Copper Modulates the Degradation of Copper Chaperone for Cu,Zn Superoxide Dismutase by the 26 S Proteosome*

Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the was II was used to the digoxigenin-labeled CCS-specific probe by random priming using the full-length cDNA as a of probe, hybridization of membrane, and detection of

Copper is an essential trace metal and a co-factor for a number of metalloenzymes that function to reduce molecular oxygen. Because of its reactive nature, free copper can participate in reactions that adversely modify proteins, lipids, and nucleic acids, which can have detrimental effects on the viability of the cell. Therefore, elaborate mechanisms have evolved to control the uptake and distribution of copper so that in the cell free copper is virtually nonexistent (1).
Copper chaperones are a family of proteins that scavenge for copper in the cell and facilitate incorporation of the metal into specific proteins through direct interaction with their target (2)(3)(4). By directly inserting copper into their target, copper chaperones prevent free copper ions from engaging in deleterious reactions in the cell. The copper chaperone HAH1/ATOX1 delivers copper to the P-type intracellular membrane-bound copper transporters ATP7A and ATP7B (5,6). Mutations in ATP7A and ATP7B have been associated with diseases of copper metabolism, namely Menkes syndrome (7) and Wilson's disease (8), respectively. Copper chaperones that deliver copper to the mitochondria, cytochrome c oxidase, and the nucleus have also been described (9 -13).
Cu,Zn superoxide dismutase (SOD1) 1 is an abundant homodimeric enzyme that contains one copper and one zinc atom per subunit. SOD1 functions as an antioxidant, eliminating toxic superoxide anion radicals. Recently, dominantly inherited mutations in SOD1 have been linked to the fatal motor neuron disorder, familial amyotrophic lateral sclerosis (14). It has been proposed that SOD1 mutants exert their toxic effects through some uncharacterized gain-of-function (15,16).
In vivo, insertion of copper into SOD1 is dependent on the copper chaperone for Cu,Zn superoxide dismutase (CCS). In mammals, CCS is a homodimer of ϳ33-kDa subunits and is localized mainly to the cytoplasm (2,17). Mice genetically engineered to lack CCS show a reduction in SOD1 activity due to the absence of the copper co-factor required for enzymatic activity (18). When copper is abundant, CCS is expressed at low levels, and CCS has been estimated to be up to 30-fold less abundant than SOD1 in some tissues (17).
CCS is composed of three distinct domains. The N-terminal sequence contains an HAH1/ATOX1 copper-binding domain, MXCXXC (M, methionine; X, any amino acid; C, cysteine), and this region has been reported to be required for insertion of copper into SOD1 under conditions where copper is limiting (19). The central domain is highly homologous to SOD1 and mediates interaction with SOD1. The C-terminal domain is a short sequence that is highly conserved between CCS molecules of different species and contains a CXC motif that can bind copper. Mutations within this motif abrogate copper transfer from CCS to SOD1 (19).
Despite considerable advances in our understanding of the mechanism by which copper chaperones transfer copper to their target (20,21), little is known about how the activity of these proteins is regulated. We have recently reported a dose-dependent increase in CCS protein in liver and erythrocytes of rats fed copper-deficient diets (22) suggesting that copper may control CCS expression. In this study, we have * This is Publication 578 of the Bureau of Nutritional Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  explored in more detail the mechanism regulating CCS expression and show that copper determines cellular CCS protein level by modulating the rate of CCS degradation by the 26 S proteosome.

EXPERIMENTAL PROCEDURES
Chemicals and Antibodies-The proteosome inhibitors MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal) and lactacystin were obtained from Calbiochem and Sigma, respectively. Inhibitors of the lysosomal degradation pathway chloroquine and NH 4 Cl and the cysteine protease inhibitor calpain inhibitor II (N-acetyl-leucinyl-leucinyl-methional-H) were purchased from Sigma. Protein synthesis was blocked with cycloheximide (Sigma). SOD1 and ␤-tubulin were detected with antibodies FL-154 and H-235 (Santa Cruz Biotechnology). Generation of the antibody against CCS used to detect CCS by Western blot is described elsewhere (17). CCS antibody FL-274 (Santa Cruz Biotechnology) was used to immunoprecipitate CCS in pulse-chase experiments. The specific copper chelator 2,3,2-tetraamine (Aldrich) was used to induce copper deficiency in cultured cell lines. CuSO 4 ⅐5H 2 O, ZnSO 4 ⅐7H 2 O, and FeSO 4 ⅐7H 2 O were added to culture medium from 50 mM aqueous stock solutions.
