Methods to detect protein glutathionylation.

Glutathionylation is a posttranslational modification that results in the formation of a mixed disulfide between glutathione and the thiol group of a protein cysteine residue. Glutathionylation of proteins occurs via both nonenzymatic mechanisms involving thiol/disulfide exchange and enzyme-mediated reactions. Protein glutathionylation is observed in response to oxidative or nitrosative stress and is redox-dependent, being readily reversible under reducing conditions.

Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma.

The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation.

Oxidative Stress and the ER Stress Response in a Murine Model for Early-Stage Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD.

A synthetic chalcone as a potent inducer of glutathione biosynthesis.

Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2',5'-dihydroxychalcone (2',5'-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure-activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels.

The role of glutathione in brain tumor drug resistance.

Chemotherapy is central to the current treatment modality for primary human brain tumors, but despite high-dose and intensive treatment regimens there has been little improvement in patient outcome. The development of tumor chemoresistance has been proposed as a major contributor to this lack of response. While there have been some improvements in our understanding of the molecular mechanisms underlying brain tumor drug resistance over the past decade, the contribution of glutathione (GSH) and the GSH-related enzymes to drug resistance in brain tumors have been largely overlooked.

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32).

Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis.

Rapid activation of glutamate cysteine ligase following oxidative stress.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress.

Enhanced glutathione biosynthetic capacity promotes resistance to As3+-induced apoptosis.

Trivalent arsenite (As(3+)) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As(3+)-induced apoptosis.

Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity.

The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain.

Distinct Nrf1/2-independent mechanisms mediate As 3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes.

Trivalent arsenite (As(3+)) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As(3+). Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress.

Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes.

Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis.

Neutrophils are the primary inflammatory cell in smokers' lungs, but little is known about the ability of cigarette smoke to modulate neutrophil function. Neutrophils undergo caspase-3-dependent spontaneous, as well as phagocytosis-induced, apoptosis. This study investigated the ability of cigarette smoke extract (CSE) to alter neutrophil caspase-3 activity, apoptosis, and phagocytosis.

Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress.

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta).

Glutamate-cysteine ligase attenuates TNF-induced mitochondrial injury and apoptosis.

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM.

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress.

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH.

TGFbeta1-induced suppression of glutathione antioxidant defenses in hepatocytes: caspase-dependent post-translational and caspase-independent transcriptional regulatory mechanisms.

TGFbeta1-induced hepatocyte apoptosis involves the production of reactive oxygen species. An effective cellular defense mechanism against oxidative stress is the tripeptide glutathione (GSH), and the rate-limiting step in GSH biosynthesis is catalyzed by the heterodimeric holoenzyme glutamate cysteine ligase (GCL). Here, we demonstrate that TGFbeta1-induced apoptosis in the TAMH murine hepatocyte cell line is accompanied by both the cleavage and loss of the catalytic subunit of GCL (GCLC) and the down-regulation of GCLC gene expression resulting in a reduction in GCL activity and depletion of intracellular GSH.

Cell culture model for acetaminophen-induced hepatocyte death in vivo.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro.

Caspase-3-Dependent Cleavage of the Glutamate-L-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death.

Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis.