High-affinity Copper Transporter Research Articles

A Re-Evaluation of the Role of hCTR1, the Human High-Affinity Copper Transporter, in Platinum-Drug Entry into Human Cells

Cisplatin (cDDP) is an anticancer drug used in a number of malignancies, including testicular, ovarian, cervical, bladder, lung, head, and neck cancers. Its use is limited by the development of resistance, often rationalized via effects on cellular uptake. It has been claimed that human copper transporter 1 (hCTR1), the human high-affinity copper transporter, is the major entry pathway for cDDP and related drugs via a mechanism that mimics copper. This is an unexpected property of hCTR1, a highly selective copper (I) transporter. We compared the uptake rates of copper with cDDP (and several analogs) into human embryonic kidney 293 cells overexpressing wild-type or mutant hCTR1, mouse embryonic fibroblasts that do or do not express CTR1, and human ovarian tumor cells that are sensitive or resistant to cDDP. We have also compared the effects of extracellular copper, which causes regulatory endocytosis of hCTR1, to those of cDDP. We confirm the correlation between higher hCTR1 levels and higher platinum drug uptake in tumor cells sensitive to the drug. However, we show that hCTR1 is not the major entry route of platinum drugs, and that the copper transporter is not internalized in response to extracellular drug. Our data suggest the major entry pathway for platinum drugs is not saturable at relevant concentrations and not protein-mediated. Clinical trials have been initiated that depend upon regulating membrane levels of hCTR1. If reduced drug uptake is a major factor in resistance, hCTR1 is unlikely to be a productive target in attempts to enhance efficacy, although the proteins involved in copper homeostasis may play a role.

Arabidopsis Copper Transport Protein COPT2 Participates in the Cross Talk between Iron Deficiency Responses and Low-Phosphate Signaling

Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

Mechanistic basis for overcoming platinum resistance using copper chelating agents.

Platinum-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other effective treatment options are limited. Recently, we conducted a clinical trial using a copper-lowering agent to overcome platinum drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in platinum-based chemotherapy, we showed that cDDP resistance associated with reduced expression of the high-affinity copper transporter (hCtr1), which is also a cDDP transporter, can be preferentially resensitized by copper-lowering agents because of enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by copper chelation can be explained by the mammalian copper homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a copper-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after platinum drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using copper chelation to overcome cDDP resistance in clinical investigations.

Calcium‐ and potassium‐permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production

Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH(•) ), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper-transport activity: Col-0, high-affinity copper transporter COPT1-overexpressing (C1(OE) ) seedlings, and T-DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm) stimulated a dose-dependent Gd(3+) and verapamil sensitive net Ca(2+) influx in the root apex but not in mature zone. C1(OE) also showed a fivefold higher Cu-induced K(+) efflux at the root tip level compared with Col-0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1(OE) seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd(3+) . Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH(•) -sensitive Ca(2+) and K(+) transport systems.

Role of the Human High-Affinity Copper Transporter in Copper Homeostasis Regulation and Cisplatin Sensitivity in Cancer Chemotherapy

The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.

Structural Determinants of Cisplatin and Transplatin Binding to the Met-Rich Motif of Ctr1: A Computational Spectroscopy Approach.

The cellular uptake of cisplatin and of other platinum-based drugs is mediated by the high-affinity copper transporter Ctr1. The eight-residue long peptide called Mets7 (MTGMKGMS) mimics one of extracellular methionine (Met)-rich motifs of Ctr1. It is an excellent model for investigating the interaction of platinum drugs with Ctr1 under in vitro and in vivo conditions. Some of us have shown that (i) Cisplatin loses all of its ligands upon reaction with Mets7 and the metal ion binds to the three Met residues and completes its coordination shell with a fourth ligand that can be a chloride or a water/hydroxyl oxygen. (ii) Transplatin loses only the chlorido ligands, which are replaced by Met residues. Here, we provide information on the structural determinants of cisplatin/Mets7 and transplatin/Mets7 adducts by computational methods. The predictions are validated against EXAFS, NMR, and CD spectra. While EXAFS gives information restricted to the metal coordination shell, NMR provides information extended to residue atoms around the coordination shell, and finally, CD provides information about the overall conformation of the peptide. This allows us to elucidate the different reaction modes of cisplatin and transplatin toward the peptide, as well as to propose the platinated peptides [PtX](+)-(M*TGM*KGM*S) (X = Cl(-), OH(-)) and trans[Pt(NH3)2](2+)-(M*TGM*KGMS) as the most relevant species occurring in water solution.

Tmac1, a transcription factor which regulated high affinity copper transport in Trichoderma reesei

The Mac1 protein was a transcriptional activator that sensed very low concentration of copper and regulated the copper transport in Saccharomyces cerevisiae. Here, we cloned a gene from Trichoderma reesei named Tmac1, whose deduced amino acid sequence showed 29% identical to Mac1p. Furthermore, two Cys-His repeats metal binding motifs of Tmac1p, one in the 354–369C terminus and one in the 475–490C terminus were also present in Mac1p. A deletion mutant of Tmac1 was hypersensitive to the copper starvation and showed poor growth. Subsequently, the function was recovered by the gene complementation experiment. Furthermore, the Tmac1 gene fully complemented growth defects of yeast ΔMac1 mutant. The expression of Tmac1p was activated at low concentration of copper and depressed when the concentration of copper excess 1mM. Furthermore, the fluorescence intensity enhanced at copper starvation and decreased under copper excess by fusion the eGFP to the Tmac1p. It proved that the expression of Mac1p was exactly regulated by copper concentration, because eGFP and Mac1p were expressed under the control of the same one promoter. We also cloned a gene named Tctr3 with bioinformatics. With a series of experiments, we proved it was the target gene of Tmac1. To sum up, Tmac1 may encode a transcriptional activator regulated high-affinity copper transport in T. reesei.

Specificity Protein 1 (Sp1) Oscillation Is Involved in Copper Homeostasis Maintenance by Regulating Human High-Affinity Copper Transporter 1 Expression

Copper is an essential micronutrient for cell growth but is toxic in excess. Copper transporter (Ctr1) plays an important role in regulating adequate copper levels in mammalian cells. We have shown previously that expression of the human high-affinity copper transporter (hCtr1) was transcriptionally up-regulated under copper-depleted conditions and down-regulated under replete conditions; moreover, elevated hCtr1 levels suppress hCtr1 expression. Specificity protein 1 (Sp1) regulates expression of hCtr1 under copper-stressed conditions. In this study, we made the following important observations: 1) Sp1 expression is down-regulated under copper-replete conditions but up-regulated under copper-depleted conditions. These up- and down-regulations of Sp1 in turn regulate hCtr1 expression to control copper homeostasis. 2) Copper-regulated Sp1 expression involved Sp1 binding to its own promoter as demonstrated by the chromatin immunoprecipitation assay; therefore, Sp1 is also transcriptionally self-regulated via hCtr1/copper intermediation. 3) Both zinc finger and glutamine-rich transactivation domains of Sp1 are involved in the Sp1-mediated hCtr1 and Sp1 regulation by copper stresses. 4) Although Sp3 expression is also regulated by copper availability, Sp3 does not regulate hCtr1 homeostasis. Collectively, our results demonstrated that mammalian cells use Sp1 oscillation in response to copper availability to regulate copper homeostasis through hCtr1 expression in a tripartite inter-regulatory relationship. These findings have important implications in mammalian copper physiology regulation.

Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper

The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency

Copper is an essential micronutrient that functions as a redox cofactor in multiple plant processes, including photosynthesis. Arabidopsis thaliana possesses a conserved family of CTR-like high-affinity copper transport proteins denoted as COPT1-5. COPT1, the only family member that is functionally characterized, participates in plant copper acquisition. However, little is known about the function of the other Arabidopsis COPT proteins in the transport and distribution of copper. Here, we show that a functional fusion of COPT5 to the green fluorescent protein localizes in Arabidopsis cells to the prevacuolar compartment. Plants defective in COPT5 do not exhibit any significant phenotype under copper-sufficient conditions, but their growth is compromised under copper limitation. Under extreme copper deficiency, two independent copt5 knockout mutant lines exhibit severe defects in vegetative growth and root elongation, low chlorophyll content, and impairment in the photosynthetic electron transfer. All these phenotypes are rescued when the wild-type copy of the COPT5 gene is retransformed into a copt5 knockout line or when copper, but not other metals, are added to the medium. COPT5 is expressed in vascular tissues, with elevated levels in roots. Taken together, these results suggest that COPT5 plays an important role in the plant response to environmental copper scarcity, probably by remobilizing copper from prevacuolar vesicles, which could act as internal stores or recycling vesicles to provide the metal cofactor to key copper-dependent processes such as photosynthesis.

A comparison of methionine, histidine and cysteine in copper(i)-binding peptides reveals differences relevant to copper uptake by organisms in diverse environments

The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator. The relative affinities of the peptides with Cu(i) reveal a trend whereby Cys > His > Met at pH 7.4 and Cys > Met > His at pH 4.5. Ligand geometry and metric parameters were determined with X-ray absorption spectroscopy. Susceptibility of the peptides to oxidation by hydrogen peroxide and copper-catalyzed oxidative conditions was evaluated by mass spectrometry. These results support hypotheses as to why certain Cu(i) binding amino acids are preferred over others in proteins expressed at different pH and exposed to oxidative environments. The results also have implications for interpreting site-directed mutagenesis studies aimed at identifying copper binding amino acids in copper trafficking proteins.

Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I)

Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins (Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM, where X can be one of several amino acids. Model peptides corresponding to 15 different Mets motifs were synthesized and determined to selectively bind Cu(I) and Ag(I), with no discernible affinity for divalent metal ions. These are rare examples of biological thioether-only metal binding sites. Effective dissociation constant (KD) values for the model Mets peptides and Cu(I) were determined by an ascorbic acid oxidation assay and validated through electrospray ionization mass spectrometry and range between 2 and 11 microM. Affinity appears to be independent of pH, the arrangement of the motif, and the composition of intervening amino acids, all of which reveal the generality and flexibility of the MX1-2MX1-2M domain. Circular dichroism spectroscopy, 1H-NMR spectroscopy, and X-ray absorption spectroscopy were also used to characterize the binding event. These results are intended to aid the development of the still unknown mechanism of copper transport across the cell membrane.

Deregulated Copper Transport Affects Arabidopsis Development Especially in the Absence of Environmental Cycles

Copper is an essential cofactor for key processes in plants, but it exerts harmful effects when in excess. Previous work has shown that the Arabidopsis (Arabidopsis thaliana) COPT1 high-affinity copper transport protein participates in copper uptake through plant root tips. Here, we show that COPT1 protein localizes to the plasma membrane of Arabidopsis cells and the phenotypic effects of transgenic plants overexpressing either COPT1 or COPT3, the latter being another high-affinity copper transport protein family member. Both transgenic lines exhibit increased endogenous copper levels and are sensitive to the copper in the growth medium. Additional phenotypes include decreased hypocotyl growth in red light and differentially affected flowering times depending on the photoperiod. Furthermore, in the absence of environmental cycles, such as light and temperature, the survival of plants overexpressing COPT1 or COPT3 is compromised. Consistent with altered circadian rhythms, the expression of the nuclear circadian clock genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) is substantially reduced in either COPT1- or COPT3-overexpressing plants. Copper affects the amplitude and the phase, but not the period, of the CCA1 and LHY oscillations in wild-type plants. Copper also drives a reduction in the expression of circadian clock output genes. These results reveal that the spatiotemporal control of copper transport is a key aspect of metal homeostasis that is required for Arabidopsis fitness, especially in the absence of environmental cues.

Thesis Abstract Biochemical and molecular studies of copper metabolism during the infection process of the human pathogenic fungus Paracoccidioides brasiliensis

The fungus Paracoccidioides brasiliensis is a human pathogen with a wide distribution in Latin America. The fungus causes paracoccidioidomycosis when mycelia reach the lungs. The success of the infection depends on the acquisition of essential micronutrients such as copper, which is required as a cofactor for a variety of enzymes important in several biological processes, such as respiration, growth and iron uptake. Previous studies of the Laboratory of Molecular Biology showed that a high-affinity copper transporter (PbCTR3) is a molecule that is highly expressed and probably necessary for the establishment of infection by P. brasiliensis. In the present study, the genomic and cDNA sequences encoding PbCTR3 of P. brasiliensis were isolated and characterized. The cDNA consists of 582 bp and encodes a protein of 193 amino acids, with a predicted molecular mass of 21.5 kDa and pI of 8.6. The genomic sequence has four exons interrupted by three introns. In silico analysis was performed on the database of the structural genome of P. brasiliensis (http://www.broad.mit.edu/ annotation/genome/paracoccidioides_brasiliensisMultiHome.html), where genes involved in maintaining the homeostasis of copper have been identified and used to design a model of copper homeostasis in P. brasiliensis. The transcriptional behavior of Pbctr3 and genes involved in copper homeostasis were examined during exposure of yeast cells of P. brasiliensis to conditions of copper and iron depletion, by real time qRT-PCR. A significant change was found in the transcription level of these genes in the absence of copper, as well as in the combined absence of both metals. qRT-PCR was used to analyze the expression of Pbctr3 and Pbcrp (which encodes a protein responsive to copper) in yeast cells of P. brasiliensis derived from infected lungs and spleen at different times of infection. Pbctr3 and Pbcrp were super-regulated during experimental infection. Taken together, these data suggest the importance of PbCTR3 and of copper/iron uptake during the infection process.