Cell Culture-The rat H4IIE and human HepG2 hepatoma cell lines were cultured in William's Medium E supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin at 37°C in 5% CO 2 . Cells were seeded in 6-cm dishes at 30 -60% confluency and incubated 16 h prior to experimental treatments.
Animals and Test Diets-The animal protocol and test diets have been described in more detail elsewhere (22). Briefly, weanling (21-dayold) male Wistar rats (Charles River Canada) were randomly assigned to one of two diet groups (n ϭ 10/group) and housed individually in wire-bottom stainless steel cages. Rats had free access to demineralized drinking water and one of two test diets nutritionally complete but varying in copper content. Diets were dry-ashed, dissolved in 3 N HCl, and copper content determined by flame atomic absorption spectrophotometry (5100 PC, PerkinElmer Life Sciences). Copper-normal and copper-deficient diets contained 5.3 Ϯ 0.06 and 0.34 Ϯ 0.01 mg of copper/kg diet, respectively. After 6 weeks of feeding the diets, rats were killed by exsanguination while under 3% isoflurane anesthesia. Livers were collected and immediately frozen in liquid nitrogen and stored at Ϫ80°C until analysis.
Northern Blot Analysis-Total RNA was isolated from rat liver using TRIzol reagent (Invitrogen). 30 g of total RNA was separated in a 1% agarose gel under denaturing conditions and transferred to a nylon membrane using a vacuum blotter (Boekel Scientific). RNA was crosslinked to the membrane using a UV cross-linker (UVP Laboratory Products). The rat CCS-specific probe was generated by first PCRamplifying and gel-purifying the rat CCS cDNA from rat liver total RNA using the SuperScript TM One-step reverse transcription-PCR with Platinum® Taq kit (Invitrogen) with CCS-specific primers (forward primer, 5Ј-ATGGCTTCGAAGTCGGGGGACGGTGGAACT-3Ј; reverse primer, 5Ј-TCAGAGGTGAGCAGGGGGTTGGGCTGAGTC-3Ј). CCS cDNA was confirmed by restriction digest. The digoxigenin High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science) was used to generate the digoxigenin-labeled CCS-specific probe by random priming using the full-length rat cDNA as a template. Generation of probe, hybridization of membrane, and detection of CCS mRNA was performed as per manufacturer's instructions.
Protein Extracts and Western Blotting-Rat livers were homogenized in 0.2% Triton X-100, supernatant recovered following centrifugation (13,000 ϫ g, 10 min), and protein concentration determined by the BCA method (23). For H4IIE and HepG2 cells, cells were washed once with ice-cold PBS and then gently scraped from the dish in PBS. Cells were spun down (2000 ϫ g, 2 min); PBS was removed, and 0.2% Triton X-100 was added to lyse the cells. Cellular debris was pelletted (13,000 ϫ g, 5 min), and protein concentration of the supernatant was determined. Proteins (25 g) were separated over 8 -16% Tris-glycine gradient gels (Invitrogen) under denaturing and reducing conditions and electroblotted onto nitrocellulose membranes (Invitrogen). Membranes were blocked in TBS/Tween (20 mM Tris, 150 mM NaCl, 0.1% Tween (v/v), pH 7.5) supplemented with 0.5% nonfat dry milk (Bio-Rad) at room temperature (RT) for 1 h. Blots were probed with the indicated antibodies for 1 h at RT at a 1:200 dilution in blocking solution. Membranes were washed with TBS/Tween and probed with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at RT at a 1:5000 dilution in blocking solution. Blots were washed, and proteins were detected by enhanced chemiluminescence with ECL Western blotting detection reagents (Amersham Biosciences) and exposure to Hyperfilm ECL (Amersham Biosciences). Film was scanned, and band intensities were determined using Scion Image software (Scion Corp.) at exposures within the linear response range of the film.
