In silico modeling of the Menkes copper-translocating P-type ATPase 3rd metal binding domain predicts that phosphorylation regulates copper-binding.

The Menkes (ATP7A) P(1B)-type ATPase is a transmembrane copper-translocating protein. It contains six similar high-affinity metal-binding domains (MBDs) in the N-terminal cytoplasmic tail that are important for sensing intracellular copper and regulating ATPase function through the transfer of copper between domains. Molecular characterization of copper-binding and transfer is predominantly dependent on NMR structures derived from E. coli expression systems. A limitation of these models is the exclusion of post-translational modifications. We have previously shown that the third copper-binding domain, MBD3, uniquely contains two phosphorylated residues: Thr-327, which is phosphorylated only in the presence of elevated copper; and Ser-339, which is constitutively phosphorylated independent of copper levels. Here, using molecular dynamic simulations, we have incorporated these phosphorylated residues into a model based on the NMR structures of MBD3. Our data suggests that constitutively phosphorylated Ser-339, which is in a loop facing the copper-binding site, may facilitate the copper transfer process by exposing the CxxC copper-binding region of MBD3. Copper-induced phosphorylation of Thr327 is predicted to stabilize this change in conformation. This offers new molecular insights into how cell signaling (phosphorylation) can affect MBD structure and dynamics and how this may in turn affect copper-binding and thus copper-translocation functions of ATP7A.

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.

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.

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.

The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites.

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.

Functional studies on the Wilson copper P-type ATPase and toxic milk mouse mutant.

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the ATP7B gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the Atp7B for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.

Copper inhibits beta-amyloid production and stimulates the non-amyloidogenic pathway of amyloid-precursor-protein secretion.

Previous studies have demonstrated that amyloid precursor protein (APP) can bind and reduce Cu(II) to Cu(I), leading to oxidative modification of APP. In the present study we show that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of amyloid Abeta peptide (Abeta) both in parental CHO-K1 and in copper-resistant CHO-CUR3 cells, which have lower intracellular copper levels. Copper also caused an increase in the secretion of the APP ectodomain, indicating that the large decrease in Abeta release was not due to a general inhibition in protein secretion. There was an increase in intracellular full-length APP levels which paralleled the decrease in Abeta generation, suggesting the existence of two distinct regulating mechanisms, one acting on Abeta production and the other on APP synthesis. Maximal inhibition of Abeta production and stimulation of APP secretion was achieved in CHO-K1 cells at about 10 microM copper and in CHO-CUR3 cells at about 50 microM copper. This dose 'window of opportunity' at which copper promoted the non-amyloidogenic pathway of APP was confirmed by an increase in the non-amyloidogenic p3 fragment produced by alpha-secretase cleavage. Our findings suggest that copper or copper agonists might be useful tools to discover novel targets for anti-Alzheimer drugs and may prove beneficial for the prevention of Alzheimer's disease.

Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice.

The pathological process in Alzheimer's disease (AD) involves amyloid beta (Abeta) deposition and neuronal cell degeneration. The neurotoxic Abeta peptide is derived from the amyloid precursor protein (APP), a member of a larger gene family including the amyloid precursor-like proteins, APLP1 and APLP2. The APP and APLP2 molecules contain metal binding sites for copper and zinc. The zinc binding domain (ZnBD) is believed to have a structural rather than a catalytic role. The activity of the copper binding domain (CuBD) is unknown, however, APP reduces copper (II) to copper (I) and this activity could promote copper-mediated neurotoxicity. The expression of APP and APLP2 in the brain suggests they could have an important direct or indirect role in neuronal metal homeostasis. To examine this, we measured copper, zinc and iron levels in the cerebral cortex, cerebellum and selected non-neuronal tissues from APP (APP(-/-)) and APLP2 (APLP2(-/-)) knockout mice using atomic absorption spectrophotometry. Compared with matched wild-type (WT) mice, copper levels were significantly elevated in both APP(-/-) and APLP2(-/-) cerebral cortex (40% and 16%, respectively) and liver (80% and 36%, respectively). Copper levels were not significantly different between knockout and WT cerebellum, spleen or serum samples. There were no significant differences observed between APP(-/-), APLP2(-/-) and WT mice zinc or iron levels in any tissue examined. These findings indicate APP and APLP2 expression specifically modulates copper homeostasis in the liver and cerebral cortex, the latter being a region of the brain particularly involved in AD. Perturbations to APP metabolism and in particular, its secretion or release from neurons may alter copper homeostasis resulting in increased Abeta accumulation and free radical generation. These data support a novel mechanism in the APP/Abeta pathway which leads to AD.

The Alzheimer's disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures.

The amyloid precursor protein (APP) of Alzheimer's disease can reduce copper (II) to copper (I) in a cell-free system potentially leading to increased oxidative stress in neurons. We used neuronal cultures derived from APP knock-out (APP(-/-)) and wild-type (WT) mice to examine the role of APP in copper neurotoxicity. WT cortical, cerebellar, and hippocampal neurons were significantly more susceptible than their respective APP(-/-) neurons to toxicity induced by physiological concentrations of copper but not by zinc or iron. There was no difference in copper toxicity between APLP2(-/-) and WT neurons, demonstrating specificity for APP-associated copper toxicity. Copper uptake was the same in WT and APP(-/-) neurons, suggesting APP may interact with copper to induce a localized increase in oxidative stress through copper (I) production. This was supported by significantly higher levels of copper-induced lipid peroxidation in WT neurons. Treatment of neuronal cultures with a peptide corresponding to the human APP copper-binding domain (APP142-166) potentiated copper but not iron or zinc toxicity. Incubation of APP142-166 with low-density lipoprotein (LDL) and copper resulted in significantly increased lipid peroxidation compared to copper and LDL alone. Substitution of the copper coordinating histidine residues with asparagines (APP142-166(H147N, H149N, H151N)) abrogated the toxic effects. A peptide corresponding to the zinc-binding domain (APP181-208) failed to induce copper or zinc toxicity in neuronal cultures. These data support a role for the APP copper-binding domain in APP-mediated copper (I) generation and toxicity in primary neurons, a process that has important implications for Alzheimer's disease and other neurodegenerative disorders.

Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K(m) and V(max) values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

Molecular mechanisms of copper homeostasis.

Copper is an essential trace element which plays a pivotal role in cell physiology as it constitutes a core part of important cuproenzymes. Novel components of copper homeostasis in humans have been identified recently which have been characterised at the molecular level. These include copper-transporting P-type ATPases, Menkes and Wilson proteins, and copper chaperones. These findings have paved the way towards better understanding of the role of copper deficiency or copper toxicity in physiological and pathological conditions.

Direct evidence that mitochondrial iron accumulation occurs in Friedreich ataxia.

Friedreich ataxia (FRDA) is due to mutations in the FRDA gene (FRDA). When the gene homologous to FRDA is knocked out in yeast, there is accumulation of iron in mitochondria and reduced respiratory function. So far, there is only indirect evidence to support the hypothesis that FRDA is due to accumulation of mitochondrial iron leading to increased production of free radicals. We show here that mitochondrial iron is significantly higher in fibroblasts from patients with FRDA than in control fibroblasts. This is the first direct evidence that the findings in yeast are reproducible in cells from patients with FRDA.

Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Mo blo) and brindled (Mo br) mouse mutants.

Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A ( MNK ) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus ( Mo, Atp7a, Mnk ) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship between the copper transport function and copper-dependent trafficking of Mnk.

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network.

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK ( ATP7A ) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans -Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.

Correction of the copper transport defect of Menkes patient fibroblasts by expression of the Menkes and Wilson ATPases.

Menkes' disease is a fatal, X-linked, copper deficiency disorder that results from defective copper efflux from intestinal cells and inadequate copper delivery to other tissues, leading to deficiencies of critical copper-dependent enzymes. Wilson's disease is an autosomally inherited, copper toxicosis disorder resulting from defective biliary excretion of copper, which leads to copper accumulation in the liver. The ATP7A and ATP7B genes that are defective in patients with Menkes' and Wilson's diseases, respectively, encode transmembrane, P-type ATPase proteins (ATP7A or MNK and ATP7B or WND, respectively) that function to translocate copper across cellular membranes. In this study, the cDNAs derived from a normal human ATP7A gene and the murine ATP7B homologue, Atp7b, were separately transfected into an immortalized fibroblast cell line obtained from a Menkes' disease patient. Both MNK and WND expressed from plasmid constructs were able to correct the copper accumulation and copper retention phenotype of these cells. However, the two proteins responded differently to elevated extracellular copper levels. Although MNK showed copper-induced trafficking from the trans-Golgi network to the plasma membrane, in the same cell line the intracellular location of WND did not appear to be affected by elevated copper.

ATP-dependent copper transport by the Menkes protein in membrane vesicles isolated from cultured Chinese hamster ovary cells.

The Menkes (MNK) protein is a vital component of copper homeostasis in mammalian cells. In this paper we provide the first biochemical evidence that the MNK protein functions as a copper-translocating P-type ATPase in mammalian cells. The enzyme activity in membrane vesicles prepared from Chinese hamster ovary cells overexpressing MNK was ATP-dependent, correlated with the amount of MNK and followed Michaelis-Menten kinetics with respect to copper. The copper transport was observed only under reducing conditions suggesting MNK transports Cu(I). This study opens the way to detailed structure-function studies and assessment of functional MNK derived from patients with Menkes disease.

Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1).

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.

Acylphosphate formation by the Menkes copper ATPase.

The Menkes ATPase is the product of the MNK gene, defective in some inherited human disorders of copper metabolism. We here show the formation of an acylphosphate intermediate by the murine MNK homologue in membranes from normal and copper resistant Chinese hamster ovary cells. In the latter, fivefold higher levels of acylphosphate were formed. Challenging these cells with copper, which induces relocation of the MNK ATPase from the trans-Golgi network to the plasma membrane, did not influence acylphosphate formation. The kinetics of phosphorylation, metal dependence, and sensitivity to inhibitors were investigated. The results show that the MNK ATPase is an active P-type ATPase and provide a direct functional test for this enzyme.