Oxidized proteins and their contribution to redox homeostasis

Abstract

Proteins are major target for radicals and other oxidants when these are formed in both intra- and extracellular environments in vivo. Formation of lesions on proteins may be highly sensitive protein-based biomarkers for oxidative damage in mammalian systems. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. ROS scavenging activities of intact proteins are weaker than those of misfolded proteins or equivalent concentrations of their constituent amino acids. Protein oxidation and enhanced proteolytic degradation, therefore, have been suggested to cause a net increase in ROS scavenging capacity. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, may contribute to the observed accumulation and damaging actions of oxidized proteins during ageing and in pathologies such as diabetes, arteriosclerosis and neurodegenerative diseases. Protein oxidation may play a controlling role in cellular remodelling and cell growth. There is some evidence that antioxidant supplementation may protect against protein oxidation, but additional controlled studies of antioxidant intake to evaluate the significance of dietary/pharmacological antioxidants in preventing physiological/pathological oxidative changes are needed.     

Heme oxygenase and iron: from bacteria to humans

Abstract

Iron and iron-containing prosthetic groups are involved in many fundamental processes that constitute life. One of the biologically most important iron-containing groups is heme, in which an iron atom is co-ordinated to a protoporphyrin ring. Heme proteins have a wide range of functions, participating in a vast repertoire of biochemical reactions. Due to its abundance, heme also serves as an important source of iron. Enzymatic degradation of heme usually involves its oxidative cleavage by heme oxygenase. Not surprisingly, heme oxygenase activity is present in organisms across different domains and kingdoms. This review summarises the current knowledge in the dynamic relationship between heme oxygenase and iron in metabolism and in the clinical context.     

Glutathione reductase is expressed at high levels in pancreatic islet cells

Abstract

Reactive oxygen species are, at least partly, involved in the diabetogenic agent-induced dysfunction of pancreatic β-cells because the expression of antioxidative and redox proteins is low. We examined the levels of antioxidant/redox proteins, peroxiredoxins-1, -4, and -6 and glutathione reductase (GR), by immunohistochemistry and found that the expression of GR was very high in pancreatic islet cells compared to exocrine cells. When diabetes was induced by an intravenous injection of streptozotocin, the pre-administration of 1,3-bis[2-chloroethyl]-1-nitrosourea, an irreversible inhibitor of GR, made islet cells more vulnerable to streptozotocin. These data point to a pivotal role of the glutathione redox system in pancreatic islet cells against diabetogenic stress.     

Role of N-acetylcysteine and cystine in glutathione synthesis in human erythrocytes

Abstract

Glutathione is an intracellular antioxidant that often becomes depleted in pathologies with high oxidative loads. We investigated the provision of cysteine for glutathione synthesis to the human erythrocyte (red blood cell; RBC). Almost all plasma cysteine exists as cystine, its oxidized form. In vitro, extracellular cystine at 1.0 mM sustained glutathione synthesis in glutathione-depleted RBCs, at a rate of 0.206 ± 0.036 μmol (L RBC)^?1min^?1 only 20% of the maximum rate obtained with cysteine or N-acetylcysteine. In plasma-free solutions, N-acetylcysteine provides cysteine by intracellular deacetylation but to achieve maximum rates of glutathione synthesis by this process in vivo, plasma N-acetylcysteine concentrations would have to exceed 1.0 mM, which is therapeutically unattainable. ¹H-NMR experiments demonstrated that redox exchange reactions between NAC and cystine produce NAC-cysteine, NAC-NAC and cysteine. Calculations using a mathematical model based on these results showed that plasma concentrations of N-acetylcysteine as low as 100 μM, that are attainable therapeutically, could potentially react with plasma cystine to produce ～50 μM cysteine, that is sufficient to produce maximal rates of glutathione synthesis. We conclude that the mechanism of action of therapeutically administered N-acetylcysteine is to reduce plasma cystine to cysteine that then enters the RBC and sustains glutathione synthesis.     

The effect of polyphenolic extract from pine bark,Pycnogenol® on the level of glutathione in children suffering from attention deficit hyperactivity disorder (ADHD)

Abstract

Attention deficit hyperactivity disorder (ADHD) belongs to the neurodevelopmental disorders characterized by impulsivity, distractibility and hyperactivity. In the pathogenesis of ADHD genetic and non-genetic factors play an important role. It is assumed that one of non-genetic factors should be oxidative stress. Pycnogenol^®, an extract from the pine bark, consists of bioflavonoids, catechins, procyanidins and phenolic acids. Pycnogenol^® acts as powerful antioxidant, chelating agent; it stimulates the activities of some enzymes, like SOD, eNOS, and exhibits other biological activities.

Aim: The aim of this randomized, double-blind, placebo-controlled trial was to investigate the influence of administered Pycnogenol^® or placebo on the level of reduced (GSH) and oxidized (GSSG) glutathione in children suffering from ADHD and on total antioxidant status (TAS). This is the first investigation of the redox glutathione state in relation to ADHD.

Results: One month of Pycnogenol^® administration (1 mg/kg body weight/day) caused a significant decrease in GSSG and a highly significant increase in GSH levels as well as improvement of GSH/GSSG ratio in comparison to a group of patients taking a placebo. TAS in children with ADHD was decreased in comparison with reference values. Pycnogenol^® administration normalizes TAS of ADHD children.     

Demethylation of theophylline (1,3-dimethylxanthine) to 1-methylxanthine: the first step of an antioxidising cascade

Abstract

The reaction of theophylline with HO^? radical, produced by photolytic methods at pH 7, was studied in aqueous solution and the products characterised by HPLC and GC-MS. In addition to the expected 1,3-dimethyluric acid, the formation of 1-methylxanthine and, to a lesser extent, of 3-methylxanthine was observed. Theoretical calculations confirmed the preferred formation of the former compound. Both demethylated products were also observed upon reaction of theophylline with O^?– radical anion at pH～13, and 1-methylxanthine was consumed faster than 3-methylxanthine after its formation. Molecular oxygen had no significant effect on the formation of the mono-methylxanthine derivatives. A reaction mechanism for the demethylation of theophylline by oxidising radicals is proposed. This demethylation reaction can play an important role in the protection of biological targets against oxidative stress as the first step of an antioxidising cascade.     

Advances in our understanding of peroxiredoxin,a multifunctional,mammalian redox protein

Abstract

Organisms living under aerobic conditions have developed various anti-oxidative mechanisms to protect them from damage by reactive oxygen species (ROS). A novel family of anti-oxidative proteins, designated as peroxiredoxin (Prx), has been identified in the past two decades and currently comprises six members in mammals. They share a common reactive Cys residue in the N-terminal region, and are capable of serving as a peroxidase and involve thioredoxin and/or glutathione as the electron donor. Prx1 to Prx4 have an additional Cys residue in the conserved C-terminal region, and are cross members as judged by the amino acid sequence similarity. Prx5 also contains an additional Cys in its C-terminal region which is less conserved. On the other hand, Prx6 has only one unique Cys. These Prx family members are distributed in the cytosol, mitochondria, peroxisome and plasma, all of which are potential sites of ROS production. In addition to their role as a peroxidase, however, a body of evidence has accumulated to suggest that individual members also serve divergent functions which are associated with various biological processes such as the detoxification of oxidants, cell proliferation, differentiation and gene expression. It would be expected that these functions might not necessarily depend on peroxidase activity and, therefore, it seems likely that the divergence is due to unique molecular characteristics intrinsic to each member. A comparative study of the divergence would lead to a better understanding of the biological significance of the Prx family.     

Contribution of hexose-6-phosphate dehydrogenase to NADPH content and redox environment in the endoplasmic reticulum

Abstract

Background: Hexose-6-phosphate dehydrogenase (H6PD) has been considered to be a main source of NADPH in the endoplasmic reticulum. It provides reducing equivalents to 11-hydroxysteroid dehydrogenase type 1 for in situ re-activation of glucocorticoids. H6PD null mice indeed show signs of glucocorticoid deficiency, but also suffer from a skeletal myopathy mainly affecting fast twitch muscles, in which the unfolded protein response (UPR) is activated. Thus, H6PD may have additional functions in muscle.

Materials and methods: To determine the contribution of H6PD to total microsomal NADPH content, we measured NADPH in microsomes from liver and quadriceps, gastrocnemius and soleus muscles. To evaluate the effect of H6PD deficiency on microsomal thiol-disulfide redox environment, we measured reduced and oxidized glutathione and free protein thiols.

Results and conclusions: H6PD deficiency decreased but did not eliminate NADPH content in liver and soleus microsomes. Thus there must be other sources of NADPH within the endoplasmic/sarcoplasmic reticulum. Levels of reduced glutathione and free protein thiols were decreased in gastrocnemius muscle from null mice, indicating a more oxidative environment. Such alterations in redox environment may underlie the myopathy and UPR activation in H6PD null mice.

General significance: H6PD plays a role in maintaining normal NADPH levels and redox environment inside the endoplasmic reticulum. Intrinsic differences in ER metabolism may explain the differing effects of H6PD deficiency in different tissues.     

Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells

Abstract

Andrographolide (ANDRO), a diterpenoid lactone isolated from the traditional herbal plant Andrographis paniculata, was reported to induce apoptosis in hepatoma Hep3B cells in our previous study (Ji LL, Liu TY, Liu J, Chen Y, Wang ZT. Andrographolide inhibits human hepatoma-derived Hep3B cells growth through the activation of c-Jun N-terminal kinase. Planta Med 2007; 73: 1397–1401). The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in ANDRO-induced apoptosis. ANDRO initially increased intracellular GSH levels which then decreased later, while inhibition of cellular GSH synthesis by L-Buthionine-(S,R)-sulfoximine (BSO) augmented ANDRO-induced cytotoxicity and apoptosis in Hep3B cells. On the other hand, the thiol antioxidant dithiothreitol (DTT) rescued ANDRO-depleted cellular GSH, and abrogated ANDRO-induced cytotoxicity and apoptosis. Furthermore, BSO pretreatment augmented ANDRO-decreased expression of antioxidant protein thioredoxin 1 (Trx1), while DTT reversed this decrease. Further results showed that ANDRO increased the activity of the GSH-related antioxidant enzyme glutathione peroxidase (GPx) and the production of intracellular reactive oxygen species (ROS). Taken together, this study demonstrates that the intracellular redox system plays important roles in regulating the cytotoxicity of ANDRO on hepatoma Hep3B cells.