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PubMed1997

CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper-inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to -11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA-repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY-DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10(-9) M and 5 x 10(-9) M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 microM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.

Adenosine Triphosphatases↗
PubMed1994

Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions.

Wilson disease is an autosomal recessive disorder of copper transport. Disease symptoms develop from the toxic build-up of copper primarily in the liver, and subsequently in the brain, kidney, cornea and other tissues. A candidate gene for WD (ATP7B) has recently been identified based upon apparent disease-specific mutations and a striking amino acid homology to the gene (ATP7A) responsible for another human copper transport disorder, X-linked Menkes disease (MNK). The cloning of WD and MNK genes provides the first opportunity to study copper homeostasis in humans. A preliminary analysis of the WD gene is presented which includes: isolation and characterization of the 5'-end of the gene; construction of a genomic restriction map; identification of all 21 exon/intron boundaries; characterization of extensive alternative splicing in brain; prediction of structure/function features of the WD and MNK proteins which are unique to the subset of heavy metal-transporting P-type ATPases; and comparative analysis of the six metal-binding domains. The analysis indicates that WD and MNK proteins belong to a subset of transporting ATPases with several unique features presumably reflecting their specific regulation and function. It appears that the mechanism of alternative splicing serves to regulate the amount of functional WD protein produced in brain, kidney, placenta, and possibly in liver.

Adenosine Triphosphatases↗
PubMed1995

Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux.

Three copper-resistant variants of cultured Chinese hamster ovary (CHO) cells were isolated and each was shown to accumulate less intracellular copper than the parental cells when grown in copper-supplemented media. The reduced copper accumulation was related to enhanced copper efflux. As cultured cells from patients with Menkes disease (mutations in MNK; ATP7A gene) accumulate copper, probably due to defective copper efflux, we investigated the possible role of the MNK gene in the molecular basis of copper resistance. We found increased MNK mRNA and MNK protein in all three resistant variants. The MNK protein, which has not been previously demonstrated experimentally in mammalian cells, was observed to have an apparent molecular weight of 178 kDa on SDS gels. The degree of increase in MNK mRNA and protein correlated well with the level of copper resistance and extent of copper efflux. By Southern blot and FISH analysis we determined that the molecular basis for overexpression of MNK was genomic amplification of the MNK gene. These data, combined with the clinical and cellular phenotype in Menkes disease, provide strong evidence that the MNK protein is involved in transmembrane copper efflux, and demonstrate a new system of gene amplification in mammalian cells.

Adenosine Triphosphatases↗
PubMed1996

The toxic milk mouse is a murine model of Wilson disease.

Wilson disease (WD) is an autosomal recessive defect of copper transport characterized by massive accumulation of copper in the liver, which can lead to liver failure. Mutations in a copper transporting ATPase (WND or ATP7B) have been shown to cause the disease. The toxic milk mouse mutant (tx) accumulates copper in the liver in a manner similar to that observed in patients with WD. However, some physiological differences between tx mice and human WD patients have cast doubts on whether this mutant mouse is a valid model for WD. In this paper we report the isolation of cDNA clones encoding the murine homologue of WND. The predicted amino acid sequence is 1462 amino acids and contains the same functional domains identified in human and rat WND. As in the rat, the fourth metal binding domain is apparently non-functional. Similar levels of a 7.5 kb WND mRNA were detected in liver and kidney from normal and tx mice, indicating that transcription of this gene was unaffected in the mutant mice. The coding sequence of WND cDNA from the tx mouse liver identified a single nucleotide difference between the normal DL mouse and the tx which is predicted to change methionine 1356 in the eighth transmembrane domain to valine. This methionine is conserved in all copper ATPases including those from bacteria and yeast. The conclusion that this is the causative mutation is supported by the recent mapping of tx and WND to the same region of mouse chromosome 8. Thus the tx mouse is presented as a valid model for studies of the role of WND in copper transport and for investigation of different treatment strategies for WD.

Adenosine Triphosphatases↗
PubMed1997

Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network.

We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

Adenosine Triphosphatases↗
PubMed1997

Mutation analysis provides additional proof that mottled is the mouse homologue of Menkes' disease.

