M Lo Bello

Three-dimensional structure of class π glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 Å resolution☆

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitors glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

STRUCTURAL FLEXIBILITY MODULATES THE ACTIVITY OF HUMAN GLUTATHIONE TRANSFERASE P1-1 : ROLE OF HELIX 2 FLEXIBILITY IN THE CATALYTIC MECHANISM

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the σ-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k°cat/kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for KGSHm, while KCDNBm is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and KGSHm seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and KGSHm, thereby suggesting a possible correlation between kcat, KGSHm, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of KGSHm, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.

Antioxidant enzymes in blood of patients with Friedreich's ataxia

Background and Aims: Increased generation of reactive oxygen species and mitochondrial dysfunction may underlie the pathophysiology of Friedreichs ataxia, the most common inherited ataxia, due to GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Because iron overload would cause oxidative stress in Friedreichs ataxia, we investigated the enzyme antioxidant system in the blood of 14 patients by determining superoxide dismutase, glutathione peroxidase, and glutathione trasferase catalytic activities. We also studied the glutathione S-transferase genotype polymorphism in order to evaluate its possible influence on enzyme activity. Methods: Blood samples were obtained from 14 unrelated patients with Friedreichs ataxia and 21 age matched healthy subjects. Antioxidant enzyme determinations were spectrophotometrically assayed using specific substrates; the glutathione S-transferase genotype polymorphism was analysed by endonuclease restriction mapping of exon 5 and 6 amplification products. Results: There was a significant elevation of the superoxide dismutase/glutathione peroxidase activity ratio (0.037 (0.01) v 0.025 (0.008) of controls) and an 83% rise of glutathione transferase specific activity (0.22 (0.1) v 0.12 (0.03) nmol/min/mg protein) in blood of patients with Friedreichs ataxia than in the controls. The genotype polymorphism of glutathione S-transferase enzyme did not show any relevant differences when compared to that of healthy subjects. Conclusions: Data show an impairment in vivo of antioxidant enzymes in patients with Friedreichs ataxia and provide evidence of an increased sensitivity to oxidative stress, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.

Proton release on binding of glutathione to alpha, Mu and Delta class glutathione transferases.

Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k(1)>/=(2-5)x10(6) M(-1).s(-1)], which is slowly converted into the final Michaelis complex (k(2)=1100-1200 s(-1)). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.

Silencing of GSTP1, a prostate cancer prognostic gene, by the estrogen receptor-β and endothelial nitric oxide synthase complex.

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-β and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERβ/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERβ antagonist or its natural ligand 5α-androstane-3β,17β-diol, eNOS inhibitors or ERβ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERβ/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERβ/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERβ/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERβ/eNOS function by 3β-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.