Pulse-Chase Experiments-H4IIE cells were grown in William's Medium E containing 10% FBS and 40 M 2,3,2-tetraamine for 24 h. Cells (at 80% confluence) were then starved with Dulbecco's modified Eagle's medium lacking methionine and cystine (Sigma) and supplemented with 2% dialyzed FBS (Sigma) for 1 h. Medium was removed, and the cells were incubated with the same medium containing 100 Ci/ml Redivue Pro-mix L-35 S in vitro cell labeling mix for 4 h (Amersham Biosciences). Cells were washed and incubated with complete William's Medium E supplemented with 2 mM of unlabeled L-methionine/L-cysteine and containing either 40 M 2,3,2-tetraamine or 20 M CuSO 4 . Cells were harvested at various times and lysed in buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA) containing Complete Mini protease inhibitor mixture tablets (Roche Diagnostics). 400 g of total protein extract was precleared for 1 h at 4°C with protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) and then incubated for 16 h with CCS antibody FL-274. Protein A/G PLUS-agarose beads were added for 1 h, and after washing, immunoprecipitated proteins were subjected to SDS-PAGE and the gel exposed to film. (22) that rats fed low copper diets show a dose-dependent up-regulation of CCS protein in liver and erythrocytes that parallels closely the reduction in SOD1 activity and changes in other indices associated with copper deficiency. These results suggest that cellular copper levels may control CCS expression. In this study we sought to determine the mechanism by which CCS expression is regulated and the involvement of copper in this process. An antibody raised against a C-terminal region of human CCS was used to determine CCS expression by Western blot in livers from rats fed a normal or copper-deficient diet for 6 weeks. By using this antibody, a single band that migrated at ϳ33 kDa in size was detected (Fig. 1A), which is in agreement with the expected molecular weight of CCS and previously reported data using this same antibody (17). Quantification of CCS immunoreactive protein revealed approximately a 9-fold increase in CCS expression in copper-deficient rats compared with rats fed a normal copper diet (Fig. 1B). These results are consistent with our previous data using a different antibody to CCS (22). In contrast to CCS, SOD1 protein levels were modestly reduced in liver of copper-deficient rats (Fig. 1A).

CCS Expression Is Regulated by a Post-transcriptional Mechanism-We have reported recently
The increase in CCS protein in copper-deficient rats could indicate increased transcription of the CCS gene or alternatively increased mRNA stability. To test these possibilities, Northern blot analysis was performed with total RNA isolated from the same livers used to measure CCS protein expression. Northern blotting using a CCS-specific probe revealed a single transcript of ϳ900 bp in size ( Fig. 2A). Quantification of CCS signal and normalization to the 18 S rRNA showed that CCS mRNA accumulated to similar levels in copper-normal and copper-deficient rats (Fig. 2B) excluding increased CCS gene transcription and mRNA stability as the mechanism underlying increased CCS expression. High quality of the RNA was confirmed by assessing the integrity of the 18 S and 28 S rRNAs ( Fig. 2A, bottom panel). Increased CCS protein content without any change in transcript levels suggests that CCS expression is likely controlled by a post-translational mechanism.
Copper Deficiency Induces CCS Expression in Rodent and Human Liver Cell Lines-To test whether copper is involved in controlling CCS expression, we examined whether CCS protein levels would change in response to copper deficiency in cultured cell lines. To induce copper deficiency, we used the specific copper chelator 2,3,2-tetraamine that has been shown previously to induce copper deficiency in various cell lines (24 -26).
From our previous work we found that copper-deficient rats showed the largest increase in CCS expression in liver tissue. Therefore, we chose to use the H4IIE rat liver cell line to examine CCS expression in response to 2,3,2-tetraamine treatment. H4IIE cells were grown in basal medium, medium supplemented with copper, or medium containing 10, 20, or 40 M 2,3,2-tetraamine for 48 h. Treatment of H4IIE cells with medium containing up to 40 M 2,3,2-tetraamine showed no adverse effects on cell growth or viability. 2 Following treatment, cells were harvested, and CCS expression was determined by Western blot. CCS protein was increased to over 4-fold with 10 M 2,3,2-tetraamine and up to 6-fold in cells treated with 40 M 2,3,2-tetraamine compared with cells cultured in medium supplemented with 10 M CuSO 4 (Fig. 3).
To determine whether 2,3,2-tetraamine treatment would induce CCS expression in human cells, we performed the same experiment with HepG2 cells, a well characterized human hepatoma cell line (Fig. 3). In these cells, CCS protein showed a more modest dose-dependent increase compared with H4IIE cells cultured in the same medium (Fig. 3). CCS expression was increased almost 3-fold with 40 M 2,3,2-tetraamine compared with cells cultured in medium supplemented with 10 M CuSO 4 (Fig. 3). ␤-Tubulin expression was unaffected by 2,3,2-tetraamine treatment and shows equal protein loading (Fig. 3). Note that CCS expression was often lower in cells cultured with basal medium supplemented with low micromolar (10 M) con-centrations of CuSO 4 compared with the same cells grown in basal medium alone, indicating that the basal medium used in our experiments is deficient in copper to an extent that incubation with medium supplemented with copper down-regulates CCS protein (Fig. 3).
Up-regulation of CCS Is Specific for Copper Deficiency-To confirm increased CCS content in cells treated with 2,3,2tetraamine was a result of copper deficiency and not deficiency of other metals, we performed experiments to examine the metal specificity of CCS up-regulation induced by 2,3,2-tetraamine. Elevated levels of CCS were induced in H4IIE cells by treating with 2,3,2-tetraamine. Equal amounts of CuSO 4 , FeSO 4 , or ZnSO 4 were then added directly to the cells and the cells incubated for 16 h. CCS level was increased 2-fold in cells cultured in the presence of 2,3,2-tetraamine compared with cells grown in basal medium (Fig. 4). Addition of CuSO 4 to the cells reduced CCS expression to levels comparable with cells cultured with basal medium alone (Fig. 4). Addition of equimolar amounts of FeSO 4 and ZnSO 4 did not reduce CCS protein, and levels were comparable with those detected in cells treated solely with 2,3,2-tetraamine (Fig. 4). In similar experiments, addition of CuSO 4 but not FeSO 4 or ZnSO 4 stimulated a decrease in CCS protein in HepG2 cells pretreated with 2,3,2tetraamine. 2 Together, these results demonstrate that up-regulation of CCS in H4IIE and HepG2 cells induced by 2,3,2tetraamine is specific for copper deficiency, and addition of copper to 2,3,2-tetraamine-treated cells promotes a decrease in CCS protein level.

Copper Promotes a Rapid Decrease in CCS Protein Level in
Copper-deficient Cells-To examine the rate of change in CCS protein level stimulated by copper, we performed a time course experiment. Elevated levels of CCS were induced in H4IIE cells with 2,3,2-tetraamine. Copper was then added directly to the cells, and the cells were analyzed for CCS expression at 0, 1, 3, 6, and 10 h following addition of copper (Fig. 5). As a reference, cells cultured in basal medium or medium supplemented with 10 M CuSO 4 were included in the experiment. CCS protein level in cells supplemented with copper was lower than in cells grown in basal medium (Fig. 5A). Addition of CuSO 4 to 2,3,2tetraamine-treated cells induced a rapid decrease in CCS protein level (Fig. 5, A and B). Decreased CCS protein could be detected as early as 1 h following addition of copper (Fig. 5, A  and B). By 10 h in the copper-rich medium, CCS protein level fell to 35% (Fig. 5B).
Copper Stimulates CCS Degradation by the 26 S Proteosome-The rapid decrease in CCS protein upon switching cells from copper-deficient to copper-rich medium suggests that high copper concentrations increase the turnover rate of CCS. The 26 S proteosome is a large multisubunit protease that controls expression of many short lived regulatory proteins (27)(28)(29). To determine whether copper-stimulated down-regulation of CCS was dependent on degradation by the 26 S proteosome, we tested whether the potent proteosome inhibitor MG132 could block the decrease in CCS protein in response to copper. H4IIE cells treated with 2,3,2-tetraamine were incubated in the presence of MG132 or vehicle (Me 2 SO) for 1 h prior to addition of CuSO 4 to the cells. CCS protein level was determined at 0, 1, 3, and 5 h following addition of copper (Fig. 6). Cells treated with Me 2 SO showed the expected reduction in CCS protein in response to copper (Fig. 6, A and B). CCS protein level decreased to 57% by 5 h (Fig. 6B) which is comparable with that observed for untreated cells (Fig. 5B). In contrast, treatment of cells with MG132 blocked the copper-stimulated decrease in CCS expression. More than 92% of CCS protein still remained in the MG132-treated cells after 5 h in copper-rich medium (Fig. 6B).
The reduction in copper-stimulated down-regulation of CCS in the presence of MG132 strongly suggested involvement of the 26 S proteosome in controlling CCS expression. To confirm involvement of the proteosome in regulating CCS expression, we tested the ability of the highly specific proteosome inhibitor lactacystin (30) to block copper-induced CCS down-regulation in a similar experiment (Fig. 7). H4IIE cells treated with lactacystin were completely resistant to copper-stimulated CCS down-regulation (Fig. 7, A and B). In contrast, CCS protein in cells treated with Me 2 SO decreased to 44% by 10 h of incubation in copper-rich medium (Fig. 7B).
In separate experiments, cells were cultured in medium supplemented with 10 M CuSO 4 and treated with lactacystin or Me 2 SO. Cells were then incubated with cycloheximide to block protein synthesis. After 1 h of incubation with cycloheximide, CCS expression was analyzed at 0, 2, and 4 h. CCS protein fell to 79% by 2 h and 44% by 4 h for Me 2 SO-treated cells (Fig. 7C).
In contrast, lactacystin completely blocked CCS degradation and showed a slight increase in CCS expression at 2 and 4 h (Fig. 7C). The time-dependent increase in CCS expression for lactacystin-treated cells likely reflects a decrease in total cellular protein upon blocking protein synthesis, resulting from degradation of proteins by proteosome-independent pathways. Because the amount of CCS was measured in a fixed amount of total protein (25 g), a decrease in total protein in the absence of CCS degradation would be reflected by an increase in CCS content.
The block in CCS down-regulation with lactacystin treatment after inhibiting protein synthesis shows that the proteosome directly or indirectly regulates CCS expression by promoting CCS degradation. To examine whether other protein degradation pathways participate in CCS turnover, we tested if the general acidification inhibitors of the lysosomal degradation pathway NH 4 Cl and chloroquine and the cysteine protease inhibitor calpain inhibitor II (N-acetyl-leucinyl-leucinyl-methional-H) affect CCS degradation in response to copper (Fig. 8). H4IIE cells treated with 2,3,2-tetraamine were incubated with calpain inhibitor II, NH 4 Cl, or chloroquine for 1 h prior to adding CuSO 4 . CCS expression was determined at 0, 2, and 6 h after addition of copper. Treatment with calpain inhibitor II, NH 4 Cl, or chloroquine had no effect on the rate of copper-induced CCS downregulation compared with untreated cells (Fig. 8B). To ensure that chloroquine and ammonium chloride increased lysosomal pH in our cells under our conditions, we compared fluorescence intensity of the pH-sensitive probes LysoSensor Green and Ly-soSensor Yellow/Blue (Molecular Probes) in cells treated with the inhibitors to that of untreated cells. Lysosomal staining intensity in cells treated for 1 h with chloroquine or ammonium chloride was significantly altered compared with untreated cells indicating increased lysosomal pH. 2 Taken together, these results show that copper stimulates CCS down-regulation mainly by the 26 S proteosome.
Copper Deficiency Increases CCS Stability-To investigate further the effect of copper on the degradation of CCS, H4IIE cells cultured in medium supplemented with copper or 2,3,2tetraamine were treated with cycloheximide to block protein synthesis (Fig. 9). After 1 h of incubation with cycloheximide, cells were examined for CCS expression at 0, 2, 4, and 7 h. Cells grown in copper-supplemented medium showed a sharp decrease in CCS protein, as levels fell to 35% by 7 h (Fig. 9, A and  B). CCS expression in cells cultured in the presence of 2,3,2tetraamine remained constant, showing little change in CCS protein level even 7 h after blocking protein synthesis (Fig. 9, A  and B).
To determine the influence of copper on the half-life of CCS, we performed a pulse-chase experiment (Fig. 10). H4IIE cells were cultured in the presence of 40 M 2,3,2-tetraamine for 24 h. Cells were metabolically labeled for 4 h with 35 S-labeled methionine and cysteine and then chased for 0, 4, 14, and 18 h or 0, 2, 4, and 12 h with medium containing 40 M 2,3,2tetraamine or 20 M CuSO 4 , respectively. The half-life of CCS in cells grown in copper-rich or copper-deficient media was determined to be ϳ6 and 22 h, respectively (Fig. 10B). Together, these data demonstrate that the degradation of CCS is more rapid when copper is abundant than when it is scarce.

DISCUSSION
The importance of proper handling of copper is underscored by the severe symptoms associated with genetic disorders related to abnormal copper metabolism such as Menkes syndrome and Wilson's disease (31,32). In addition, mutations in the human SCO2 gene that encodes for a copper chaperone required for cytochrome c oxidase assembly have been linked recently to a fatal infantile mitochondrial disorder characterized by hypertrophic cardiomyopathy and encephalopathy (33,34).
Copper homeostasis is dependent on the coordinate interaction of cell-surface transporters that mediate copper uptake, membrane-bound intercellular transporters that regulate cellular copper efflux, and copper chaperones that deliver copper to specific targets within the cell. The P-type transporters ATP7A and ATP7B have been shown to redistribute from the trans-Golgi network to the cell surface and cytoplasmic vesicles, respectively, under condition of high copper where they function to rid the cell of excess copper (35)(36)(37)(38). A recent study examining expression and localization of the human high affinity copper uptake transporter, ctr1, has revealed that cells exposed to elevated levels of copper reduce cell-surface expression of ctr1 and decrease its steady-state level, suggesting a reduction in cellular copper uptake (39). Studies addressing the copper-dependent regulation of copper transporters have improved considerably our understanding of the mechanisms involved in regulating copper concentrations in the cell; however, to date, little is known about how the distribution of copper to cellular compartments and enzymes by copper chaperones is influenced by copper status. Our previous work (22) has shown that inducing copper deficiency in rats by feeding low copper diets significantly up-regulates (up to 10-fold) CCS protein levels in various tissues, suggesting that copper may control CCS activity. This study was aimed at investigating the mechanism regulating CCS expression and the role that copper plays in this process.
We have shown that increased CCS protein induced by copper deficiency is not accompanied by an increase in CCS mRNA expression, implying a post-transcriptional mechanism for CCS regulation. Exposing H4IIE and HepG2 cells to the specific copper chelator 2,3,2-tetraamine revealed a dose-dependent increase in CCS protein consistent with our previous results observed in liver and erythrocytes of rats fed diets varying in copper content (22). CCS up-regulation induced by 2,3,2tetraamine was determined to be specific for copper deficiency as addition of copper to these cells reduced CCS protein to background levels. In addition, adding similar amounts of iron or zinc to the cells had no effect on CCS expression, further supporting copper deficiency as the sole cause for increased CCS protein level with 2,3,2-tetraamine treatment.
Even though 2,3,2-tetraamine treatment elevated CCS levels in both H4IIE and HepG2 cells, the magnitude of induction was less pronounced in HepG2 cells. At this time we cannot say for certain whether the difference in response is due to species differences or specific properties of the cell lines. In this regard, discrepancies in the regulation of ctr1 have also been reported with different cell types (39 -41). It is likely that different tissues have evolved to respond differently to copper availability and the response may depend on the specific copper requirements for that cell type.
We observed that switching cells from a copper-deficient to a copper-rich environment rapidly stimulated a decrease in CCS protein level. A decrease in CCS protein was detected as early as 1 h following addition of copper. The rapid decrease in CCS protein is consistent with the rapid influx of copper reported for cells in culture (40). These results are also in agreement with the rapid induction of SOD1 activity observed when yeast are switched from copper-deficient to copper-rich conditions (42). Given that CCS functions by scavenging for copper and delivering the metal specifically to SOD1, elevated levels of CCS likely increase the efficiency of copper transfer to SOD1. The elevated levels of CCS in copper-deficient cells may allow for the rapid insertion of copper into apoSOD1 when it becomes available. Once SOD1 is fully active there would be no need for elevated CCS levels to increase efficiency of copper delivery to the enzyme, and hence CCS expression is quickly decreased.
Blocking protein synthesis and monitoring CCS degradation over time in cells cultured in copper-deficient or copper-rich medium revealed that the turnover of CCS is much faster when copper is abundant. When cells were cultured in copper-rich medium, CCS levels decreased to 35% in 7 h after blocking protein synthesis with cycloheximide. In contrast, cells grown in copper-deficient medium induced by the presence of 2,3,2tetraamine showed a resistance to degradation of CCS. Furthermore, pulse-chase experiments showed an extended halflife for CCS when cells were cultured in copper-deficient (ϳ22 h) as opposed to copper-rich medium (ϳ6 h). However, in contrast to the rapid decrease in CCS expression upon switching cells from copper-deficient to copper-rich medium, CCS upregulation upon incubating cells in copper-deficient medium was considerably slower. We observed a steady increase in CCS protein level in cells cultured in the presence of 2,3,2-tetraamine over the course of 60 h, which could be reduced to background levels in about 12-14 h upon adding copper to the cells. 2 Together, our results are consistent with a model in which low cellular copper levels increase CCS stability which promotes its accumulation over time.
Many cytosolic proteins whose function is dependent on their rapid regulation in expression are degraded by the 26 S proteosome, a large multisubunit protease. In our experiments, we determined that the potent proteosome inhibitor MG132 and the specific proteosome inhibitor lactacystin blocked copperstimulated down-regulation of CCS protein. In addition, lactacystin treatment completely blocked CCS degradation when protein synthesis was inhibited with cycloheximide. We also found that calpain inhibitor II, a cysteine protease inhibitor, and general acidification inhibitors of the lysosomal degradation pathway did not affect copper-induced degradation of CCS. These results demonstrate that copper regulates CCS expression by promoting its degradation predominantly by the 26 S proteosome. Interestingly, a recent study (43) has shown that CCS mutants with a mutation that is similar to a mutation in SOD1 that causes SOD1-mediated familial amyotrophic lateral sclerosis form aggregates, and aggregate formation could be exacerbated by treatment of cells with an inhibitor of the proteosome. Given that copper modulates CCS degradation by the proteosome, it would be interesting to test whether aggregation of these CCS mutants is affected by copper status of the cell.
The stability of many proteins is altered by changes in conformation of the protein. When copper is abundant it is likely that CCS can bind copper more readily than when cellular copper levels are reduced. It is therefore possible that copperloaded CCS adopts a conformation that renders the protein less stable, increasing its rate of degradation by the proteosome. This is consistent with biochemical analyses that have shown that binding of copper causes an allosteric conformational change in CCS (19). Alternatively, increased stability of CCS in copper-deficient cells may be mediated by a stable interaction with apoSOD1 or other proteins. Also from our data we cannot exclude a model where the proteosome regulates CCS expression indirectly by modulating the activity of a protein that is sensitive to copper status of the cell and that influences the stability of CCS.
In summary, we have shown that copper regulates CCS expression level by modulating its rate of degradation by the 26 S proteosome. To our knowledge, this is the first report describing copper-dependent regulation of any of the copper chaperones identified in higher eukaryotes. Further studies examining how the expression and activity of other copper chaperones and their respective enzymes are influenced by copper deficiency will provide valuable information regarding the utilization of copper when it is limiting. Nonetheless, our results provide new insight into the mechanisms involved in maintaining cellular copper homeostasis and suggest a novel role for CCS in prioritizing the distribution of copper when it is scarce.