Marzia Nuccetelli

Nitrosylation of Human Glutathione Transferase P1-1 with Dinitrosyl Diglutathionyl Iron Complex in Vitro and in Vivo

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.

Orexinergic system dysregulation, sleep impairment, and cognitive decline in Alzheimer disease.

IMPORTANCE Nocturnal sleep disruption develops in Alzheimer disease (AD) owing to the derangement of the sleep-wake cycle regulation pathways. Orexin contributes to the regulation of the sleep-wake cycle by increasing arousal levels and maintaining wakefulness. OBJECTIVES To study cerebrospinal fluid levels of orexin in patients with AD, to evaluate the relationship of orexin cerebrospinal fluid levels with the degree of dementia and the cerebrospinal fluid AD biomarkers (tau proteins and β-amyloid 1-42), and to analyze potentially related sleep architecture changes measured by polysomnography. DESIGN, SETTING, AND PARTICIPANTS We conducted a case-control study from August 1, 2012, through May 31, 2013. We included 48 drug-naive AD patients referred to the Neurological Clinic of the University Hospital of Rome Tor Vergata. Based on the Mini-Mental State Examination score, 21 patients were included in mild AD group (score, ≥21), whereas 27 were included in the moderate to severe AD group (score, <21). The control group consisted of 29 nondemented participants of similar age and sex. EXPOSURE Laboratory assessment of cerebrospinal fluid levels of orexin, tau proteins, and β-amyloid 1-42 and polysomnographic assessment of sleep variables. MAIN OUTCOMES AND MEASURES Levels of orexin, tau proteins, and β-amyloid 1-42; macrostructural variables of nocturnal sleep (total sleep time, sleep efficiency, sleep onset and rapid eye movement [REM] sleep latencies, non-REM and REM sleep stages, and wakefulness after sleep onset); and Mini-Mental State Examination scores. RESULTS Patients with moderate to severe AD presented with higher mean (SD) orexin levels compared with controls (154.36 [28.16] vs 131.03 [26.55]; P < .01) and with more impaired nocturnal sleep with respect to controls and patients with mild AD. On the other hand, in the global AD group, orexin levels were positively correlated with total tau protein levels (r = 0.32; P = .03) and strictly related to sleep impairment. Finally, cognitive impairment, as measured by the Mini-Mental State Examination, was correlated with sleep structure deterioration. CONCLUSIONS AND RELEVANCE Our results demonstrate that, in AD, increased cerebrospinal fluid orexin levels are related to a parallel sleep deterioration, which appears to be associated with cognitive decline. Therefore, the orexinergic system seems to be dysregulated in AD, and its output and function appear to be overexpressed along the progression of the neurodegenerative process. This overexpression may result from an imbalance of the neurotransmitter networks regulating the wake-sleep cycle toward the orexinergic system promoting wakefulness.

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.

Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2,4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N., Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G. (1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines.

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1–1 (GST P1–1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35 °C, glutathione (GSH) binding to GST P1–1 displays positive cooperativity, whereas negative cooperativity occurs below 25 °C. This binding mechanism minimizes changes of GSH affinity for GST P1–1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1–1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.

Aβ1–42 Detection in CSF of Alzheimer's disease is influenced by temperature: Indication of reversible Aβ1–42 aggregation?

Amyloid-beta 1-42 (Abeta1-42), peptide detectable in cerebrospinal fluid (CSF), has been extensively studied as diagnostic marker for Alzheimers disease; however, results are variable. We investigated whether Abeta1-42 detection in CSF may be affected by handling temperature after lumbar puncture. CSF was collected from patients affected by probable AD (n=27), other dementias (OD) (n=24), or other neurological disorders without cognitive impairment (OND) (n=23). After lumbar puncture, CSF samples were either maintained at 37 degrees C, or handled according to standard procedures and centrifuged at 4 degrees C for 10 min; thereafter, one aliquot was further stored at 4 degrees C and another at 37 degrees C, before freezing all samples 90 min later at -80 degrees C, pending analysis. Abeta1-42 and total tau were determined using a commercially available sandwich enzyme-linked immunosorbent assay ELISA. Reduced Abeta1-42 and increased total tau CSF levels were confirmed as characteristic hallmarks of the OD and AD groups, providing standard measurement in samples stored at 4 degrees C before freezing. However, avoiding cooling or reheating CSF from 4 to 37 degrees C before freezing strikingly increased the Abeta1-42 concentration detectable in the AD group (P<0.01), but not in control groups. The results indicate that a pool of Abeta1-42 cannot be detectable in the CSF of AD patients, because standard preanalytical cooling masks in some ways the epitope recognized by Abeta1-42 specific antibodies. Moreover, our study suggests that low temperature could induce Abeta1-42 conformational changes and multimeric aggregates in probable AD, but, more importantly, Abeta1-42 aggregation could be reversible.

Screening of antinuclear antibodies: comparison between enzyme immunoassay based on nuclear homogenates, purified or recombinant antigens and immunofluorescence assay

Abstract Current clinical practice considers antinuclear antibody (ANA) testing as a screening test; this has a major impact on laboratory work with a growing volume of analyses that need to be performed rapidly, to maintain good specificity and sensitivity. Ongoing discussions have been raised in order to identify the best technology to use in ANA screening, taking into account both clinical and economical implications. The aim of our study was to compare three different enzyme immunoassays (EIA) with immunofluorescence (IF) assay in order to identify which test is better for use as a screening test. The study was performed on 473 sera and the three different EIA tests were based on nuclear homogenates from HeLa cells, purified antigens from HEp-2 cells and recombinant antigens, respectively. The concordance between EIA-ANA and IF-ANA techniques, determined by the κ statistic, was acceptable, but not complete, and discrepancies between both EIA-positive/IF-negative samples and IF-positive/EIA-negative were found. Both methods show interesting diagnostic abilities, however, the IF-ANA assay seems to be the first choice test in a well-standardized immunofluorescence laboratory with experienced microscopists, whereas the EIA test might be useful especially in large-scale ANA screening.

CSF beta-amyloid levels are altered in narcolepsy: a link with the inflammatory hypothesis?

Narcolepsy is characterized by hypocretin deficiency due to the loss of hypothalamic orexinergic neurons, and is associated with both the human leucocyte antigen DQB1*06:02 and the T cell receptor polymorphism. The above relationship suggests autoimmune/inflammatory processes underlying the loss of orexinergic neurons in narcolepsy. To test the autoimmune/inflammatory hypothesis by means of cerebrospinal fluid (CSF) levels of beta‐amyloid1–42 and/or total tau proteins in a sample of narcoleptic patients, we analysed 16 narcoleptic patients and 16 healthy controls. Beta‐amyloid1–42 CSF levels were significantly lower in narcoleptic patients compared with healthy controls. We also documented pathologically low levels of CSF beta‐amyloid1–42 (<500 pg mL−1) in six of 16 narcoleptic patients (37.5%). We hypothesize that the significant decrease of the CSF beta‐amyloid1–42 levels in narcoleptic patients may support both the inflammatory/autoimmune hypothesis as the basis of the pathogenesis of narcolepsy and the prevalence of an ‘amyloidogenic’ pathway caused by the deficiency of the alpha‐secretases enzymes.

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S‐nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1‐1 (hGSTP1‐1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild‐type hGSTP1‐1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild‐type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S‐nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix α2 of hGST P1‐1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild‐type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix α2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S‐nitrosylation of Cys47 by GSNO is a likely physiological scenario.