Daisaku Yoshihara

Oxidative modification to cysteine sulfonic acid of Cys111 in human copper-zinc superoxide dismutase.

Copper-zinc superoxide dismutase (SOD1) plays a protective role against oxidative stress. On the other hand, recent studies suggest that SOD1 itself is a major target of oxidative damage and has its own pathogenicity in various neurodegenerative diseases, including familial amyotrophic lateral sclerosis. Only human and great ape SOD1s among mammals have the highly reactive free cysteine residue, Cys111, at the surface of the SOD1 molecule. The purpose of this study was to investigate the role of Cys111 in the oxidative damage of the SOD1 protein, by comparing the oxidative susceptibility of recombinant human SOD1 modified with 2-mercaptoethanol at Cys111 (2-ME-SOD1) to wild-type SOD1. Wild-type SOD1 was more sensitive to oxidation by hydrogen peroxide-generating fragments, oligomers, and charge isomers compared with 2-ME-SOD1. Moreover, wild-type SOD1, but not 2-ME-SOD1, generated an upper shifted band in reducing SDS-PAGE even by air oxidation. Using mass spectrometry and limited proteolysis, this upper band was identified as an oxidized subunit of SOD1; the sulfhydryl group (Cys-SH) of Cys111 was selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and to cysteine sulfonic acid (Cys-SO3H). The antibody raised against a synthesized peptide containing Cys111-SO3H reacted with only the Cys111-peroxidized SOD1 by Western blot analysis and labeled Lewy body-like hyaline inclusions and vacuole rims in the spinal cord of human SOD1-mutated amyotrophic lateral sclerosis mice by immunohistochemical analysis. These results suggest that Cys111 is a primary target for oxidative modification and plays an important role in oxidative damage to human SOD1, including familial amyotrophic lateral sclerosis mutants.

Antioxidants: Benefits and risks for long-term health

The oxidative modification hypothesis postulates that oxidative stress is one of the major factors in aging and the development of age-related disorders, including cardiovascular diseases. In this scenario, the oxidative modification of lipids, proteins and nucleic acids in vascular walls contributes to the etiology of cardiovascular disease, implying that consumption or therapeutic use of antioxidants could prevent the onset of such pathological disorders. Because of this, a number of studies have been conducted to address the question of whether cardiovascular diseases can be modulated by antioxidant treatment or consumption. Although some of the earliest data, collected in animal studies and epidemiologic studies have shown a measure of success, numerous clinical trials indicate that this approach is of minimal or no benefit. These conclusions represent a challenge to design more sensitive antioxidant trials in order to confirm or alter these conclusions. The focus of this review is on the benefits and disadvantages associated with the use of antioxidants, such as vitamins C and E, polyphenols, or antioxidant therapies, including hormone replacement therapy and iron reduction therapy, on overall vascular health.

The role of O-linked GlcNAc modification on the glucose response of ChREBP

The carbohydrate response element-binding protein (ChREBP) functions as a transcription factor in mediating the glucose-activated gene expression of multiple liver enzymes, which are responsible for converting excess carbohydrate to storage fat. ChREBP is translocated into the nucleus in response to high glucose levels, and then up-regulates transcriptional activity. Although this glucose activation of ChREBP is generally observed only in liver cells, overexpression of wild type max-like protein X (Mlx), but not an inactive mutant Mlx, resulted in the exhibition of the ChREBP functions also in a human kidney cell line. Because high glucose conditions induce the glycosylation of cellular proteins, the effect of O-linked GlcNAc modification on ChREBP functions was examined. Treatment with an O-GlcNAcase inhibitor (PUGNAc), which increases the O-linked GlcNAc modification of cellular proteins, caused an increase in the glucose response of ChREBP. In contrast, treatment with a glutamine fructose amidotransferase inhibitor (DON), which decreases O-GlcNAcylation by inhibiting the hexosamine biosynthetic pathway, completely blocked the glucose response of ChREBP. These results suggest that the O-linked glycosylation of ChREBP itself or other proteins that regulate ChREBP is essential for the production of functional ChREBP.

Hydrogen peroxide enhances LPS-induced nitric oxide production via the expression of interferon beta in BV-2 microglial cells

Activated microglia produces inflammatory cytokines and nitric oxide (NO) that involved in neuronal injury and neurodegenerative diseases. We report herein, that H(2)O(2) intensifies the LPS-triggered expression of iNOS in the microglia cell line, BV-2, resulting in an enhancement in the production of NO. The NO production induced by a combination of LPS and H(2)O(2) was blocked by the addition of an anti-interferonβ (IFNβ) neutral antibody, suggesting that IFNβ levels are correlated with the LPS/H(2)O(2)-induced production of NO. However, although the expression of IFNβ was induced by H(2)O(2) treatment alone, neither the expression of iNOS mRNA nor the production of NO were induced. In addition, the expression of IFN receptor (IFNR) was induced by LPS but not by H(2)O(2). These data indicate that although H(2)O(2) alone cannot induce iNOS expression because of the insufficient expression of IFNR, in the presence of LPS, H(2)O(2) enhances iNOS expression via the expression of IFNβ. Our findings suggest that H(2)O(2) produced by activated microglia further enhances NO production in various inflammatory states.