Menkes' disease (MD) and occipital horn syndrome (OHS) are allelic X-linked disorders caused by mutations in the copper ion transporting ATPase, ATP7A. Genetic, phenotypic and biochemical data suggest that mottled mutants in the mouse, which range in severity and phenotype, are caused by mutations in Atp7a, the mouse homologue of ATP7A. As the only causal mutation in Atp7a has been reported in one very mild allele thought to be a model for OHS, Atp7aMo-blo (mottled blotchy), we sequenced the entire 4.5 kb coding region of three other mottled mutants, two of which are thought to be models for classical MD (AtpaMo-br, AtpaMo-13H) and one with a slightly milder phenotype (Atp7aMo-vbr). Although no causal mutation was found in Atp7aMo-13H, mutations which can be predicted to affect Atp7a function were identified in Atp7aMo-br and Atp7aMo-vbr. A 6 bp deletion of nucleotides 2478-2483, which can be predicted to affect the correct processing of the protein, was found in Atp7aMo-br and an A3189-->C nucleotide change, which results in lysine-->threonine amino acid substitution in the phosphorylation domain, was found in Atp7aMo-vbr. Thus we provide further proof that mottled mutants will provide excellent models for MD as well as OHS.

Adenosine Triphosphatases↗
PubMed1997

The mottled mouse as a model for human Menkes disease: identification of mutations in the Atp7a gene.

Mutations in the Atp7a gene, the mouse homologue of the MNK (ATP7A) gene, have been suggested to be responsible for the mottled phenotype. To date, despite considerable effort, changes associated with the mottled mutations have been detected in only two such mutants. In this study, we identify changes in the level of Atp7a transcript and mutations which could explain the mottled phenotype in nine out of the 10 mutants analysed. The fluorescence-assisted mismatch analysis method used here has proved particularly well suited for mRNA scanning of heterozygous carrier animals, because of its ability to detect mutations even in the presence of an excess of wild-type mRNA. The three new underlying mutations identified at the Atp7a locus include a splice mutation and two missense mutations. While the spectrum of mutations detected in the Atp7a murine gene provides an explanation for at least part of the wide phenotypic variation observed in mottled mutant mice, there is a singular absence of deletions which are associated with a sizeable fraction of human Menkes syndrome cases.

Adenosine Triphosphatases↗
PubMed2026

A SINE-like insertion in intron 13 of the ATP7A gene is associated with a mild form of Menkes-like disease in a Cavalier King Charles Spaniel.

A 7-month-old intact male Cavalier King Charles Spaniel was presented for persistent glucosuria despite normoglycemia, failure to thrive, chronic diarrhea, and cerebellar ataxia. Fanconi syndrome was diagnosed, but the neurologic abnormalities were not fully explained. As a consequence of the early onset Fanconi syndrome, a hereditary process was suspected. Whole genome sequencing identified a private hemizygous SINE-like insertion into the ATP7A gene, at the end of intron 13, near the start of exon 14. In humans, variants in ATP7A are associated with Menkes disease, a disorder of copper metabolism associated with a spectrum of clinical signs including progressive neurodegeneration and connective tissue abnormalities. Clinically affected dogs with variants in ATP7A have not been reported previously. Although this case appears to represent a mild phenotypic presentation of Menkes-like disease, it raises the possibility that copper disorders aside from copper-associated hepatitis might exist in dogs. Further genetic screening and phenotypic characterization of rare genetic variants associated with copper metabolism would be beneficial to expand our knowledge of copper disorders in dogs and allow potential early intervention and modeling for metabolic diseases in humans.

Animals↗
PubMed2025

Changes in the FXR-cistrome and alterations in bile acid physiology in Wilson disease.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder that results in excessive hepatic copper, causing hepatic steatosis, inflammation, fibrosis, cirrhosis, and liver failure. Previous studies have revealed dysregulation of many farnesoid X receptor (FXR) metabolic target genes in WD, including the bile salt exporter pump, the major determinant of bile flow. METHODS: We tested the hypothesis that the FXR-cistrome is decreased in Atp7b-/- mice in accord with dysregulated bile acid homeostasis. RESULTS: FXR binding within Atp7b-/- mouse livers displayed surprising complexity: FXR binding was increased in distal intergenic regions but decreased in promoter regions in Atp7b-/- versus wild-type mice. Decreased FXR occupancy in Atp7b-/- versus wild-type mice was observed in hepatocyte metabolic and bile acid homeostasis pathways, while enrichment of FXR binding was observed in pathways associated with cellular damage outside of hepatocytes. Indeed, disparate FXR occupancy was identified in parenchymal and non-parenchymal marker genes in a manner that suggests decreased FXR activity in parenchymal cells, as expected, and increased FXR activity in non-parenchymal cells. Consistent with altered FXR function, serum and liver bile acid concentrations were higher in Atp7b-/- mice than in wild-type mice. Comparison of bile acid profiles in the serum of WD patients with "liver," "neurological," or "mixed" disease versus healthy controls also revealed increases in specific bile acids in WD-liver versus healthy controls. CONCLUSIONS: We identified novel FXR-occupancy across the genome that varied in parenchymal and non-parenchymal cells, demonstrating complex FXR regulation of metabolic and hepatocellular stress pathways in Atp7b-/- mice. Dynamic changes in FXR activity support our novel finding of altered bile acid metabolism in Atp7b-/- mice and WD patients.

Animals↗
PubMed1995

Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport.

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, ORF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611-amino-acid sequence of ORF1, a P-type ATPase (designated hpCopA), had striking homology (29-38%) with several bacterial P-type ATPase and contained the potential functional domains conserved in P-type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion-transporting protein, and contains the heavy metal-binding motif. Disruption of ORF1 with a chloramphenicol-resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alteration of susceptibility to Ni2+, Cd2d+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation-transporting system which is associated with copper export out of the H. pylori cells.

Adenosine Triphosphatases↗
PubMed1995

H714Q mutation in Wilson disease is associated with late, neurological presentation.

Wilson disease is an autosomal recessive copper storage disease resulting from an inability of the liver to excrete copper. Patients can present at a young age, generally with symptoms of liver copper intoxication, or later on, generally with neurological symptoms. The gene for Wilson disease has recently been cloned. Five mutations have been described so far, but only one is found frequently, H714Q. We analysed 38 Dutch symptomatic Wilson disease patients for the H714Q mutation and correlated this finding with age and symptoms at presentation. Ten patients homozygous for the H714Q mutation presented at a mean age of 20.3 (SD 6.1) years, with either neurological symptoms or a Kayser-Fleischer ring. Six patients with a H714Q mutation in one chromosome and an unknown mutation in the other chromosome presented at a mean age of 17.8 (SD 5.8) years, with either neurological or hepatic symptoms. With the exception of one, all 22 patients with an uncharacterised mutation in both chromosomes presented with liver involvement, at a mean age of 9.9 (SD 2.4) years. The difference in age at presentation between the H714Q/H714Q group and the patients with an unknown mutation was highly significant (p < 0.0001). This suggests that the H714Q mutation represents a relatively mild mutation, possibly with some residual function in the copper transporting protein, resulting in a slower build up of copper.

Adenosine Triphosphatases↗
PubMed1996

ATP-dependent copper transporter, in the Golgi apparatus of rat hepatocytes, transports Cu(II) not Cu(I).

The Wilson disease adenosinetriphosphatase (ATPase; ATP7B) is believed to bind copper as Cu(I). We provide evidence to suggest that the ATPase actually transports Cu as Cu(II). When the copper is presented to rat liver microsomes as Cu(I), virtually all uptake is ATP independent. If the copper is presented as copper oxalate [Cu(II)], total uptake is reduced to approximately 10% of Cu(I) levels, but ATP-dependent uptake rises, both as a proportion of total uptake and in absolute terms. The reducing agent vitamin C and the Cu(I) chelator bathocuproine both override the effect of oxalate. The data indicate that there are two transporters in the microsomes, an ATP-independent Cu(I) transporter and an ATP-dependent Cu(II) pump. The activity of the Cu(I) transporter correlates most strongly with alkaline phosphatase, suggesting that it is derived from plasma membrane contamination. Cu(II) ATP-dependent transport correlates only with beta-1, 4-galactosyltransferase, which indicates that it is located in the Golgi apparatus.

Adenosine Triphosphatases↗
PubMed2024

Macrothrombocytopenia with leukocyte inclusions in a patient with Wilson disease: a case report and literature review.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder caused by homozygous or compound heterozygous mutations in ATP7B. Clinical manifestations primarily involve liver and nervous system lesions, with rarely observed hematologic manifestations. CASE PRESENTATION: In the present case, a patient with WD presented with thrombocytopenia, giant platelets, and D&#xf6;hle-like cytoplasmic inclusions in the leukocytes. Initially, the May-Hegglin anomaly was considered; however, whole-exome sequencing did not reveal any mutation in the MYH9 gene but a heterozygous mutation was found in (C.2804&#xa0;C&#x2009;>&#x2009;T, p.T935M) in the ATP7B gene. After two years, the patient developed tremors in his hands, lower limb stiffness, and foreign body sensation in the eyes. Additionally, Kayser-Fleischer rings in the corneal limbus were detected by slit-lamp examination. Copper metabolism test indicated a slight decrease in serum ceruloplasmin. Transmission electron microscopy revealed that the inclusion bodies of leukocytes were swollen mitochondria. Mass spectrometry analysis showed that the copper levels were almost 20-fold higher in the leukocytes of the patient than in those of the control group. Based on the Leipzig scoring system, a diagnosis of WD was confirmed. Zinc sulfate treatment ameliorated the patient's symptoms and enhanced platelet, serum ceruloplasmin, and albumin levels. CONCLUSIONS: In conclusion, this case represents the first documented instance of WD presenting as thrombocytopenia, giant platelets, and D&#xf6;hle-like cytoplasmic inclusions in the leukocytes. Excessive cellular copper accumulation likely underlies these findings; however, understanding precise mechanisms warrants further investigation.

Humans↗
PubMed2024

WilsonGenAI a deep learning approach to classify pathogenic variants in Wilson Disease.

BACKGROUND: Advances in Next Generation Sequencing have made rapid variant discovery and detection widely accessible. To facilitate a better understanding of the nature of these variants, American College of Medical Genetics and Genomics and the Association of Molecular Pathologists (ACMG-AMP) have issued a set of guidelines for variant classification. However, given the vast number of variants associated with any disorder, it is impossible to manually apply these guidelines to all known variants. Machine learning methodologies offer a rapid way to classify large numbers of variants, as well as variants of uncertain significance as either pathogenic or benign. Here we classify ATP7B genetic variants by employing ML and AI algorithms trained on our well-annotated WilsonGen dataset. METHODS: We have trained and validated two algorithms: TabNet and XGBoost on a high-confidence dataset of manually annotated, ACMG & AMP classified variants of the ATP7B gene associated with Wilson's Disease. RESULTS: Using an independent validation dataset of ACMG & AMP classified variants, as well as a patient set of functionally validated variants, we showed how both algorithms perform and can be used to classify large numbers of variants in clinical as well as research settings. CONCLUSION: We have created a ready to deploy tool, that can classify variants linked with Wilson's disease as pathogenic or benign, which can be utilized by both clinicians and researchers to better understand the disease through the nature of genetic variants associated with it.

Hepatolenticular Degeneration↗
PubMed1993

Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae.

We cloned an operon, copAB, from Enterococcus hirae encoding two P-type ATPases of 727 and 745 amino acids, respectively. Both enzymes display heavy metal ion binding motifs in their polar N-terminal region. With an antibody against CopB, we showed on Western blots that expression of the operon is induced by either low or high ambient copper concentrations. Disruption of the copA gene renders the cells dependent, whereas copper disruption of copB results in a copper-sensitive phenotype. CopA exhibits 35% sequence similarity to CopB and 43% similarity to the ATPase encoded by the recently cloned human Mc1 gene, a gene responsible for the Menkes inborn error of copper metabolism. Our results imply that CopA and CopB are heavy metal ion ATPases that regulate the cytoplasmic copper activity, with CopA serving in the uptake and CopB in the extrusion of copper.

Adenosine Triphosphatases↗
PubMed1996

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.

Adenosine Triphosphatases↗
PubMed1997

Identification of point mutations in 41 unrelated patients affected with Menkes disease.

Genomic DNA of 41 unrelated patients affected with the classical severe form of Menkes disease was investigated for point mutations in the ATP7A gene (previously designated as the "MNK" gene). Using SSCP analysis and direct sequencing of the exons amplified by PCR, we identified 41 different mutations, including 19 insertions/deletions, 10 nonsense mutations, 4 missense mutations, and 8 splice-site alterations. Approximately 90% of the mutations were predicted to result in the truncation of the protein (ATP7A). In 20 patients the mutations were within exons 7-10, and half of these mutations affected exon 8. Furthermore, five alterations were observed within the 6-bp sequence at the splice-donor site of intron 8, which would be predicted to affect the efficiency of splicing of exon 8. Although a specific function has not been attributed to the protein region encoded by this exon, this region may be important in serving as a "stalk" joining the metal-binding domains and the ATPase core. The present findings not only help us in understanding the underlying genetic defect but are invaluable data especially for carrier detection and prenatal diagnosis of this lethal disorder.

Adenosine Triphosphatases↗