SOD1 Activation Research Articles

Nmnat1 Modulates Mitochondrial Oxidative Stress by Inhibiting Caspase-3 Signaling in Alzheimer's Disease.

Nigrostriatal pathway disturbance is one of the major pathogenic factors in Alzheimer's disease (AD). Dopaminergic neuron dysfunction results in bradykinesia and akinesia (inability to initiate movement), indicating a significant risk factor for substantia nigra pars compacta lesions. Furthermore, the nicotinamide adenine dinucleotide (NAD+) is associated with Aβ toxicity decline in AD therapy. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) is an essential enzyme that preserves normal neuronal function and protects neurons from insult. This study aimed to investigate the potential therapeutic effects of Nmnat1 and its underlying mechanisms in a triple-transgenic mouse model of AD (3xTgAD). Results showed that Nmnat1 improved the substantial behavioral measures of cognitive impairments compared with the 3xTgAD control. Additionally, Nmnat1 overexpression significantly increased tyrosine hydroxylase-positive neurons and anti-apoptotic protein Bcl2 and caspase-3 expression levels in 3xTgAD mice. Nmnat1 also effectively controlled SOD1 activation. In conclusion, Nmnat1 substantially decreases multiple AD-associated pathological characteristics at least partially by the increase of caspase-3 activation.

Antioxidative effect of DJ-1 is enhanced in NG108-15 cells by DPMQ-induced copper influx.

Disruption of copper homeostasis is closely involved in neurodegenerative disorders. This study examined whether a hybrid copper-binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), is able to protect NG108-15 cells against oxidative stress. We found that treatment of cells with rotenone or hydrogen peroxide increased cellular oxidative stress and resulted in mitochondrial dysfunction and apoptosis. The cellular levels of Nrf2 and the Cu2+ chaperone DJ-1 were also decreased. These oxidative detrimental effects were all inhibited when cells were cotreated with DPMQ. DPMQ increased cellular Cu2+ content, DJ-1 protein level, superoxide dismutase (SOD) activity, and Nrf2 nuclear translocation under basal state. The activity of SOD decreased under redox imbalance and this decrease was blocked by DPMQ treatment, while the protein level of SOD1 remained unaltered regardless of the oxidative stress and DPMQ treatment. Using endogenous proteins, coimmunoprecipitation showed that DJ-1 bound with SOD1 and Nrf2 individually. The amount of Nrf2, bound to DJ-1, consistently reflected its cellular level, while the amount of SOD1, bound to DJ-1, was potentiated by DPMQ, being greater in the basal state than under redox imbalance. Simultaneous inclusion of nonpermeable Cu2+ chelator tetrathiomolybdate or triethylenetetramine during DPMQ treatment blocked all aforementioned effects of DPMQ, showing that the dependency of the effect of DPMQ on extracellular Cu2+. In addition, silencing of DJ-1 blocked the protection of DPMQ against oxidative stress. Taken all together, our results suggest that DPMQ stabilizes DJ-1 in a Cu2+-dependent manner, which then brings about SOD1 activation and Nrf2 nuclear translocation; these together alleviate cellular oxidative stress.

Copper\u2013zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1

Copper–zinc superoxide dismutase (Sod1) is a critical antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochemical examination of each step in the pathway determined that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addition of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in solution. This heterotrimer can also be made by the addition of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import.

Exploring the Extended Biological Functions of the Human Copper Chaperone of Superoxide Dismutase 1

The human copper chaperone of SOD1 (designated as CCS) was discovered more than two decades ago. It is an important copper binding protein and a homolog of Saccharomyces cerevisiae LYS7. To date, no studies have systematically or specifically elaborated on the functional development of CCS. This review summarizes the essential information about CCS, such as its localization, 3D structure, and copper binding ability. An emphasis is placed on its interacting protein partners and its biological functions in vivo and in vitro. Three-dimensional structural analysis revealed that CCS is composed of three domains. Its primary molecular function is the delivery of copper to SOD1 and activation of SOD1. It has also been reported to bind to XIAP, Mia40, and X11α, and other proteins. Through these protein partners, CCS is implicated in several vital biological processes in vivo, such as copper homeostasis, apoptosis, angiogenesis and oxidative stress. This review is anticipated to assist scientists in systematically understanding the latest research developments of CCS for facilitating the development of new therapeutics targeting CCS in the future.

The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation

Copper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O2). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1·CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1·CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."

P-102 - The involvement of the anti-oxidant protein DJ-1 in SOD1 maturation pathway: implications for neurobehavioral deficits in Drosophila Melanogaster

The Parkinson's disease associated protein DJ-1 is as a multitasking protein with anti-oxidant activity. Indeed, DJ-1 has been shown to modulate signaling pathways involved in anti-oxidant defense. Nevertheless, no consensus has been found on its effective cellular function. Our group has demonstrated, in vitro, that DJ-1 can induce the activation of SOD1, the first line of defense against reactive oxygen species. SOD1 and DJ-1 are known to play an important role against oxidative stress. Although SOD1 activation normally relies on a dedicated copper chaperone, it has been shown to be partially activated also through an alternative pathway, to which DJ-1 may participate. To better clarify this mechanism in vivo we utilize Drosophila Melanogaster as model organism, which possesses homologue pathways for anti-oxidant responses. For this study, we are evaluating the behavioral traits of wild-type and mutant flies under normal conditions and under mild oxidative insults, assessing lifespan and complex locomotor features. Although DJ-1 loss does not affect fly lifespan under normal conditions, its absence compromises survival under paraquat exposure. Therefore, our work aims to elucidate the anti-oxidant role of DJ-1, focusing on its participation to SOD1 activation.

Quantifying the Interaction between Copper-Zinc Superoxide Dismutase (Sod1) and its Copper Chaperone (Ccs1).

Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1•Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1SH). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.

An essential role of N-terminal domain of copper chaperone in the enzymatic activation of Cu/Zn-superoxide dismutase

Cu/Zn-superoxide dismutase (SOD1) is an enzyme that disproportionates superoxide anion into hydrogen peroxide and molecular oxygen. The enzymatic activity of SOD1 requires the binding of copper and zinc ions and also the formation of a conserved intramolecular disulfide bond. In a eukaryotic cell, a copper chaperone for SOD1 (CCS) has been known to supply a copper ion and also introduce the disulfide bond into SOD1; however, a mechanism controlling the CCS-dependent activation of SOD1 remains obscure. Here, we characterized CCS isolated from a human liver fluke, Clonorchis sinensis, and found that an N-terminal domain of CCS was essential in supplying a copper ion in SOD1. Regardless of the presence and absence of the N-terminal domain, CCS was able to bind a cuprous ion at the CxC motif of its C-terminal domain with quite high affinity (Kd~10−17). The copper-bound form of full-length CCS successfully activated C. sinensis SOD1, but that of CCS lacking the N-terminal domain did not. Nonetheless, the N-terminally truncated CCS with the bound copper ion was found to correctly introduce the disulfide bond into SOD1. Based upon these results, we propose that the N-terminal domain of CCS has roles in the release of the copper ion bound at the C-terminal domain of CCS to SOD1.

Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.

The combination of the active principles of Podophyllum hexandrum supports early recovery of the gastrointestinal system via activation of Nrf2-HO-1 signaling and the hematopoietic system, leading to effective whole-body survival in lethally irradiated mice

This study is aimed at the development of a safe radioprotective formulation to minimize human sufferings during accidental nuclear exposures. In the current study, a combination of three active principles, namely podophyllotoxin, podophyllotoxin beta-D-glucoside, and rutin (G-002M), isolated from Podophyllum hexandrum rhizomes, has been evaluated for its radioprotective potential and mode of action. Total body protection studies have demonstrated that a single prophylactic dose of G-002M delivered more than 85% survival in mice exposed to a lethal (9 Gy) dose of gamma radiation, and significantly protected the radiosensitive hematopoietic and gastrointestinal organs. Studies have also revealed a reduction in free radical generation, lipid peroxidation, protein carbonylation, and cell death in mouse intestine after G-002M treatment, while GSH was observed to be enhanced in the same tissue. Redox-sensitive transcription factor (Nrf2) activation and subsequent upregulation of heme oxygenase-1 (HO-1) and SOD-1 revealed the cytoprotective role of G-002M. A histological examination of the jejunum pretreated with the formulation also demonstrated less damage to the villi, crypts, and the mucosal layers. These observations reiterated that the reduction in the ROS levels, protection of cellular macromolecules, and activation of the antioxidant signaling pathway may have been the principle factors involved in G-002M- mediated protection against radiation-induced tissue impairment. The potentially safe and effective radioprotective characteristics of this new combination are encouraging for further studies for human application.

P163 - Nitric oxide-mediated antioxidative mechanism in yeast: The transcription factor Mac1-dependent activation of the superoxide dismutase Sod1

P163 - Nitric oxide-mediated antioxidative mechanism in yeast: The transcription factor Mac1-dependent activation of the superoxide dismutase Sod1

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis.

Yeast copper–zinc superoxide dismutase can be activated in the absence of its copper chaperone

Copper-zinc superoxide dismutase (Sod1) is an abundant intracellular enzyme that catalyzes the disproportionation of superoxide to give hydrogen peroxide and dioxygen. In most organisms, Sod1 acquires copper by a combination of two pathways, one dependent on the copper chaperone for Sod1 (CCS), and the other independent of CCS. Examples have been reported of two exceptions: Saccharomyces cerevisiae, in which Sod1 appeared to be fully dependent on CCS, and Caenorhabditis elegans, in which Sod1 was completely independent of CCS. Here, however, using overexpressed Sod1, we show there is also a significant amount of CCS-independent activation of S. cerevisiae Sod1, even in low-copper medium. In addition, we show CCS-independent oxidation of the disulfide bond in S. cerevisiae Sod1. There appears to be a continuum between CCS-dependent and CCS-independent activation of Sod1, with yeast falling near but not at the CCS-dependent end.

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker's yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80% similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS-SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.

The involvement of GSH in the activation of human Sod1 linked to FALS in chronologically aged yeast cells

Mutations in Cu, Zn-superoxide dismutase (Sod1) have been associated with familial amyotrophic lateral sclerosis, an age-related disease. Because several studies suggest that oxidative stress plays a central role in neurodegeneration, we aimed to investigate the role of the antioxidant glutathione (GSH) in the activation of human A4V Sod1 during chronological aging. Transformation of wild-type and A4V hSod1 into a gsh null mutant and in its parental strain of Saccharomyces cerevisiae indicated that during aging, the number of viable cells was strongly influenced by A4V hSod1 mainly in cells lacking GSH. Activity of hSod1 increased in response to aging, although the increase observed in A4V hSod1 was almost 60% lower. Activation of hSod1 (A4V and WT) did not occur after aging, in cells lacking GSH, but could still be observed in the absence of Ccs1. Furthermore, no increase in activity could be seen in grx1 and grx2 null mutants, suggesting that glutathionylation is essential for hSod1 activation. The A4V mutation as well as the absence of GSH, reduced hSod1 activity, and increased oxidative damage after aging. In conclusion, our results point to a GSH requirement for hSod1 Ccs1-independent activation as well as for protection of hSod1 during the aging process.

Pharmacologic Induction of Heme Oxygenase-1 Plays a Protective Role in Diabetic Retinopathy in Rats

We investigated the protective effects of HO-1, induced by hemin, in the retinas of streptozotocin (STZ)-induced diabetic rats, and document the possible anti-inflammatory, anti-apoptotic, and anti-proliferative mechanisms underlying the protection. Sprague-Dawley (SD) rats were induced to diabetes by intraperitoneal injection of STZ (60 mg/kg). Later, some of the rats were given intraperitoneal injections of hemin (20 mg/kg) to induce expression of HO-1. The protective effects of hemin were evaluated by examining the hemoglobin concentration (Hb) and the glycosylated hemoglobin (HbA1c) level of blood from the rats, further calculating the numbers of TUNEL positive cells in retinal ganglion cell (RGC) layer and diI-labeled RGCs. We also documented the expressions of HO-1, HIF-1α, SOD-1, VEGF, p53, and bcl-2 by Western blot analysis and real-time quantitative PCR. Expressions of Nrf2, tERK 1/2, and pERK 1/2 were detected only by Western blot analysis. HO-1, Nrf2, pERK, and GFAP proteins were detected by immunofluorescence. The Hb level was higher in hemin-treated rat blood than nontreated diabetic group, while the HbA1c level was lower. Hemin significantly activated HO-1 expression in the retinas of diabetic rats, combined with accordant changes of Nrf2/pERK protein expression, and upregulated the expression of GFAP in retina. Retinal ganglion cells displayed greater sensitivity to apoptosis when the HO-1 level was lower. Overexpression of HO-1 was associated with an increase in the activation of SOD-1 and bcl-2, and a decrease of the expression of HIF-1α, VEGF, and p53. HO-1 is an important positive modulator of the Nrf2/ERK-related signaling. Overexpression of HO-1 by hemin induction protected retinal ganglion cells in diabetic retinopathy through anti-inflammatory, anti-apoptotic, and anti-proliferative effects.

SOD1 (Copper/Zinc Superoxide Dismutase) Deficiency Drives Amyloid β Protein Oligomerization and Memory Loss in Mouse Model of Alzheimer Disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.

Melatonin and steroid hormones activate intermembrane Cu,Zn-superoxide dismutase by means of mitochondrial cytochrome P450

Melatonin and steroid hormones are cytochrome P450 (CYP or P450; EC 1.14.14.1) substrates that have antioxidant properties and mitochondrial protective activities. The mitochondrial intermembrane space (IMS) Cu,Zn-superoxide dismutase (SOD1) is activated after oxidative modification of its critical thiol moieties by superoxide anion (O2•−). This study was aimed at investigating the potential association between the hormonal protective antioxidant actions in mitochondria and the regulation of IMS SOD1 activity. Melatonin, testosterone, dihydrotestosterone, estradiol, and vitamin D induced a sustained activation over time of SOD1 in intact mitochondria, showing a bell-shaped enzyme activation dose response with a threshold at 50nM and a maximum effect at 1μM concentration. Enzyme activation was not affected by furafylline, but it was inhibited by omeprazole, ketoconazole, and tiron, thereby supporting the occurrence of a mitochondrial P450 activity and O2•− requirements. Mitochondrial P450-dependent activation of IMS SOD1 prevented O2•−-induced loss of aconitase activity in intact mitochondria respiring in State 3. Optimal protection of aconitase activity was observed at 0.1μM P450 substrate concentration, evidencing a likely oxidative effect on the mitochondrial matrix by higher substrate concentrations. Likewise, enzyme activation mediated by mitochondrial P450 activity delayed CaCl2-induced loss of transmembrane potential and decreased cytochrome c release. Omeprazole and ketoconazole abrogated both protecting mitochondrial functions promoted by melatonin and steroid hormones.

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.

Requirement of glutathione for Sod1 activation during lifespan extension

It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1-dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre-treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild-type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension.