Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

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

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 natur

A new schedule of fotemustine in temozolomide-pretreated patients with relapsing glioblastoma

In the present study we investigated the feasibility and effectiveness of a new biweekly schedule of fotemustine (FTM) in patients with recurrent glioblastoma, after at least one previous treatment. The primary endpoint was progression-free survival at 6 months; secondary objectives were clinical response, overall survival, disease-free survival, and toxicity. Forty patients (median age 52.8 years; median Karnofsky Performance Status at progression 90) underwent second-line chemotherapy with FTM. Selected patients were previously treated with a standard radiotherapy course with concomitant temozolomide (TMZ). After tumor relapse or progression proven by magnetic resonance imaging (MRI), all patients underwent chemotherapy with FTM, given intravenously at dose of 80 mg/m2 every 2 weeks for five consecutive administrations (induction phase), and then every 3 weeks at 100 mg/m2 as maintenance. A total of 329 infusions were administered; the median number of cycles administered was 8. All patients completed the induction phase, and 29 patients received at least one maintenance infusion. Response to treatment was assessed using MacDonald criteria. One complete response [2.5%, 95% confidence interval (CI): 0–10%], 9 partial responses (22.5%, 95% CI: 15–37%), and 16 stable diseases (40%, 95% CI: 32–51%) were observed. Median time to progression was 6.7 months (95% CI: 3.9–9.1 months). Progression-free survival at 6 months was 61%. Median survival from beginning of FTM chemotherapy was 11.1 months. The schedule was generally well tolerated; the main toxicities were hematologic (grade 3 thrombocytopenia in two cases). To the best of our knowledge, this is the first report specifically dealing with the use of a biweekly induction schedule of FTM. The study demonstrates that FTM has therapeutic efficacy as single-drug second-line chemotherapy with a favorable safety profile

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

International audienceNegative cooperativity in enzyme reactions - in which the first event makes subsequent events less favorable - is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (though smaller values of nH are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role - safeguarding DNA from DNDGIC - display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs beside the well-known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity

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 a- 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 4264 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 disease

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) 1. BioL Chem. 282, 6364 - 637 1) prompted us to reconsi-der 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

Reactivation rate of GSTP1-1.

<p>Purified GSTP1-1 (20 pmoles) was incubated with HOSCN (10 µM, final concentration) for 20 min, 25°C. Then the samples were incubated with DTT at different concentrations for 2.5 min, 37°C in potassium phosphate buffer 0.1 M, pH 8.4, and the activity was measured. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inactivation of GSTP1-1 by GSSG and CysSSCys.

<p>(A) GSTP1-1 (20 pmoles) incubated at 25°C with variable amounts of oxidized glutathione or oxidized cysteine in 70 µl of 0.1 M potassium phosphate buffer, pH 7.0. For comparison the same amount of GSTP1-1 was incubated with 70 µl saliva. (B) pH dependence of the inactivation by cystine (100 µM). The activity was evaluated after 5 min of incubation. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inhibition of purified GSTP1-1 by H₂O₂.

<p>GSTP1-1 (20 pmoles) was incubated at 25°C with two different concentrations of H₂O₂, 10 µM, corresponding to the physiological level (open triangle) and 100 µM (open square) in 70 µl potassium phosphate buffer, pH 7.0. For comparison GSTP1-1 (20 pmoles) was also incubated with 70 µl of saliva (open circle). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inactivation of GSTP1-1 by human defensins (HNP-1 and HNP-2).

<p>GSTP1-1 (90 pmoles) was incubated at 25°C with HNP-1 (0.54 nmoles) (open triangle), HNP-2 (open diamond) (0.54 nmoles) and both HNP-1 0.27 nmoles and HNP-2 (0.27 nmoles) (open square) in 70 µl (final volume) of potassium phosphate buffer, pH 7.0 (these defensin levels reproduce the average concentration of these proteins in saliva). An identical amount of GSTP1-1 was also incubated with 70 µl saliva (open circle). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p