Characterization of Ctr family genes and the elucidation of their role in the life cycle of Neurospora crassa

Transcriptional analysis using qRT-PCR of 62 metal ion transporters during conidial germination of Neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the Ctr family. Herein we characterised the Ctr family genes ( tcu- 1, tcu- 2 and tcu- 3) and deciphered their role in various developmental phases of the N. crassa life cycle. Cross complementation assays in copper uptake mutant of Saccharomyces cerevisiae revealed that tcu-1, tcu-2 and tcu-3 are functional homologs of S. cerevisiae copper transporters. Expression studies of Ctr family members in various developmental phases of N. crassa showed differential expression pattern for high-affinity copper transporter, TCU1. Functional analysis of their gene knockout mutants showed that tcu- 1 is essential for saprophytic conidial germination, vegetative growth and perithecia development under copper limited conditions while conidiation remained unaffected.

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP(C)) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP(C) proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP(C)-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP(C) in mediating copper uptake by clathrin-dependent endocytosis.

Regulation of copper transport in Arabidopsis thaliana: a biochemical oscillator?

Plants are among the most versatile higher eukaryotes in accommodating environmental copper availability to largely variable demands. In particular, copper deficiency in soils is a threat for plant survival since it mostly affects reproductive structures. One of the strategies that plant cells use to overcome this situation is to increase copper levels by expressing high-affinity copper transporters delivering the metal to the cytosol. In this minireview, we discuss recent advances in the structure, function, and regulation of the CTR/COPT family of copper transporters, and pay special attention to the Arabidopsis thaliana counterparts. These are constituted by transmembrane polypeptides, containing several copper-binding sequences of functional and/or regulatory value, and assembling as trimers. Copper deficiency activates the expression of some members of the COPT family via the interaction of the SPL7 transcription factor with reiterative GTAC motifs present in their promoters. Interestingly, the regulation of the synthesis of these transporters by copper itself constitutes a negative-feedback loop that could cause a sustained oscillation in the cytosolic copper levels. We analyze the theoretical conditions required for this hypothetical copper oscillation and the potential advantages of synchronization with other cycles. Diverse data in other organisms point to the relationship between copper homeostasis and circadian cycles.

Effect of a Deficiency of Ceruloplasmin Copper in Blood Plasma on Copper Metabolism in the Brain

Copper deficiency in adult rats was induced by addition of silver chloride to the feed. The concentrations of silver, copper, iron, and zinc and relative activity of genes for copper transporting proteins and copper enzymes were measured in the cortex, cerebellum, hippocampus, amygdala, pituitary gland, and hypothalamus. Silver was accumulated only in the hypothalamic-pituitary system. These changes were accompanied by a decrease in the concentration of copper and increase in the contents of iron and zinc. Activity of genes for copper transport enzymes (high-affinity copper transporter; and two copper transport ATPases, ATP7A and ATP7B) and copper enzymes that were formed in the intracellular secretory pathway did not decrease in the brain of rats with copper deficiency. Relative activity of genes for intracellular copper enzymes (Cu(2+)/Zn(2+) superoxide dismutase and subunit IV of cytochrome c oxidase), concentration of immunoreactive polypeptides of superoxide dismutase, and enzymatic activity of superoxide dismutase remained unchanged under these conditions.

Mechanistic Comparison of Human High-Affinity Copper Transporter 1-Mediated Transport between Copper Ion and Cisplatin

The human high-affinity copper transporter (hCtr1) plays an important role in the regulation of intracellular copper homeostasis. hCtr1 is involved in the transport of platinum-based antitumor agents such as cisplatin (CDDP); however, the mechanisms that regulate hCtr1-mediated transport of these agents have not been well elucidated. We compared the mechanisms of hCtr1-mediated transport of copper and CDDP. We found that replacements of several methionine residues that are essential for hCtr1-mediated copper transport conferred a dominant-negative effect on the endogenous hCtr1's function, resulting in reduced rates of Cu(I) and CDDP transport and increased resistance to the toxicities of copper and CDDP treatments. Kinetic constant analyses revealed that although these mutations reduced maximal transport rates (V(max)) for Cu(I) and CDDP, reduction of K(m) only for Cu(I) but not for CDDP was observed. Mutation in Gly167, which is located in the third transmembrane domain and is involved in helix packing of hCtr1, also conferred dominant-negative property of Cu(I) transport but not of CDDP transport. Deleting the N-terminal 45 amino acids that contain two methionine-rich motifs resulted in cytoplasmic localization of the hCtr1 and abolished the dominant-negative function of these mutants. Nonetheless, these mutations did not affect the capacities of hCtr1 oligomerization induced by copper or CDDP, suggesting a distinct structural requirement between metal transport and oligomerization. Finally, we also observed that expressing the dominant-negative hCtr1 mutants up-regulates endogenous hCtr1 mRNA expression, consistent with our previous report that intracellular copper homeostasis and homeostatic levels of hCtr1 mRNA are mutually regulated.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic- l-amino-acid decarboxylase ( Pbddc), lumazine synthase ( Pbls) and a homologue of the high affinity copper transporter ( Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RT-PCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis.