Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).     

Uptake of copper from plasma proteins in cells where expression of CTR1 has been modulated

Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1?μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.     

Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro

MDCK cells expressing an inducible duodenal cytochrome b-green fluorescent protein (Dcytb-EGFP) fusion construct were used to investigate the function of Dcytb. The Dcytb-EGFP protein was targeted correctly to the plasma membrane, and cells displayed increased ferric and cupric reductase activities, which were greatly reduced in the presence of doxycycline. The data suggests that Dcytb plays a physiological role in both iron and copper uptake, through divalent metal transporter 1 (DMT1) and copper transporter 1, respectively. In support of this hypothesis, we show that 59Fe uptake was significantly enhanced in Dcytb-EGFP expressing MDCK cells which endogenously express DMT1.     

Composition of proteoglycans in the aortas of copper-deficient rats

Copper deficiency adversely affects the extracellular matrix of the arterial wall, leading to cardiovascular lesions. To study the lesions resulting from copper deficiency, the composition of proteoglycans from aortas of copper-deficient rats was compared with proteoglycans of aortas from copper-supplemented rats. Copper deficiency in rats was verified by copper levels in adrenal glands (mean +/- SE, 0.37 +/- 0.07 vs 1.03 +/- 0.17 micrograms/g wet wt in supplemented rats). The proteoglycans were isolated from the aorta by extraction with 4 M guanidine-HCl and by digestion of the tissue with elastase. The proteoglycans were purified by CsCl isopycnic centrifugation and fractionated by gel filtration. The fractions were characterized for molecular size and glycosaminoglycan composition. Total uronate in the aortas from copper-deficient rats was 25% greater than in aortas from copper-supplemented rats, and the proteoglycans from copper-deficient rat aortas were of greater molecular size. Among the glycosaminoglycans the concentration (microgram/mg tissue) of isomeric chondroitin sulfates, particularly dermatan sulfate, was greater in copper-deficient animals than in copper-supplemented animals. These observations are similar to earlier findings in experimental atherosclerosis and to a response of cardiovascular connective tissue to injury.     

The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c(6) and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper- conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper-deficient conditions, the crd strains fail to accumulate photosystem I and light-harvesting complex I, and they contain reduced amounts of light-harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate-bridged di-iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper- or oxygen-deficient cells, and Crd1 function is required for adaptation to these conditions.     

The effects of copper deficiency on the pyridinium crosslinks of mature collagen in the rat skeleton and cardiovascular system

The 3-hydroxypyridinium crosslinks of collagen were quantified in tissues of the skeleton and cardiovascular system of normal and copper-deficient rats. The copper-deficient rats used in this study displayed retarded growth, cardiac hypertrophy, anemia, and lowered liver copper concentrations. Quantification of the crosslinks by high performance liquid chromatography indicated that there were lower concentrations of collagen crosslinks in the hearts of copper-deficient animals, a finding that was manifest in both right and left ventricles. This was in contrast to the collagen of the aorta where no alteration in crosslink concentration was observed. The femoral diaphysis of copper-deficient rats also had lower amounts of collagen crosslinks than copper-supplemented animals, whereas crosslinking in the tibial diaphysis and articular cartilage was relatively unaffected by copper deficiency. These results are discussed with reference to the cardiac and skeletal abnormalities that occur in copper-deficient animals.     

Different induction of 70,000-Da heat shock protein and metallothionein in HeLa cells by copper.

To clarify the physiological roles of heat shock proteins induced by copper, we studied the synthesis of these proteins and metallothionein, as well as the level and nature of copper incorporated into HeLa cells. Incubation in medium containing 200 microM cupric sulfate and above induced the synthesis of 70,000-Da heat shock protein (hsp70) in these cells. However, the synthesis of hsp70 did not increase in the presence of less than 200 microM cupric sulfate. On the other hand, the synthesis of metallothionein increased due to 100 microM cupric sulfate. The uptake of copper into the cells depended on the cupric sulfate concentration in the medium. To analyze the nature of the intracellular copper, cell extracts were separated by gel filtration chromatography into three fractions: the high molecular weight, metallothionein, and low molecular weight fractions. No copper was found in the low molecular weight fraction of control cells, but appeared distinctly at 200 microM cupric sulfate and above. Copper in the high molecular weight fraction also began to increase at 200 microM cupric sulfate and above, whereas in the metallothionein fraction it began to increase even at 50 to 100 microM cupric sulfate. Furthermore, inhibition of cell growth was also observed at 200 microM cupric sulfate and above but not at 100 microM and below.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.     

Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.     

Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit approximately 30% residual copper transport activity that is saturable, with a K(m) of approximately 10 microm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.     

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 utilization of copper when it is scarce.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

Regulation of copper absorption by copper availability in the Caco-2 cell intestinal model

Relatively little is known about the individual steps in intestinal copper absorption and whether or how they may be regulated. Polarized Caco-2 cell monolayers with tight junctions offer an already tested model in which to study intestinal metal transport. This model was used to examine potential effects of cellular copper availability on copper absorption. Uptake and transport were determined on application of (64)Cu(II) to the brush border. In the range of 0.2-2 micro M, uptake was dose dependent and was approximately 20% of dose/90 min. Overall transport of (64)Cu across the basolateral surface was approximately 0.3%. When cellular copper levels were depleted 40% by 18-h pretreatment with the specific copper chelator triethylenetetraamine, uptake and overall transport were markedly increased, going to 80 and 65% of dose, respectively. Cellular retention of (64)Cu fell fourfold, from 6 to 1.5%. Depletion of copper with the chelator was rapid and preceded initial changes in uptake and overall transport by 4 h. A lesser depletion of cellular copper (13%) failed to enhance copper uptake but doubled the rate of overall transport, as measured with (64)Cu and by atomic absorption. As previously reported, preexposure of the cells to excess copper (10 micro M, 18 h) also enhanced copper uptake ( approximately 3-fold). In contrast, ascorbate (10-1,000 micro M) failed to significantly alter uptake and transport of 1 micro M (64)Cu. Our findings are consistent with the concepts that, in the low physiological range, copper availability alters the absorption capacity of the intestine to support whole body homeostasis and that basolateral transport is more sensitively regulated than uptake.