Menkes Disease Protein Research Articles

An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal‐binding domains of the Menkes protein

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.

A Novel Role for the Immunophilin FKBP52 in Copper Transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.

Wilson disease

Wilson disease

Supplying Copper to the Cuproenzyme Peptidylglycine α-Amidating Monooxygenase

We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.

Function and regulation of the mammalian copper-transporting ATPases: insights from biochemical and cell biological approaches.

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase.

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.

Transport and detoxification systems for transition metals, heavy metals and metalloids in eukaryotic and prokaryotic microbes

Transition metals, heavy metals and metalloids are usually toxic in excess, but a number of transition metals are essential trace elements. In all cells there are mechanisms for metal ion homeostasis that frequently involve a balance between uptake and efflux systems. This review will briefly describe ATP-coupled resistance pumps. ZntA and CadA are bacterial P-type ATPases that confers resistance to Zn(II), Cd(II) and Pb(II). Homologous copper pumps include the Menkes and Wilson disease proteins and CopA, an Escherichia coli pump that confers resistance to Cu(I). For resistance to arsenicals and antimonials there are several different families of transporters. In E. coli the ArsAB ATPase is a novel system that confers resistance to As(III) and Sb(III). Eukaryotic arsenic resistance transporters include Acr3p and Ycf1p of Saccharomyces cerevisiae. These systems provide resistance to arsenite [As(III)]. Arsenate [As(V)] detoxification involves reduction of As(V) to As(III), a process catalyzed by arsenate reductase enzymes. There are three families of arsenate reductases, two found in bacterial systems and a third identified in S. cerevisiae.

Wilson disease.

Wilson disease is an autosomal recessive inherited disorder of copper metabolism, characterized by the accumulation of copper in the body due to defective biliary copper excretion from hepatocytes. Recently, novel components involved in copper metabolism, including Menkes disease protein (ATP7A), Wilson disease protein (ATP7B), and copper chaperones, have been identified. It has been demonstrated that ATP7B functions in copper secretion into the plasma, coupled with ceruloplasmin synthesis and biliary copper excretion. However, the precise intracellular localization of ATP7B has been disputed. Various mutations of ATP7B have been reported in patients with Wilson disease, and investigations of genotype-phenotype correlations are now being conducted in the patients. These recent findings provide us with information on the molecular pathogenesis of Wilson disease, as well as the biological mechanisms of copper homeostasis. In this review, recent advances in this field are briefly summarized.

Role of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity.

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4). These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins.

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.

A Strong Loss-of-Function Mutation in RAN1 Results in Constitutive Activation of the Ethylene Response Pathway as Well as a Rosette-Lethal Phenotype

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Characterization of the Interaction between the Wilson and Menkes Disease Proteins and the Cytoplasmic Copper Chaperone, HAH1p

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex.

Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by β-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K 0.5, determined to be 65 μM and 19 μM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an ‘all-or-none’ cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.

Intracellular localization of the Menkes and Wilson's disease proteins and their role in intracellular copper transport.

Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. In excess, copper is a very toxic ion and therefore efficient regulation of its metabolism is required. This is dramatically illustrated by the genetic disorders X-linked Menkes disease and autosomal recessive Wilson's disease. In 1993, both the Menkes and Wilson's genes were isolated and it was found that these genes encode homologous cation copper transporting P-type ATPase proteins. The Menkes protein (ATP7A) is expressed in most tissues, except liver. In contrast, the Wilson's protein (ATP7B) is abundantly expressed in liver. Intracellular localization of those proteins was investigated. Both ATP7A and ATP7B are localized in the trans-Golgi network and post-Golgi vesicular compartment (PGVC) in the cell. This intracellular localization was altered by the copper content present in the cell. This result may support the hypothesis that ATP7A and ATP7B are involved in cellular copper transport and those proteins could be suitable models for elucidating intracellular copper metabolism.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane.

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Molecular mechanisms of copper metabolism and the role of the Menkes disease protein.

Menkes disease is an X-linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births. The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood. The symptoms of Menkes disease result from a deficiency of serum copper and copper-dependent enzymes. A candidate gene for the disease has been isolated and designated MNK. The MNK gene codes for a P-type cation transporting ATPase, based on homology to known P-type ATPases and in vitro experimentation. cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments. The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal-binding repeats in the amino-terminal domain. The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease. Each of the Menkes protein amino-terminal repeats contains a conserved -X-Met-X-Cys-X-X-Cys- motif (where X is any amino acid). These metal-binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes. An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented. This forms the basis for a discussion of Menkes disease and the protein deficit in this disease.

The cell biology of the Menkes disease protein.

Copper is a trace element which is readily converted between cuprous and cupric forms under physiological conditions. This redox property has been harnessed in biological systems where copper forms an integral component of enzymes whose catalytic function involves electron exchange. However, this same property of copper also makes it toxic when present within the cell at elevated levels. Consequently, intracellular copper levels must be carefully controlled, presumably by regulated transport mechanisms. Disruptions of some of these mechanisms can have a genetic basis as illustrated in the case of both Menkes disease and Wilson disease (Danks, 1995).KeywordsChinese Hamster Ovary CellWilson DiseaseCopper TransportCopper HomeostasisMenkes DiseaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A Golgi localization signal identified in the Menkes recombinant protein.

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.

Investigation of the copper binding sites in the Menkes disease protein, ATP7A. SSIEM Award. Society of the Study of Inborn Errors of Metabolism.

P. Y. JENSEN1,2,*, N. BONANDER2, B. G. KARLSSON2, N. HORN3, Z. TUŽ MER3,4 and O. FARVER1 of Analytical and Pharmaceutical Royal Danish School 1Department Chemistry, T he of of Biochemistry and Biophysics, Pharmacy, Copenhagen, Denmark ; 2Department L undberg University of T echnology and Institute, Chalmers GoŽ teborg University, John F. Kennedy 4Present GoŽ teborg, Sweden ; 3 T he Institute, Glostrup, Denmark ; address : of Medical of Copenhagen, Panum Department Genetics, University Institute, Denmark

Eukaryotic Expression Vectors That Replicate to Low Copy Number in Bacteria: Transient Expression of the Menkes Protein

A set of low copy number plasmid vectors for mammalian gene expression has been constructed. These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell. The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression. A cDNA encoding the Menkes disease protein was cloned into two of these vectors, and transient expression studies in COS-7 cells showed that both constitutive and inducible expression was possible. This set of expression vectors will provide a useful tool for the manipulation, inEscherichia coli,of mammalian genes or cDNAs that are unstable in the high copy number vectors that are currently available.