Wilson ATPase Research Articles

Update on the Diagnosis and Management of Wilson Disease

Exciting developments relating to Wilson disease (WD) have taken place with respect to both basic biological and clinical research. This review critically examines some of these findings and considers their implications for current thinking about WD. It is not a comprehensive review of WD as a clinical disorder. The structure of the gene product of ATP7B, abnormal in WD, is being worked out in detail, along with a broader description of how the protein ATP7B (Wilson ATPase) functions in cells including enterocytes, not only in relation to copper disposition but also to lipid synthesis. Recent population studies raise the possibility that WD displays incomplete penetrance. Innovative screening techniques may increase ascertainment. New strategies for diagnosing and treating WD are being developed. Several disorders have been identified which might qualify as WD-mimics. WD can be difficult to diagnose and treat. Insights from its pathobiology are providing new options for managing WD.

Chapter 12 - Wilson disease in children

Wilson disease (WD) is an inherited disorder mainly of hepatocellular copper disposition, due to dysfunction of the Wilson ATPase, a P1B-ATPase encoded by the gene ATP7B. In children, as in older age brackets, clinical disease is highly diverse. Although hepatic disease is the common presentation in children/adolescents, neurologic, psychiatric, and hematologic clinical presentations do occur. Very young children may have clinically evident liver disease due to WD. Early diagnosis, preferably when the child/adolescent is asymptomatic, is most likely to result in near-normal longevity with generally good health so long as the patient tolerates effective medication, is adherent to the lifelong treatment regimen, and has consistent access to the medication. Apart from a lively index of clinical suspicion on the part of physicians, biochemical tests including liver tests, serum ceruloplasmin, and basal 24-hour urinary copper excretion and genotype determination are key to diagnosis. Oral chelation treatment remains central to medical management, although zinc appears to be an attractive option for the presymptomatic child. Pediatric patients presenting with Wilsonian fulminant hepatic failure must be differentiated from those with decompensated cirrhosis, since the latter may respond to intensive medical interventions and not require liver transplantation. Recently identified WD-mimic disorders reveal important aspects of WD pathogenesis.

The puzzle posed by COMMD1, a newly discovered protein binding Cu(ii)

Copper is critically important for cellular metabolism. It plays essential roles in developmental processes, including angiogenesis. The liver is central to mammalian copper homeostasis: biliary excretion is the major route of excretion for ingested copper and serves to regulate the total amount of copper in the organism. An extensive network of proteins manipulates copper disposition in hepatocytes, but comparatively little is known about this protein system. Copper exists in two oxidation states: most extracellular copper is Cu(II) and most, if not all, intracellular copper is Cu(I). Typical intracellular copper-binding proteins, such as the Cu-transporting P-type ATPases ATP7B (Wilson ATPase) and ATP7A (Menkes ATPase), bind copper as Cu(I). Accordingly, the recent discovery that the ubiquitous protein COMMD1 binds Cu(II) exclusively raises the question as to what role Cu(II) may play in intracellular processes. This issue is particularly important in the liver and brain. In humans, Wilson’s disease, due to mutations in ATP7B, exhibits progressive liver damage from copper accumulation; in some Bedlington terriers, mutations in COMMD1 are associated with chronic copper-overloaded liver disease, clinically distinct from Wilson’s disease. It seems unlikely that Cu(II), which generates reactive oxygen species through the Fenton reaction, has a physiological role intracellularly; however, Cu(II) might be the preferred state of copper for elimination from the cell, such as by biliary excretion. We argue that COMMD1 participates in the normal disposition of copper within the hepatocyte and we speculate about that role. COMMD1 may contribute to the mechanism of biliary excretion of copper by virtue of binding Cu(II). Additionally, or alternatively, COMMD1 may be an important component of an intracellular system for utilizing Cu(II), or for detecting and detoxifying it.

Protein‐Based Capacitive Biosensors: a New Tool for Structure‐Activity Relationship Studies

AbstractThe present work reports a new application of a protein‐based capacitive biosensor as an in vitro assay for the selectivity study of the bacterial periplasmic protein MerP and four MerP variants. The modified MerP proteins were produced by site‐directed mutagenesis of the heavy metal associated motif (HMA). The MerP and modified MerPs selectivity for copper, zinc, cadmium and mercury bivalent ions were investigated and compared. The variations in the proteins affinity were related to the primary structure of the HMA motifs. Key amino acids for copper coordination of metalloproteins that contain the metal binding sequence Gly‐Met‐Thr‐Cys‐xxx‐xxx‐Cys were identified. The results brought insights valid for Menkes and Wilson ATPases. The protein‐based capacitive biosensors were a simple and useful tool for studying structure‐activity relationships of proteins.

Sulfate Acts as Phosphate Analog on the Monomeric Catalytic Fragment of the CPx-ATPase CopB from Sulfolobus solfataricus

The crystal structure of the catalytic fragment of a Sulfolobus solfataricus P-type ATPase, CopB-B, was determined with a 2.6 Å resolution. CopB-B is the major soluble fragment of the archaeal CPx-ATPase CopB and is comprized of a nucleotide and a phosphorylation domain. In the crystalline state two molecules of CopB-B are in close contact to each other, although the presence of dimers in free solution could be ruled out by analytical ultracentrifugation. The overall architecture of CopB-B is similar to that of other P-type ATPases such as Ca-ATPase. Short peptide segments are linking the nucleotide binding to the phosphorylation domain. CopB-B exhibits 33% sequence identity (of 216 aligned residues) with the respective fragment of the Archaeoglobus fulgidus ATPase CopA. The CopB-B nucleotide-binding domain has the most primitive fold yet identified for this enzyme class. It is 24% identical to the nucleotide-binding domain of the disease-related Wilson ATPase ATP7B (80 structurally aligned residues). Structural superposition with Ca-ATPase suggests a putative nucleotide-binding site in CopB-B. The phosphorylation domain of CopB-B is structurally related to the corresponding part of Ca-ATPase in the anion-bound E2 state. In CopB-B crystals, a bound sulfate anion was identified at the phosphate-binding location. In solution state, the potential binding of CopB-B to phosphate was probed with 32P i. Bound phosphate could be readily displaced by orthovanadate at submillimolar concentration as well as by sulfate at millimolar concentration. It is possible therefore to assign the structure of the sulfate-bound phosphorylation domain of CopB-B to a state related to the E2·P i intermediate state of the catalytic cycle.

Purification and functional reconstitution of the human Wilson copper ATPase, ATP7B

Wilson disease is a disorder of copper metabolism, due to inherited mutations in the Wilson copper ATPase gene ATP7B. To purify and study the function of the ATPase, the enzyme was truncated by five of the six metal binding domains and endowed with an N-terminal histidine-tag for affinity purification. This construct, Δ1–5WNDP, was able to functionally complement a yeast strain defective in its native copper ATPase CCC2. Δ1–5WNDP was purified by Ni-affinity chromatography and reconstituted into proteoliposomes. This allowed, for the first time, the functional study of the Wilson ATPase in a purified, reconstituted system.

Expression of Menkes ATPase and Wilson ATPase in different regions of the adult rat brain

Expression of Menkes ATPase and Wilson ATPase in different regions of the adult rat brain

Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH 3CO–Lys–Gly–Met–Thr–Cys–Ala–Ser–Cys–Val–His–Asn–Lys–CONH 2), and 2K8p (CH 3CO–Lys–Leu–Cys–Ile–Ala–Cys–Pro–Cys–Ser–Lys–CONH 2), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH 3CO–Lys–Leu–Ser–Ile–Ala–Cys–Pro–Cys–Ser–Lys–CONH 2), 2s (CH 3CO–Lys–Leu–Cys–Ile–Ala–Ser–Pro–Cys–Ser–Lys–CONH 2) and 3s (CH 3CO–Lys–Leu–Cys–Ile–Ala–Cys–Pro–Ser–Ser–Lys–CONH 2), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.

The Enterococcus hirae paradigm of copper homeostasis: copper chaperone turnover, interactions, and transactions.

The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. The cop operon is induced by copper, which allows uncompromised growth in up to 5 mM ambient copper. Copper uptake appears to be accomplished by the CopA ATPase, a member of the heavy metal CPx-type ATPases and closely related to the human Menkes and Wilson ATPases. The related CopB ATPase extrudes copper when it reaches toxic levels. Intracellular copper routing is accomplished by the CopZ copper chaperone. Using surface plasmon resonance analysis, it was demonstrated that CopZ interacts with the CopA ATPase where it probably becomes copper loaded. CopZ in turn can donate copper to the copper responsive repressor CopY, thereby releasing it from DNA. In high copper, CopZ is proteolyzed. Cell extracts were found to contain a copper activated proteolytic activity that degrades CopZ in vitro. This post-translational control of CopZ expression presumably serves to avoid the accumulation of detrimental Cu-CopZ levels.

Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Structure–function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000 → Arg, changing the conserved Cys-Pro-Cys (‘CPC’) motif, was mimicked in CopB. The corresponding Cys-396 → Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069 → Gln, was introduced into CopB as His-480 → Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues.

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000-->Arg, changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

How cells handle copper: A view from microbes

Copper is an essential element, yet toxic to cells. It can damage biomolecules by radical formation and, therefore, the intracellular copper needs to be carefully controlled. In the Gram-positive bacterium Enterococcus hirae, copper homeostasis is effected by the cop operon. It allows growth under copper limiting conditions, but also offers resistance to 8 mM copper. The cop operon consists of the four genes copY, copZ, copA, and copB. CopA and copB encode copper ATPases. CopA serves in copper uptake under copper limitation, whereas CopB expels copper when copper is excessive. CopA and CopB are closely related to the human Menkes and Wilson ATPases. Mutation in these human copper ATPases lead to inherited defects of copper metabolism, known as Menkes and Wilson disease, respectively. Copper ATPases and other heavy metal ATPases form a new, evolutionarily distinct subgroup of the P-type ATPases, called CPx-type ATPases. The cop operon of E. hirae is regulated by the copper responsive repressor CopY. Copper donated to CopY by the copper chaperone CopZ releases it from the promoter. CopZ belongs to a family of conserved metal chaperones that transfer copper to copper-transporting ATPases in humans and yeast (HAH1, Atx1), and mercury to a mercury uptake system in bacteria (MerP). CopZ-like motifs are also found in the N-termini of the CPx-type ATPases and mercuric reductases. A model of copper homeostasis and the role of copper chaperones in mammalian cells based on findings in yeast and E. hirae is presented. J. Trace Elem. Exp. Med. 12:347–360, 1999. © 1999 Wiley-Liss, Inc.

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.

Genetic and molecular basis for copper toxicity

Recent studies resulted in the cloning of the genes responsible for Menkes syndrome and Wilson disease. Despite the distinct clinical phenotypes of these disorders, each gene encodes a highly homologous member of the cation-transport P-type ATPase family. The remarkable evolutionary conservation of these proteins in bacteria, yeast, plants, and mammals reveals a fundamental protein structure essential for copper export in all life forms. Characterization of a molecular defect in the rat homologue of the Wilson ATPase in the Long-Evans Cinnamon rat identifies an animal model of Wilson disease and will permit experimental analysis of the precise role of this ATPase in copper transport, the effects of specific inherited mutations on transport function, and the cellular and molecular mechanisms of tissue injury resulting from copper accumulation. Finally, recent molecular genetic analysis of a distinct group of patients with low serum ceruloplasmin and basal ganglia symptoms identified a series of mutations in the ceruloplasmin gene. The presence of these mutations in conjunction with the clinical and pathologic findings clarifies the essential biological role of this abundant copper protein in metal metabolism and identifies aceruloplasminemia as a novel autosomal recessive disorder of iron metabolism.