Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Differential regulation of the Menkes and Wilson disease copper transporters by hormones: an integrated model of metal transport in the placenta

Copper (Cu) plays a critical role in the developing foetus, but virtually nothing is known concerning the regulation of its uptake and metabolism in the placenta. In this issue of the Biochemical Journal, Hardman and colleagues, using a model of placental trophoblasts in culture, identify differential hormonal regulation of two copper-transporting ATPases; namely, those responsible for Menkes disease (ATP7A; MNK) and Wilson disease (ATP7B; WND). Insulin and oestrogen, which are essential during gestation, up-regulate MNK and this leads to trafficking of the MNK protein from the Golgi to the basolateral membrane, resulting in increased Cu efflux. At the same time, insulin decreased WND levels, and this leads to intracellular sequestration of the protein to a perinuclear region that reduces apical Cu release. As such, this results in a concerted flux of Cu from the basolateral surface of the trophoblast that would potentially be used by the developing foetus. An integrated model of vectorized Cu transport is proposed, which involves co-ordinated expression of transporters, organelle interactions and probable protein-protein interactions. The findings have wider implications for considering general models of intracellular metal transport.     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

The multi-layered regulation of copper translocating P-type ATPases

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.     

Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.     

A comparison of the mutation spectra of Menkes disease and Wilson disease

The genes for two copper-transporting ATPases, ATP7A and ATP7B, are defective in the heritable disorders of copper imbalance, Menkes disease (MNK) and Wilson disease (WND), respectively. A comparison of the two proteins shows extensive conservation in the signature domains, with amino acid identities outside of the conserved domains being limited. The mutation spectra of MNK and WND were compared to confirm and refine further regions critical for normal function. Mutations were found to be relatively widespread; however, the majority was concentrated within defined functional domains and membrane-spanning segments, reinforcing the importance of these regions for protein function. Of the total published point mutations in ATP7A, 23.0% are splice-site, 20.7% nonsense, 17.2% missense, and 39.1% small insertions/deletions. There is a high prevalence (58.2%) of missense mutations in ATP7B. For the other mutations in ATP7B, 7.4% are splice-site, 7.4% nonsense, and 27.0% small insertions/deletions. A region of possible importance is the intervening sequence between the last copper-binding domain and the first transmembrane helix, as this region has a high percentage of MNK mutations. Similarly, the region containing the ATP-binding domain has 24.6% of all WND mutations. The study of mutation locations is useful for defining critical regions or residues and for efficient molecular diagnosis.Electronic Supplementary Material Supplementary material is available for this article if you access the article at .     

Menkes copper-translocating P-type ATPase (ATP7A): biochemical and cell biology properties,and role in Menkes disease

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.     

Cu(I) binding and transfer by the N terminus of the Wilson disease protein

Wilson and Menkes diseases are genetic disorders of copper metabolism caused by mutations in the Wilson (WND) and Menkes (MNK) copper-transporting P1B-type ATPases. The N termini of these ATPases consist of six metal binding domains (MBDs). The MBDs interact with the copper chaperone Atox1 and are believed to play roles in catalysis and in copper-mediated cellular relocalization of WND and MNK. Although all six MBDs have similar folds and bind one Cu(I) ion via a conserved CXXC motif, biochemical and genetic data suggest that they have distinct functions. Most studies aimed at characterizing the MBDs have employed smaller polypeptides consisting of one or two domains. The role of each MBD is probably defined by its environment within the six-domain N terminus, however. To study the properties of the individual domains within the context of the intact Wilson N terminus (N-WND), a series of variants in which five of the six metal binding CXXC motifs are mutated to SXXS was generated. For each variant, the Cu(I) binding affinity and the ability to exchange Cu(I) with Atox1 were investigated. The results indicate that Atox1 can deliver Cu(I) to and remove Cu(I) from each MBD, that each MBD has stronger Cu(I) retention properties than Atox1, and that all of the MBDs as well as Atox1 have similar K(Cu) values of (2.2-6.3) x 10(10) m(-1). Therefore, the specific role of each MBD is not conferred by its position within the intact N-WND but may be related to interactions with other domains and partner proteins.     

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.     

Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

The Menkes P-type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper-translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P-type ATPase has a role in copper efflux has come from studies using copper-resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper-resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady-state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady-state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.     

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230Asp, within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.     

Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl beta-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 microM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 microM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.     

Insulin therapy and its consequences for the mother,foetus, and newborn in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a disease characterised by glucose intolerance and first diagnosed in pregnancy. This condition relates to an anomalous placental environment and aberrant placental vascular function. GDM-associated hyperglycaemia changes the placenta structure leading to abnormal development and functionality of this vital organ. Aiming to avoid the GDM-hyperglycaemia and its deleterious consequences in the mother, the foetus and newborn, women with GDM are firstly treated with a controlled diet therapy; however, some of the women fail to reach the recommended glycaemia values and therefore they are passed to the second line of treatment, i.e., insulin therapy. The several protocols available in the literature regarding insulin therapy are variable and not a clear consensus is yet reached. Insulin therapy restores maternal glycaemia, but this beneficial effect is not reflected in the foetus and newborn metabolism, suggesting that other factors than d-glucose may be involved in the pathophysiology of GDM. Worryingly, insulin therapy may cause alterations in the placenta and umbilical vessels as well as the foetus and newborn additional to those seen in pregnant women with GDM treated with diet. In this review, we summarised the variable information regarding indications and protocols for administration of the insulin therapy and the possible outcomes on the function and structure of the foetoplacental unit and the neonate parameters from women with GDM.     

Reduction of biliverdin and placental transfer of bilirubin and biliverdin in the pregnant guinea pig.

Biliverdin was reduced to bilirubin in pregnant and foetal guinea pigs, and the 100000 g supernatant from homogenates of foetal liver, placenta and maternal liver showed high biliverdin reductase activity. The placental transport of unconjugated bilirubin and biliverdin was compared by injecting unlabelled and radiolabelled pigments into the foetal or maternal circulation and analysing blood collected from the opposite side of the placenta. Injected bilirubin crossed the placenta from foetus to mother and vice versa, but injected biliverdin did not appear to cross without prior reduction to bilirubin. The guinea-pig placenta is apparently more permeable to bilirubin than biliverdin. Reduction of biliverdin to bilirubin in the foetus may, therefore, be essential for efficient elimination of haem catabolites from the foetus in placental mammals.     

Binding of copper(I) by the Wilson disease protein and its copper chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.     

Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells

The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH₂-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper. Menkes disease; PDZ; copper; trafficking     

Structure and function of ATP7A and ATP7B proteins--Cu-transporting ATPases

Living organisms have developed refined and geneticaly controlled mechanisms of the copper metabolism and transport. ATP7A and ATP7B proteins play the key role in copper homeostasis in the organism. Both proteins are P-type Cu-transporting ATPases and use the energy of ATP hydrolysis to transfer the copper ions across the cellular membranes. Both proteins are localised in Golgi aparatus and involved in regulation of overall copper status in the body and their function is the export of excess copper from the cells and delivery of copper ions to Cu-dependent enzymes. Moreover in organism Cu-transporting ATPases are involved in absorption of dietary copper, Cu removal with the bile, placental copper transport and its secretion to the milk during lactation. Moreover it is known that Cu-transporting ATPases play a role in generation of anti-cancer drug resistance. Disturbances of ATP7A and ATP7B function caused by mutations lead to severe metabolic diseases Menkes and Wilson diseases, respectively.     

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.     

Placental essential fatty acid transport and prostaglandin synthesis

The studies reported here demonstrate two important aspects of placenta EFA transport and metabolism. (1) A mechanism exists within the placenta for the selective incorporation of 20:4 omega 6 into phosphoglycerides and the export of those phosphoglycerides to the fetal circulation. This mechanism allows the selective sequestering of 20:4 omega 6 in the fetoplacental unit and may provide the fetus with important performed structural membrane components. (2) Placental PG synthesis is directed mostly to the maternal circulation and stimulated placental PG synthesis is directed totally to the maternal circulation. This mechanism may protect the fetus from fluctuations in maternal and placental PG synthesis and may direct stimulated placental PG synthesis to a target organ, the myometrium. The perfused human placental model provides a valuable method for the study of a variety of biochemical phenomena in a whole human organ and its use may further elucidate the role of this tissue in the maintenance of pregnancy, the transport of EFA to the developing fetus and the involvement of placental PG synthesis in fetal development and parturition.     

A conditional mutation affecting localization of the Menkes disease copper ATPase. Suppression by copper supplementation

Copper is an essential co-factor for several key metabolic processes. This requirement in humans is underscored by Menkes disease, an X-linked copper deficiency disorder caused by mutations in the copper transporting P-type ATPase, MNK. MNK is located in the trans-Golgi network where it transports copper to secreted cuproenzymes. Increases in copper concentration stimulate the trafficking of MNK to the plasma membrane where it effluxes copper. In this study, a Menkes disease mutation, G1019D, located in the large cytoplasmic loop of MNK, was characterized in transfected cultured cells. In copper-limiting conditions the G1019D mutant protein was retained in the endoplasmic reticulum. However, this mislocalization was corrected by the addition of copper to cells via a process that was dependent upon the copper binding sites at the N-terminal region of MNK. Reduced growth temperature and the chemical chaperone, glycerol, were found to correct the mislocalization of the G1019D mutant, suggesting this mutation interferes with protein folding in the secretory pathway. These findings identify G1019D as the first conditional mutation associated with Menkes disease and demonstrate correction of the mislocalized protein by copper supplementation. Our findings provide a molecular framework for understanding how mutations that affect the proper folding of the MNK transporter in Menkes patients may be responsive to parenteral copper therapy.