Molecular Mechanisms of Allosteric Inhibition of Brain Glycogen Phosphorylase by Neurotoxic Dithiocarbamate Chemicals.

Dithiocarbamates (DTCs) are important industrial chemicals used extensively as pesticides and in a variety of therapeutic applications. However, they have also been associated with neurotoxic effects and in particular with the development of Parkinson-like neuropathy. Although different pathways and enzymes (such as ubiquitin ligases or the proteasome) have been identified as potential targets of DTCs in the brain, the molecular mechanisms underlying their neurotoxicity remain poorly understood. There is increasing evidence that alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. Interestingly, recent studies with N,N-diethyldithiocarbamate suggest that brain glycogen phosphorylase (bGP) and glycogen metabolism could be altered by DTCs. Here, we provide molecular and mechanistic evidence that bGP is a target of DTCs. To examine this system, we first tested thiram, a DTC pesticide known to display neurotoxic effects, observing that it can react rapidly with bGP and readily inhibits its glycogenolytic activity (kinact = 1.4 × 105 m-1 s-1). Using cysteine chemical labeling, mass spectrometry, and site-directed mutagenesis approaches, we show that thiram (and certain of its metabolites) alters the activity of bGP through the formation of an intramolecular disulfide bond (Cys318-Cys326), known to act as a redox switch that precludes the allosteric activation of bGP by AMP. Given the key role of glycogen metabolism in brain functions and neurodegeneration, impairment of the glycogenolytic activity of bGP by DTCs such as thiram may be a new mechanism by which certain DTCs exert their neurotoxic effects.

An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP.

Brain glycogen and its metabolism are increasingly recognized as major players in brain functions. Moreover, alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. In the brain, both muscle and brain glycogen phosphorylase isozymes regulate glycogen mobilization. However, given their distinct regulatory features, these two isozymes could confer distinct metabolic functions of glycogen in brain. Interestingly, recent proteomics studies have identified isozyme-specific reactive cysteine residues in brain glycogen phosphorylase (bGP). In this study, we show that the activity of human bGP is redox-regulated through the formation of a disulfide bond involving a highly reactive cysteine unique to the bGP isozyme. We found that this disulfide bond acts as a redox switch that precludes the allosteric activation of the enzyme by AMP without affecting its activation by phosphorylation. This unique regulatory feature of bGP sheds new light on the isoform-specific regulation of glycogen phosphorylase and glycogen metabolism.

Identification of cancer chemopreventive isothiocyanates as direct inhibitors of the arylamine N-acetyltransferase-dependent acetylation and bioactivation of aromatic amine carcinogens.

Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood. This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (k(i) = 200 M(-1).s(-1) and 66 M(-1).s(-1) for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals.

Protection and reversal of hepatic fibrosis by red wine polyphenols in hyperhomocysteinemic mice.

Hyperhomocysteinemia leads to several clinical manifestations and, particularly, liver disease. Lowering homocysteine through nutrition or other means might offer preventive or therapeutic benefits. Polyphenols are natural compounds known for their antioxidant and healing properties for vessels. In a previous study we have shown a beneficial effect of a red wine polyphenolic extract (PE) administration on plasma homocysteine level in cystathionine beta synthase deficient mice, a murine model of hyperhomocysteinemia. These mice also develop hepatic fibrosis. As increased matrix metalloproteinase (MMP) 2 has been shown to be involved in the development of hepatic fibrosis, we then focused on the effect of PE administration on expression and activity of MMP-2 in liver of hyperhomocysteinemic mice and its impact on hepatic fibrosis development. PE was added for four weeks to the drinking water of heterozygous cystathionine beta synthase-deficient mice fed a high-methionine diet. Effects of PE administration were examined by histological analysis with Sirius red staining, zymography, immunobloting, real-time quantitative reverse transcriptase polymerase chain reaction, peroxynitrite level, catalase activity and nicotinamide adenine dinucleotide phosphate oxidase activity. We show that administration of PE had a beneficial effect (i) on MMP-2 expression via modulation of nitrotyrosine-modified total protein level and (ii) on MMP-2 activity via modulation of its activator/inhibitor balance. We also demonstrated a reversal effect of PE supplementation on hepatic fibrosis development. Our results demonstrate a preventive action of PE administration on biomarkers of hepatic dysfunction due to hyperhomocysteinemia.