Protective role of glutathione S-transferase A4 induced in copper/zinc-superoxide dismutase knockout mice

Copper/zinc-superoxide dismutase (SOD1) plays a protective role in cells by catalyzing the conversion of the superoxide anion into molecular oxygen and hydrogen peroxide. Although SOD1 knockout (KO) mice exhibit a reduced life span and an elevated incidence of dysfunctions in old age, young SOD1 KO mice grow normally and exhibit no abnormalities. This fact leads to the hypothesis that other antioxidative proteins prevent oxidative stress, compensating for SOD1. Differently expressed genes in 3-week-old SOD1 KO and littermate wild-type mice were explored. A gene remarkably elevated in SOD1 KO mouse kidneys was identified as the glutathione S-transferase Alpha 4 gene (Gsta4), which encodes the GSTA4 subunit. The GSTA4 protein level and activity were also significantly increased in SOD1 KO mouse kidneys. The administration of an iron complex, a free radical generator, induced GSTA4 expression in wild-type mouse kidneys. Iron deposition detected in SOD1 KO mouse kidney is thought to be an inducer of GSTA4. In addition, overexpression of mouse GSTA4 cDNA in human embryonic kidney cells decreased cell death caused by both 4-hydroxynonenal and hydrogen peroxide. These findings suggest that compensatory induced GSTA4 plays a protective role against oxidative stress in young SOD1 KO mouse kidneys.

Cu,Zn-SOD deficiency induces the accumulation of hepatic collagen

Abstract Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases, and results in the development of fibrosis. Oxidative stress is thought to be one of the underlying causes of NAFLD. Copper/zinc superoxide dismutase (SOD1) is a primary antioxidative enzyme that scavenges superoxide anion radicals. Although SOD1 knockout (KO) mice have been reported to develop fatty livers, it is not known whether this lack of SOD1 leads to the development of fibrosis. Since the accumulation of collagen typically precedes liver fibrosis, we assessed the balance between the synthesis and degradation of collagen in liver tissue from SOD1 KO mice. We found a higher accumulation of collagen in the livers of SOD1 KO mice compared to wild type mice. The level of expression of HSP47, a chaperone of collagen, and a tissue inhibitor (TIMP1) of matrix metalloproteinases (a collagen degradating enzyme) was also increased in SOD1 KO mice livers. These results indicate that collagen synthesis is increased but that its degradation is inhibited in SOD1 KO mice livers. Moreover, SOD1 KO mice liver sections were extensively modified by advanced glycation end products (AGEs), which suggest that collagen in SOD1 KO mice liver might be also modified with AGEs and then would be more resistant to the action of collagen degrading enzymes. These findings clearly show that oxidative stress plays an important role in the progression of liver fibrosis.

Alterations in renal iron metabolism caused by a copper/zinc-superoxide dismutase deficiency

Abstract Copper/zinc-superoxide dismutase knockout (SOD1 KO) mice have been extensively used as an experimental animal model of pathology associated with oxidative stress. The mice spontaneously develop mild chronic hemolytic anaemia (HA). We previously reported that the kidneys of these types of mice contain massive amounts of iron. In this study, to clarify the role of the kidney for iron metabolism under HA, changes in the levels of expression and functions of iron-related proteins were examined. In SOD1 KO mice kidneys, protein levels of iron transporters, the iron-responsive element (IRE)-binding activity of IRP1 and the levels of phosphorylation of IRP1 are all increased. These findings indicate that oxidative stress caused by a SOD1 deficiency probably enhances the phosphorylation of and the conversion of IRP1 to the IRE-binding form, which may accelerate the reabsorption of iron by renal tubular cells. Kidney could play an important role in iron homeostasis under conditions of HA.

Inhibition of gene expression of heparin-binding epidermal growth factor-like growth factor by extracellular superoxide dismutase in rat aortic smooth muscle cells

Both extracellular superoxide dismsutase (EC-SOD) and heparin binding EGF like growth factor (HB-EGF) are produced in smooth muscle cells of the arterial wall, and are thought to play pathological roles in atherosclerosis with heparin binding characteristics. EC-SOD treatment clearly reduced the H2O2 induced expression of HB-EGF in rat aortic smooth muscle cells (RASMC). EC-SOD also inhibited the induction of HB-EGF by 12-O-tetradecanoylphorbol-13-acetate (TPA) in RASMC by 60%. Both H2O2 and TPA increased intracellular ROS levels, and EC-SOD inhibited ROS generation only for the case of H2O2 but not TPA. Treatment of the cells with heparin alone decreased HB-EGF expression by 20%, whereas EC-SOD alone and a co-incubation with EC-SOD and heparin suppressed the induction by 60 and 70%, respectively. These results suggest that EC-SOD is related to the EGF signaling in two ways, competition for HSPG with HB-EGF and as an ROS scavenger.

The absence of the SOD1 gene causes abnormal monoaminergic neurotransmission and motivational impairment-like behavior in mice.

Abstract Copper/zinc superoxide dismutase (SOD1), a primary anti-oxidative enzyme, protects cells against oxidative stress. We report herein on a comparison of behavioral and neurobiological changes between SOD1 knockout (KO) and wild-type mice, in an attempt to assess the role of SOD1 in brain functions. SOD1 KO mice exhibited impaired motivational behavior in both shuttle-box learning and three-chamber social interaction tests. High levels of dopamine transporter protein and an acceleration of serotonin turnover were also detected in the cerebrums of the SOD1 KO mice. These findings suggest that SOD1 deficiency disturbs monoaminergic neurotransmission leading to a decrease in motivational behavior.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.