevented not only by GSH but even by GSSG. This result indicate that the protection of the parasite against free hemin is independent of the redox status of the cell.