Electrostatic Association of Glutathione Transferase to the Nuclear Membrane

Abstract

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 S-transferases (GSTs)2 are a superfamily of enzymes that protect the cell from toxic endogenous or xenobiotic compounds. Eight different gene-independent, isoenzymatic classes have been found in mammals, and two of these, Alpha and Mu classes, are abundantly expressed in rat liver, where they represent 43 and 56%, respectively, of the entire pool of cytosolic GSTs (1). Beside their catalytic competence to conjugate GSH to many toxic substances having an electrophilic center, these enzymes also display peroxidase activity and ligandin properties (2, 3). In hepatocytes, Alpha GSTs efficiently trap a natural nitric oxide carrier, the dinitrosyl-diglutathionyl-iron complex (DNDGIC), thus preventing the irreversible inhibition of glutathione reductase caused by this compound (4). Preliminary evidence was also reported that GSTs could be associated with the nuclear membrane or compartmentalized in nuclei. The presence of GSTs on intracellular membranes is not a new finding. MGST1 is a peculiar trimeric microsomal integral membrane isoenzyme discovered and characterized many years ago (5). GSTA4-4, a specific isoenzyme able to detoxify hydroxyalkenals, displays a widespread mitochondrial, peroxisomal, and cytosolic localization, but the plasma membrane also binds detectable amounts of this enzyme (6). Furthermore, the tight association of Alpha and Mu class GSTs with the microsomal membrane fraction of rat liver was demonstrated by Morgenstern et al. (7), and about 2% of the cytosolic GSTA1-1 has been found in the microsomal membrane...

Glutathione Transferases Sequester Toxic Dinitrosyl-Iron Complexes in Cells

“…(a) In the male rat liver, Alpha and Mu GSTs are 43 and 56%, respectively (29,30). These values were confirmed for our male rat liver preparations by means of high pressure liquid chromatography (31). (b) Specific activities of Alpha and Mu GSTs are 16 and 22 units/mg, respectively.…”

“…Unexpectedly, after treatment with 10 mM KCN to displace the complex (9), we find considerable GST activity associated with these components (not shown). The accompanying paper (31) demonstrates that the EPR signal is entirely because of the DNGIC-GST species and that significant amounts of GSTs, mainly GSTA1-1 and GSTA2-2, are associated with the nucleus.…”

Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function

“…In fact, ϳ30% of the entire pool of AlphaGSTs is concentrated near the inner and outer nuclear envelope, forming, as with many other enzymes, a defense barrier for DNA termed "nuclear shield" (31,32). For this peculiar role, some Alpha GSTs developed an extraordinary affinity during evolution but, as it appears, at the expense of their catalytic efficiency.…”

Glutathione Transferase P1 Interacts Strongly with the Inner Leaflet of the Plasma Membrane

“…A peripheral association of P and Mu GST isoforms with the plasma membrane of goat sperm has been described previously (Hemachand et al, 2002). Furthermore, murine GSTA4-4 and several human GSTA isoforms were found to associate with the plasma membrane and outer nuclear membrane, respectively, through electrostatic interactions (Singh et al, 2002;Stella et al, 2007). We have found that GSTP1 is still associated with the plasma membrane after high salt treatment and alkaline carbonate extraction, ruling out the possibilities of electrostatic and/or peripheral interaction of GSTP1 with the plasma membrane.…”