Progestins induce catalase activities in breast cancer cells through PRB isoform: correlation with cell growth inhibition.

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H(2)O(2)), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H(2)O(2) detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.

Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants.

Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and beta-naphthylamine (beta-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H(2)O(2) or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

Cystathionine beta synthase deficiency induces catalase-mediated hydrogen peroxide detoxification in mice liver.

Cystathionine beta synthase deficiency induces hyperhomocysteinemia which is considered as a risk factor for vascular diseases. Studies underlined the importance of altered cellular redox reactions in hyperhomocysteinemia-induced vascular pathologies. Nevertheless, hyperhomocysteinemia also induces hepatic dysfunction which may accelerate the development of vascular pathologies by modifying cholesterol homeostasis. The aim of the present study was to analyze the modifications of redox state in the liver of heterozygous cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia. In this purpose, we quantified levels of reactive oxygen and nitrogen species and we assayed activities of main antioxidant enzymes. We found that cystathionine beta synthase deficiency induced NADPH oxidase activation. However, there was no accumulation of reactive oxygen (superoxide anion, hydrogen peroxide) and nitrogen (nitrite, peroxynitrite) species. On the contrary, hepatic hydrogen peroxide level was decreased independently of an activation of glutathione-dependent mechanisms. In fact, cystathionine beta synthase deficiency had no effect on glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. However, we found a 50% increase in hepatic catalase activity without any variation of expression. These findings demonstrate that cystathionine beta synthase deficiency initiates redox disequilibrium in the liver. However, the activation of catalase attenuates oxidative impairments.

Identification of the xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 as a new target of cisplatin in breast cancer cells: molecular and cellular mechanisms of inhibition.

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that plays an important role in the biotransformation of aromatic drugs and carcinogens. NAT1 activity has long been associated with susceptibility to various cancers. Evidence for a role of NAT1 in malignant progression has also been obtained, particularly for breast and prostate cancer. Cisplatin is widely used in chemotherapy against human cancers, and it is thought to act principally by forming DNA adducts. However, recent studies have suggested that some of the pharmacological and/or toxicological effects of cisplatin may be due to the direct targeting and inhibition of certain cellular enzymes. We show here that the exposure of breast cancer cells, known to express functional NAT1 enzyme, to therapeutically relevant concentrations of cisplatin impairs the catalytic activity of endogenous NAT1. Endogenous NAT1 was also found to be inactivated, in vivo, in the tissues of mice treated with cisplatin. Mechanistic studies with purified human NAT1 indicated that this inhibition resulted from the irreversible formation of a cisplatin adduct with the active-site cysteine residue of the enzyme. Kinetic studies suggested that NAT1 interacts rapidly with cisplatin, with a second-order rate inhibition constant of 700 M(-1) min(-1). This rate constant is one the highest ever reported for the reaction of cisplatin with a biological macromolecule. Few enzymes have been clearly shown to be inactivated by cisplatin. We provide here molecular and cellular evidence suggesting that NAT1 is one of the targets of cisplatin in cells.

Nitration of a critical tyrosine residue in the allosteric inhibitor site of muscle glycogen phosphorylase impairs its catalytic activity.

Muscle glycogen phosphorylase (GP) is a key enzyme in glucose metabolism, and its impairment can lead to muscle dysfunction. Tyrosine nitration of glycogen phosphorylase occurs during aging and has been suggested to be involved in progressive loss of muscle performance. Here, we show that GP (in its T and R form) is irreversibly impaired by exposure to peroxynitrite, a biological nitrogen species known to nitrate reactive tyrosine residues, and to be involved in physiological and pathological processes. Kinetic and biochemical analysis indicated that irreversible inactivation of GP by peroxynitrite is due to the fast (k(inact)=3 x 10(4) M(-1) s(-1)) nitration of a unique tyrosine residue of the enzyme. Endogenous GP was tyrosine nitrated and irreversibly inactivated in skeletal muscle cells upon exposure to peroxynitrite, with concomitant impairment of glycogen mobilization. Ligand protection assays and mass spectrometry analysis using purified GP suggested that the peroxynitrite-dependent inactivation of the enzyme could be due to the nitration of Tyr613, a key amino acid of the allosteric inhibitor site of the enzyme. Our findings suggest that GP functions may be regulated by tyrosine nitration.

The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins.

Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus.