The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1−/− mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1−/− phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1−/− progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis

The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein–protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three disease-associated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development

The trace metal copper (Cu) plays an essential role in biology as a cofactor for many enzymes that include Cu, Zn superoxide dismutase, cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine β-hydroxylase. Consequently, Cu transport at the cell surface and the delivery of Cu to intracellular compartments are critical events for a wide variety of biological processes. The components that orchestrate intracellular Cu trafficking and their roles in Cu homeostasis have been elucidated by the studies of model microorganisms and by the characterizations of molecular basis of Cu-related genetic diseases, including Menkes disease and Wilson disease. However, little is known about the mechanisms for Cu uptake at the plasma membrane and the consequences of defects in this process in mammals. Here, we show that the mouse Ctr1 gene encodes a component of the Cu transport machinery and that mice heterozygous for Ctr1 exhibit tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. Mice completely deficient for Ctr1 exhibit profound growth and developmental defects and die in utero in mid-gestation. These results demonstrate a crucial role for Cu acquisition through the Ctr1 transporter for mammalian Cu homeostasis and embryonic development.

Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis1

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa polypeptide exhibiting significant similarity and identity to various bacterial metal transporters. Potential ATX1 homologues were also identified in multicellular eukaryotes, including the plants Arabidopsis thaliana and Oryza sativa and the nematode Caenorhabditis elegans. In yeast cells, ATX1 evidently acts in the transport and/or partitioning of copper, and this role in copper homeostasis appears to be directly relevant to the ATX1 suppression of oxygen toxicity: ATX1 was incapable of compensating for SOD when cells were depleted of exogenous copper. Strains containing a deletion in the chromosomal ATX1 locus were generated. Loss of ATX1 function rendered both mutant and wild-type SOD strains hypersensitive toward paraquat (a generator of superoxide anion) and was also associated with an increased sensitivity toward hydrogen peroxide. Hence, ATX1 protects cells against the toxicity of both superoxide anion and hydrogen peroxide.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5′ flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at −646 and −395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at −395 bp. There was no reaction at −395 bp with anti-Sp1 antibody nor with either antibody at −646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.

Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca2+-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca2+]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca2+]cyt and inhibition of growth that was similar to the effect of the Ca2+ channel blocker La3+ and the Ca2+ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca2+ channel blockers verapamil and nifedipine did not induce a decrease in [Ca2+]cyt in these cells and also failed to inhibit growth. Al and La3+, but not verapamil or nifedipine, reduced the rate of Mn2+ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca2+- and Mn2+-permeable channels. These results suggest that Al may act to block Ca2+ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein

We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.

The Doa4 Deubiquitinating Enzyme Is Required for Ubiquitin Homeostasis in Yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Δ mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Δ cells. This loss of viability and several other defects of doa4Δ cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.