Copper-transporting ATPase Research Articles

NMR backbone resonance assignments of the N, P domains of CopA, a copper-transporting ATPase, in the apo and ligand bound states

Copper-transporting ATPase, a member of P-type ATPase family, plays a key role in the homeostasis of cellular copper levels. Here, the backbone assignments of the directly connected N and P domains (292 residues, 31kDa) of Cu-transporting ATPase in the ligand free and the AMPPCP-bound states are reported in solution. The NMR assignments pave the way for binding and dynamics studies of this enzyme to better understand its function.

Modifying factors and phenotypic diversity in Wilson's disease

Wilson's disease (WD) is a human disorder of copper homeostasis caused by mutations in the copper-transporting ATPase ATP7B. WD is characterized by copper accumulation, predominantly in the liver and brain, hepatic pathology, and wide differences between patients in the age of onset and the spectrum of symptoms. Several factors contribute to the phenotypic variability of WD. The WD-causing mutations produce a wide range of changes in stability, activity, intracellular localization, and trafficking of ATP7B; the nonpathogenic genetic polymorphisms may contribute to the phenotype. In Atp7b(-/-) mice, a mouse model of WD, an abnormal intracellular distribution of copper in the liver triggers distinct changes in the transcriptome; these mRNA profiles might be used to more specifically define disease progression. The major effect of accumulating copper on lipid metabolism and especially cholesterol homeostasis in mice and humans suggests the importance of fat and cholesterol metabolism as modifying factors in WD.

Peptide-Based Approach to Study Cytosolic Domain Interactions in a Bacterial Copper-Transporting ATPase

Copper-transporting PIB-type ATPases (CopA-family) fulfill key roles in copper homeostasis by pumping copper across bacterial or intracellular and cytoplasmic eukaryotic membranes. Mutations of the copper-transporting ATPase gene cause Wilson and Menkes diseases. CopA consists of 8 transmembrane helices and three cytosolic domains; Nucleotide binding (N), Phosphorylation (P) and Actuator (A) domains. We have used synthetic peptides to identify minimal structural motifs and physical mechanisms of cytosolic domain interactions.Based on the crystal structure[1], we have designed decameric-peptides whose sequences are derived from the putative interaction site of the A-domain with the PN domain of CopA from Legionella pneumophila. The interaction of the synthetic A-domain peptide with the individually expressed PN domain was measured by Isothermal Titration Calorimetry(ITC), Circular Dichroism (CD), and stopped-flow fluorescence techniques. Preliminary ITC results show a favorable TΔS, indicating that binding of the peptide carrying the native sequence is driven by hydrophobic interactions with stoichiometry(N=1). Time-resolved fluorescence measurements with a tryptophan-carrying actuator peptide show association and dissociation rate constants with the PN domain of 22000M−2s−1 and 0.7M−1s−1, respectively. This corresponds to a Kd of the peptide for the PN-domain of 32μM. In the presence of the non-hydrolyzable nucleotide AMPPNP the best kinetic fit was obtained with rates of 20000M−2s−1 and 1.0M−1s−1, indicating an almost unaffected affinity of the peptide for the nucleotide-loaded PN-domain. In agreement with the kinetic and isothermal data, the deca-peptide also shifted the melting temperature of the PN domain from 330 to 332K as monitored by CD spectroscopy.In summary, we have good evidence that the actuator peptide binds in a predominantly hydrophobic mechanism to the PN domain in the 30-50μM Kd range with little dependence on the occupancy of the nucleotide binding site of the PN domain.1)Gourdon,P. et.al.2011 Nature475, 59.

Profile of whole blood gene expression following immune stimulation in a wild passerine.

BackgroundImmunoecology aims to explain variation among hosts in the strength and efficacy of immunological defences in natural populations. This requires development of biomarkers of the activation of the immune system so that they can be collected non-lethally and sampled from small amounts of easily obtainable tissue. We used transcriptome profiling in wild greenfinches (Carduelis chloris) to detect whole blood transcripts that most profoundly indicate upregulation of antimicrobial defences during acute phase response. The more general aim of this study was to obtain a functional annotation of a substantial portion of the greenfinch transcriptome that would enable to gain access to more specific genomic tools in subsequent studies. The birds received either bacterial lipopolysaccharide or saline injections and RNA-seq transcriptional profiling was performed 12 h after treatment to provide initial functional annotation of the transcriptome and assess whole blood response to immune stimulation.ResultsA total of 66,084 transcripts were obtained from de novo Trinty assembly, out of which 23,153 could be functionally annotated. Only 1,911 of these were significantly upregulated or downregulated. The manipulation caused marked upregulation of several transcripts related to immune activation. These included avian-specific antimicrobial agents avidin and gallinacin, but also some more general host response genes, such as serum amyloid A protein, lymphocyte antigen 75 and copper-transporting ATPase 1. However, links with avian immunity for most differentially regulated transcripts remained rather hypothetical, as a large set of differentially expressed transcripts lacked functional annotation.ConclusionsThis appears to be the first large scale transcriptional profiling of immune function in passerine birds. The transcriptomic data obtained suggest novel markers for the assessment of the immunological state of wild passerines. Characterizing the function of those possible novel infection markers would assist future vertebrate genome annotation. The extensive sequence information collected enables to identify possible target and housekeeping genes needed to gain access to more specific genomic tools in future studies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-533) contains supplementary material, which is available to authorized users.

Nephrotic syndrome after treatment with D-penicillamine in a patient with Wilson’s disease

Wilson’s disease is an inherited autosomal recessive disorder of copper balance leading to accumulation of copper mainly in liver and brain result from absent or reduced function of copper-transporting P-type ATPase. Copper is an essential trace element but in Wilson’s disease it accumulate to the point of toxicity. D-penicillamine is a classic drug for treatment of Wilson’s disease. Its major effect is to promote the urinary copper excretion. The use of D-penicillamine in the therapy of Wilson’s disease is known to be complicated by the development of various glomerular diseases. In this report we describe the development of nephrotic syndrome after 2 years treatment with D-penicillamine in a 31-year-old male undergoing treatment for Wilson’s disease, with a prompt regression at the discontinuation of the drug. We present this case to draw attention to the rare complication as nephrotic syndrome in patients with Wilson’s disease under D-penicillamine treatment and possible underlying causes. It is strongly necessary the therapy and clinical condition of patients with Wilson’s disease to be monitoring regularly – we recommended monthly.

A copper-responsive gene cluster is required for copper homeostasis and contributes to oxidative resistance in Deinococcus radiodurans R1.

Excess copper is toxic to organisms, and therefore, copper homeostasis is important for the limitation of its cellular levels. However, copper homeostasis has not been studied to date in the bacteria Deinococcus radiodurans R1, which exhibits extreme resistance to various environmental stresses. We have identified a copper-responsive gene cluster that encodes CopA, which is a copper-transporting P1-type ATPase, CopZ, which is a copper metallochaperone, and CsoR, which is a copper-sensing repressor. Copper induces the transcription of genes in this cluster. Mutants lacking copA exhibited reduced copper resistance and the overaccumulation of copper compared with the wild-type strain. Additionally, both in the absence and presence of copper, the copZ mutation increased the expression of copA and led to the accumulation of lower levels of copper compared with the wild type. The bioinformatic analysis showed that CsoR in D. radiodurans R1 shares high sequence similarity and identity with the CsoR of Mycobacterium tuberculosis and Bacillus subtilis. We also demonstrated through DNase I footprinting and electrophoretic mobility shift assays that CsoR binds to the promoter of the cluster and that copper ions eliminate this interaction. This implies that CsoR is the repressor of this cluster and that CopA, CopZ and CsoR participate in the regulation of copper homeostasis. Our data also indicate that after treatment with H2O2 and cumene hydroperoxide, the viability of the copA mutants was significantly reduced. This suggests that copper homeostasis plays an important role in oxidative resistance in D. radiodurans R1.

Silencing the Menkes Copper-Transporting ATPase (Atp7a) Gene in Rat Intestinal Epithelial (IEC-6) Cells Increases Iron Flux via Transcriptional Induction of Ferroportin 1 (Fpn1)1–3

The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron (59Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished.

Copper‐transporting ATPase is important for malaria parasite fertility

Homeostasis of the trace element copper is essential to all eukaryotic life. Copper serves as a cofactor in metalloenzymes and catalyses electron transfer reactions as well as the generation of potentially toxic reactive oxygen species. Here, we describe the functional characterization of an evolutionarily highly conserved, predicted copper-transporting P-type ATPase (CuTP) in the murine malaria model parasite Plasmodium berghei. Live imaging of a parasite line expressing a fluorescently tagged CuTP demonstrated that CuTP is predominantly located in vesicular bodies of the parasite. A P. berghei loss-of-function mutant line was readily obtained and showed no apparent defect in in vivo blood stage growth. Parasite transmission through the mosquito vector was severely affected, but not entirely abolished. We show that male and female gametocytes are abundant in cutp− parasites, but activation of male microgametes and exflagellation were strongly impaired. This specific defect could be mimicked by addition of the copper chelator neocuproine to wild-type gametocytes. A cross-fertilization assay demonstrated that female fertility was also severely abrogated. In conclusion, we provide experimental genetic and pharmacological evidence that a healthy copper homeostasis is critical to malaria parasite fertility of both genders of gametocyte and, hence, to transmission to the mosquito vector.

Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Intracellular copper-binding proteins (metallothionein I/II) and a copper exporter (Menkes copper-transporting ATPase) are upregulated in duodenal enterocytes from iron-deficient rats, consistent with copper accumulation in the intestinal mucosa. How copper enters enterocytes during iron deficiency is, however, not clear. Divalent metal transporter 1 (Dmt1), the predominant iron importer in the mammalian duodenum, also transports other metal ions, possibly including copper. Given this possibility and that Dmt1 expression is upregulated by iron deprivation, we sought to test the hypothesis that Dmt1 transports copper during iron deficiency. Two model systems were utilized: the Belgrade (b) rat, expressing mutant Dmt1, and an inducible Dmt1-overexpression cell culture system. Mutant rats (b/b) were fed a semipurified, AIN93G-based control diet and phenotypically normal littermates (+/b) were fed control or iron-deficient diets for ∼14 wk. An everted gut sleeve technique and a colorimetric copper quantification assay were utilized to assess duodenal copper transport. The control diet-fed +/b rats had normal hematological parameters, whereas iron-deprived +/b and b/b rats were iron deficient and Dmt1 mRNA and protein levels increased. Importantly, duodenal copper transport was similar in the control +/b and b/b rats; however, it significantly increased (∼4-fold) in the iron-deprived +/b rats. Additional experiments in Dmt1 overexpressing HEK-293 cells showed that copper (64Cu) uptake was stimulated (∼3-fold) in the presence of an iron chelator. Dmt1 transcript stabilization due to a 3′ iron-responsive element was also documented, likely contributing to increased transport activity. In summary, these studies suggest that Dmt1 enhances copper uptake into duodenal enterocytes during iron deprivation.

In silico investigation of the ATP7B gene: insights from functional prediction of non-synonymous substitution to protein structure

ATP7B is a copper-transporting ATPase that plays a key role in the regulation of copper homeostasis. Mutations in the ATP7B gene are causative for Wilson's disease, and recent reports have suggested that genetic variants are associated with susceptibility to Alzheimer's disease. Unfortunately, it is difficult to profile experimentally novel genetic variants in the ATP7B gene, because the human protein X-ray structure is not yet entirely understood. In order to investigate ATP7B non-synonymous substitutions, we used an in silico amino acid sequence-based approach. Specifically, we analyzed 337 ATP7B non-synonymous substitutions, which included Wilson's disease-causing mutations (DVs) and non Wilson's disease-causing variants (NDVs), with an algorithm that estimated a combined probability (cPdel) of an amino acidic change to be deleterious for the protein function. This approach appeared to reliably indentify the probability of DVs and NDVs to be deleterious and to profile still unknown gene variants. Specifically, after analyzing ATP7B protein domains with the cPdel method, we found results in line with the predicted-modeled domains and some new suggestions. In conclusion, a functional survey of amino acid changes in the ATP7B protein is provided herein, and we suggest that this bioinformatic method can furnish information about novel ATP7B mutations. Furthermore, the same approach can be applied to other uncharacterized proteins.

Gene expression profiling for analysis acquired oxaliplatin resistant factors in human gastric carcinoma TSGH-S3 cells: The role of IL-6 signaling and Nrf2/AKR1C axis identification

Oxaliplatin treatment is a mainstay of treatment for advanced gastrointestinal tract cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain largely obscured. We previously demonstrated that increased DNA repair capacity and copper-transporting ATPase 1 (ATP7A) level contributed to oxaliplatin resistance in the human gastric carcinoma cell line TSGH-S3 (S3). In the present study, we applied gene array technology to identify additional resistance factors in S3 cells. We found that interleukin-6 (IL-6), aldo-keto reductase 1C1 (AKR1C1), and AKR1C3 are the top 3 upregulated genes in S3 cells when compared with parent TSGH cells. Despite a higher level of endogenous IL-6 in S3, IL-6 receptor (IR-6R, gp-80, and gp-130) levels were similar between TSGH and S3 cells. The addition of exogenous IL-6, IL-6 targeted siRNA, or neutralizing antibodies neither affected Stat3 activation, a downstream target of IL-6, nor changed oxaliplatin sensitivity in S3 cells. However, manipulation of AKR1C activity with siRNA or AKR1C inhibitors significantly reversed oxaliplatin resistance. AKR1Cs are classical antioxidant response element (ARE) genes that can be transcriptionally upregulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown of Nrf2 not only decreased the levels of AKR1C1, AKR1C2, and AKR1C3 mRNA and protein but also reversed oxaliplatin resistance in S3 cells. Taken together, these results suggest that activation of the Nrf2/AKR1C axis may contribute to oxaliplatin resistance in S3 cells but that the IL-6 signaling pathway did not contribute to resistance. Manipulation of Nrf2/AKR1Cs activity may be useful for management of oxaliplatin-refractory gastric cancers.

Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia

Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2α-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2α regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

Golgi in copper homeostasis: a view from the membrane trafficking field

Copper is essential for a variety of important biological processes as a cofactor and regulator of many enzymes. Incorporation of copper into the secreted and plasma membrane-targeted cuproenzymes takes place in Golgi, a compartment central for normal copper homeostasis. The Golgi complex harbors copper-transporting ATPases, ATP7A and ATP7B that transfer copper from the cytosol into Golgi lumen for incorporation into copper-dependent enzymes. The Golgi complex also sends these ATPases to appropriate post-Golgi destinations to ensure correct Cu fluxes in the body and to avoid potentially toxic copper accumulation. Mutations in ATP7A or ATP7B or in the proteins that regulate their trafficking affect their exit from Golgi or subsequent retrieval to this organelle. This, in turn, disrupts the homeostatic Cu balance, resulting in copper deficiency (Menkes disease) or copper overload (Wilson disease). Research over the last decade has yielded significant insights into the enzymatic properties and cell biology of the copper ATPases. However, the mechanisms through which the Golgi regulates trafficking of ATP7A/7B and, therefore, maintains Cu homeostasis remain unclear. This review summarizes current data on the role of the Golgi in Cu metabolism and outlines questions and challenges that should be addressed to understand ATP7A and ATP7B trafficking mechanisms in health and disease.

Evaluation of effects of copper histidine on copper transporter 1‐mediated accumulation of platinum and oxaliplatin‐induced neurotoxicity in vitro and in vivo

The purpose of the present study was to determine whether copper histidine could inhibit copper transporter 1 (Ctr1)-mediated transport of oxaliplatin in vitro and thereby limit the accumulation of platinum and neurotoxicity of oxaliplatin in dorsal root ganglion (DRG) tissue in vivo. In HEK293 cells overexpressing rat Ctr1, copper histidine was shown to be transported by Ctr1 and to inhibit their Ctr1-mediated uptake of oxaliplatin. Pilot in vivo dose-finding studies showed that copper histidine at doses up to 2mg/kg, p.o., daily for 5days/week could be added to maximum tolerated doses of oxaliplatin (1.85mg/kg, i.p., twice weekly) for 8week combination treatment studies in female Wistar rats. After treatment, rats showed significant changes in sensory neuron size profiles in DRG tissue induced by oxaliplatin that were not altered by its coadministration with copper histidine. The expression of copper transporters (Ctr1 and copper-transporting P-type ATPase 1 (Atp7a)) in DRG tissue appeared unchanged following treatment with oxaliplatin given alone or with copper histidine. Platinum and copper tissue levels were higher in DRG than in most other tissues, but were unaltered by the addition of copper histidine to oxaliplatin treatment. In conclusion, copper histidine inhibited cellular uptake of oxaliplatin mediated by Ctr1 in vitro without altering the accumulation of platinum or neurotoxicity of oxaliplatin in DRG tissue in vivo at doses tolerated in combination with oxaliplatin treatment.

Iron Metabolism in a Rodent Model of Menkes Disease (Brindled Mice)

The Menkes copper‐transporting ATPase (Atp7a) is induced in the rodent duodenum during iron deficiency. To understand its role in intestinal iron metabolism, we thus initiated an investigation in Atp7a mutant mice (Brindled). As mutant mice die perinatally of severe copper deficiency, IP copper injections were administered at 7 and 9 days‐of‐age. Mice developed normally thereafter and were similar in size and activity level to wt littermates. Subsequently, mice were deprived of dietary iron for 3 wks and iron and copper homeostasis was studied. Several important observations arose from these analyses, including: 1) mutant mice on control diet were copper deficient and anemic with no changes in iron status; 2) mutant mice on iron‐deficient diet were iron and copper deficient and more significantly anemic; 3) the transcriptional response to iron deficiency in enterocytes was seen only in mutant mice on low Fe diet, suggesting that the HIF2α‐mediated induction of iron and copper homeostasis‐related genes is triggered by iron levels and not hypoxia per se; and 4) Brindled mice had increased hepatic copper levels during iron deprivation, leading to a concomitant increase in serum ferroxidase activity, while no such changes were noted in control or low Fe wt mice. This study has thus revealed novel aspects of the iron‐copper connection and is the first to investigate iron homeostasis in a model of genetic copper deficiency.Grant Funding Source: NIH

An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

Antioxidant protein 1 (Atox1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, Atox1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of Atox1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Accumulating data suggest that cellular functions of Atox1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of transcription, and antioxidant defense. The conserved metal binding site of Atox1, CxGC, differs from the metal-binding sites of copper-transporting ATPases and has a physiologically relevant redox potential that equilibrates with the GSH:GSSG pair. Tight relationship appears to exist between intracellular copper levels and glutathione (GSH) homeostasis. The biochemical properties of Atox1 place it at the intersection of cellular networks that regulate copper distribution and cellular redox balance. Mechanisms through which Atox1 facilitates copper export and contributes to oxidative defense are not fully understood. The current picture of cellular redox homeostasis and copper physiology will be enhanced by further mechanistic studies of functional interactions between the GSH:GSSG pair and copper-trafficking machinery.

COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.

Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis.

Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

Copper stabilizes the Menkes copper-transporting ATPase (Atp7a) protein expressed in rat intestinal epithelial cells

Iron deficiency decreases oxygen tension in the intestinal mucosa, leading to stabilization of hypoxia-inducible transcription factor 2α (Hif2α) and subsequent upregulation of genes involved in iron transport [e.g., divalent metal transporter (Dmt1) and ferroportin 1 (Fpn1)]. Iron deprivation also alters copper homeostasis, reflected by copper accumulation in the intestinal epithelium and induction of an intracellular copper-binding protein [metallothionein (Mt)] and a copper exporter [Menkes copper ATPase (Atp7a)]. Importantly, Atp7a is also a Hif2α target. It was, however, previously noted that Atp7a protein expression was induced more strongly than mRNA in the duodenum of iron-deprived rats, suggesting additional regulatory mechanisms. The current study was thus designed to decipher mechanistic aspects of Atp7a regulation during iron deprivation using an established in vitro model of the mammalian intestine, rat intestinal epithelial (IEC-6) cells. Cells were treated with an iron chelator and/or copper loaded to mimic the in vivo situation. IEC-6 cells exposed to copper showed a dose-dependent increase in Mt expression, confirming intracellular copper accumulation. Iron chelation with copper loading increased Atp7a mRNA and protein levels; however, contrary to our expectation, copper alone increased only protein levels. This suggested that copper increased Atp7a protein levels by a posttranscriptional regulatory mechanism. Therefore, to determine if Atp7a protein stability was affected, the translation inhibitor cycloheximide was utilized. Experiments in IEC-6 cells revealed that the half-life of the Atp7a protein was ~41 h and, furthermore, that intracellular copper accumulation increased steady-state Atp7a protein levels. This investigation thus reveals a novel mechanism of Atp7a regulation in which copper stabilizes the protein, possibly complementing Hif2α-mediated transcriptional induction during iron deficiency.

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.