Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.     

Copper handling machinery of the brain

Copper plays an indispensable role in the physiology of the human central nervous system (CNS). As a cofactor of dopamine-β-hydroxylase, peptidyl-α-monooxygenase, superoxide dismutases, and many other enzymes, copper is a critical contributor to catecholamine biosynthesis, activation of neuropeptides and hormones, protection against reactive oxygen species, respiration and other processes essential for normal CNS function. Copper content in the CNS is tightly regulated, and changes in copper levels in the brain are associated with a wide spectrum of pathologies. However, the mechanistic understanding of copper transport in the CNS is still in its infancy. Little is known about copper distribution among various cell types or cell-specific regulation of copper homeostasis, despite the fact that the molecules mediating copper transport and distribution in the brain (CTR1, Atox1, CCS, ScoI/II, ATP7A and ATP7B) have been identified and their importance in CNS function increasingly understood. In this review, we summarize current knowledge about copper levels and uses in the CNS and describe the molecules involved in maintaining copper homeostasis in the brain.     

Carnosine and taurine treatments decreased oxidative stress and tissue damage induced by d-galactose in rat liver

d-galactose (GAL) causes aging-related changes and oxidative stress in the organism. We investigated the effect of carnosine (CAR) or taurine (TAU), having antioxidant effects, on hepatic injury and oxidative stress in GAL-treated rats. Rats received GAL (300 mg/kg; s.c.; 5 days/week) alone or together with CAR (250 mg/kg/daily; i.p.; 5 days/week) or TAU (2.5 % w/w; in rat chow) for 2 months. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and hepatic malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH) levels and superoxide dismutase (SOD), glutathione peroxidase (GSH-0050x), and glutathione transferase (GST) activities were determined. Hepatic expressions of B cell lymphoma-2 (Bcl-2), Bax and Ki-67 were evaluated. Serum ALT, AST, hepatic MDA, and PC levels were observed to increase in GAL-treated rats. Hepatic Bax expression, but not Bcl-2, increased, Ki-67 expression decreased. GAL treatment caused decreases in GSH levels, SOD and GSH-Px activities in the liver. Hepatic mRNA expressions of SOD, but not GSH-Px, also diminished. CAR or TAU treatments caused significant decreases in serum ALT and AST activities. These treatments decreased apoptosis and increased proliferation and ameliorated histopathological findings in the livers of GAL-treated rats. Both CAR and TAU reduced MDA and PC levels and elevated GSH levels, SOD and GSH-Px (non significant in TAU?+?GAL group) activities. These treatments did not alter hepatic mRNA expressions of SOD and GSH-Px enzymes. Our results indicate that CAR and TAU restored liver prooxidant status together with histopathological amelioration in GAL-induced liver damage.     

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.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

Mechanisms of biosynthesis of mammalian copper/zinc superoxide dismutase

Copper/zinc superoxide dismutase (SOD1) is an abundant intracellular enzyme with an essential role in antioxidant defense. The activity of SOD1 is dependent upon the presence of a bound copper ion incorporated by the copper chaperone for superoxide dismutase, CCS. To elucidate the cell biological mechanisms of this process, SOD1 synthesis and turnover were examined following 64Cu metabolic labeling of fibroblasts derived from CCS+/+ and CCS-/- embryos. The data indicate that copper is rapidly incorporated into both newly synthesized SOD1 and preformed SOD1 apoprotein, that each process is dependent upon CCS and that once incorporated, copper is unavailable for cellular exchange. The abundance of apoSOD1 is inversely proportional to the intracellular copper content and immunoblot and gel filtration analysis indicate that this apoprotein exists as a homodimer that is distinguishable from SOD1. Despite these distinct differences, the abundance and half-life of SOD1 is equivalent in CCS+/+ and CCS-/- fibroblasts, indicating that neither CCS nor copper incorporation has any essential role in the stability or turnover of SOD1 in vivo. Taken together, these data provide a cell biological model of SOD1 biosynthesis that is consistent with the concept of limited intracellular copper availability and indicate that the metallochaperone CCS is a critical determinant of SOD1 activity in mammalian cells. These kinetic and biochemical findings also provide an important framework for understanding the role of mutant SOD1 in the pathogenesis of familial amyotrophic lateral sclerosis.     

Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu‐directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF‐B and in the liver in vivo. Copper (10 µm ) caused ATP7B to exit the trans‐Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton‐pump inhibitor bafilomycin‐A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu‐stimulated) case. Overall, loss of acidification‐impaired Cu‐regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.      

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.     

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.     

Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L.

Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4 weeks and then transferred to full-strength MS liquid medium for 3 weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200 μM) of Cu for 7 and 14 days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50 μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200 μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.     

Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat

Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.     

A Role for the ATP7A Copper-transporting ATPase in Macrophage Bactericidal Activity

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, suggesting a role for ATP7A-dependent copper transport in the bactericidal activity of macrophages. Significantly, a copper-sensitive mutant of Escherichia coli lacking the CopA copper-transporting ATPase was hypersensitive to killing by RAW264.7 macrophages, and this phenotype was dependent on ATP7A expression. Collectively, these data suggest that copper-transporting ATPases, CopA and ATP7A, in both bacteria and macrophage are unique determinants of bacteria survival and identify an unexpected role for copper at the host-pathogen interface.     

Improved β-carotene production by oxidative stress in Blakeslea trispora induced by liquid paraffin

When 3 % (v/v) liquid paraffin was added to the medium, β-carotene production increased from 397 to 715 mg l^?1 in mated cultures of Blakeslea trispora. Liquid paraffin also enhanced the oxygen concentration and induce high oxidative stress, as observed by the increase in activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). After 84 h of cultivation in the presence of liquid paraffin, the activities of SOD, CAT and POD in B. trispora increased 77, 52.5 and 76.6 %, respectively.     

Effects of Copper on Hemocyte Apoptosis,ROS Production,and Gene Expression in White Shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L^?1 Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6–48 h among shrimp exposed to 1 mg L^?1 Cu and at each time points in 5 mg L^?1 Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.     

The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson’s disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington’s disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.     

Mechanisms of the copper-dependent turnover of the copper chaperone for superoxide dismutase

The copper chaperone for superoxide dismutase (CCS) is an intracellular metallochaperone required for incorporation of copper into the essential antioxidant enzyme copper/zinc superoxide dismutase (SOD1). Nutritional studies have revealed that the abundance of CCS is inversely proportional to the dietary and tissue copper content. To determine the mechanisms of copper-dependent regulation of CCS, copper incorporation into SOD1 and SOD1 enzymatic activity as well as CCS abundance and half-life were determined after metabolic labeling of CCS-/- fibroblasts transfected with wild-type or mutant CCS. Wild-type CCS restored SOD1 activity in CCS-/- fibroblasts, and the abundance of this chaperone in these cells was inversely proportional to the copper content of the media, indicating that copper-dependent regulation of CCS is entirely post-translational. Although mutational studies demonstrated no role for CCS Domain I in this copper-dependent regulation, similar analysis of the CXC motif in Domain III revealed a critical role for these cysteine residues in mediating copper-dependent turnover of CCS. Further mutational studies revealed that this CXC-dependent copper-mediated turnover of CCS is independent of the mechanisms of delivery of copper to SOD1 including CCS-SOD1 interaction. Taken together these data demonstrate a mechanism determining the abundance of CCS that is competitive with the process of copper delivery to SOD1, revealing a unique post-translational component of intracellular copper homeostasis.