Functional Studies on the Wilson Copper P-Type ATPase and Toxic Milk Mouse Mutant

doi:10.1006/bbrc.2001.4445

Biochemical and Biophysical Research Communications

Volume 281, Issue 4, 9 March 2001, Pages 966-970

https://doi.org/10.1006/bbrc.2001.4445 Get rights and content

Abstract

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.

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Cited by (73)

Diverse biological roles of the tetrathiomolybdate anion
2021, Coordination Chemistry Reviews
Citation Excerpt :
This result indicates that TTM is a good neurological and/or hepatic drug compared to other copper-chelating drugs. Czachor et al. also noticed that TTM significantly reduced the elevated copper levels in the liver, brain and MT for tx mice [126]. LEC rat [109], and tx mice [124], indicated above, have similarilties in copper storage, but dissimilarities in phenotype.
Biological systems use proteins as scaffolds to harbor catalytic metal centers responsible for the assimilatory and energy pathways that make life possible. Therefore, metal homeostasis is essential for life. Generally, when compared to normal cells, copper overload has negative consequences, such as radical formation, up-regulation of Cu-enzyme activity by promoting the progression of disease states and major toxic effects by substituting native metal sites in [Fe-S] and Zn proteins. So, elevated copper levels are the cause of many health problems, such as Wilson disease (WD) and cancer. One of the most important therapeutic interventions of WD is anti-copper therapy, where tetrathiomolybdate (TTM = [MoS₄]^2–, coprexa) is used as a copper-sequestering drug. The discovery of the Cu-TTM interaction in ruminant nutrition had a great impact on research in humans. Currently, TTM is also being exploited as a drug for treatment of several forms of cancer. Cu-TTM based therapy reduces excess copper, decreasing harmful and toxic effects. Therefore, high concentrations of copper, as well as several Cu-enzymes involved in WD/cancer, are promising targets for anti-copper therapy. Several TTM-treated pre-clinical models, as well as humans with WD/cancer, provide successful results. Interestingly, Cu-TTM interest expanded when a metal-cofactor, [Cu(TTM)₂]^3-, was found in the orange protein (ORP) isolated from a sulfate-reducing bacteria. This review will discuss the chemical and biological occurrence of TTM, the imbalance of Cu-trafficking leading to copper overloaded human diseases (WD and cancer), preclinical and clinical aspects of TTM as an anti-copper therapy and finally TTM as a part of the metal cofactor of ORP. The aim of this review is to promote our understanding on the role of Mo-Cu antagonism in humans as an anti-copper therapy.
Animal Models of Wilson Disease
2019, Wilson Disease: Pathogenesis, Molecular Mechanisms, Diagnosis, Treatment and Monitoring
Wilson disease (WD) is an autosomal recessive disease, characterized by copper overload in the liver and brain, and exhibits a remarkable variability of phenotypic manifestations. The causative gene ATP7B has been identified; however, specific mechanisms of copper toxicity and phenotypic diversity are still enigmatic. Substantial advances in unraveling the molecular pathogenesis, copper metabolism, and WD progression have been achieved through numerous research and therapeutic studies using mammalian models of copper toxicity.
Rodent models with Atp7b gene defects include the LEC and LPP rats, inbred mouse models (toxic milk (tx) mouse and Jackson tx (tx-j) mouse), and the genetically engineered Atp7b^−/− (knockout) mouse; all develop liver disease to different extents; some also accumulate copper in the brain accompanied by slight neurological involvement, which is by far less severe than in human neurological WD. Several species of dog and North Ronaldsay sheep also show hepatic copper toxicity; however, the brain is unaffected, and the underlying genetic defect differs.
Animal models for Wilson disease
2018, Clinical and Translational Perspectives on WILSON DISEASE
Wilson disease (WD) is a multisystem disorder that manifests primarily with hepatic and neurological symptoms due to copper toxicity in liver and brain. More than two decades after identification of the defective gene, ATP7B, the underlying mechanisms of copper toxicity and the striking phenotypic diversity remain far from understood. Animal models with defects in the Atp7b homolog have provided new insights into pathophysiology of WD. Several rodent models harboring Atp7b defects have been described, which include inbred mouse models [toxic milk [tx; tx-r] mouse, Jackson tx [tx-j] mouse], the Atp7b^−/− (knockout) mouse, as well as the Long-Evans Cinnamon and LPP rats, all of which develop liver disease. Brain copper accumulation accompanied by neurologic involvement was observed in several of these animals; however, symptoms were less severe compared to humans. These models are valuable for exploration of copper metabolism, gene expression, and investigation of WD progression. To facilitate mandatory new research on novel drugs, gene, and cell therapy, animal models constitute an invaluable tool.
Cellular function of ATP7B (Wilson ATPase)
2018, Clinical and Translational Perspectives on WILSON DISEASE
The copper-transporting ATPase ATP7B (Wilson ATPase) plays an essential role in hepatocellular function. In the liver, ATP7B maintains intracellular copper concentration by promoting excretion of excess copper from hepatocytes into the bile. ATP7B also delivers copper into the lumen of the secretory pathway contributing to biosynthesis of ceruloplasmin, a major copper metalloprotein in the blood. The biosynthetic and homeostatic functions of ATP7B are regulated by copper and require targeting of the transporter to different cell compartments. Loss-of-function mutations in ATP7B cause toxic accumulation of copper in the liver producing the metabolic disorder Wilson disease. This chapter summarizes current data on the functional properties of the Wilson ATPase and on the mechanisms that regulate its activity and intracellular trafficking.
Animal models of Wilson disease
2017, Handbook of Clinical Neurology
Citation Excerpt :
This assumption was confirmed by findings of Theophilos et al. (1996), who isolated cDNA clones encoding the murine homolog of the WD gene ATP7B, identified the mutation (methionine1356valine in the eighth transmembrane domain) causing the disease, and established the tx mouse as a valid model for human WD, despite there being some dissimilarities. Further basic research using in vitro model systems for ATP7B localization and copper transport provided evidence that the Met1386Val mutation found in the tx mouse caused mislocalization as well as loss of copper transport activity (La Fontaine et al., 2001; Voskoboinik et al., 2001). Comparable results have been shown for a number of human ATB7B variants, both in vitro and in vivo (Huster et al., 2003, 2012), which revealed significant similarities between the human disease and the animal model, and underline the importance of its availability.
Wilson disease (WD) is caused by ATPase copper-transporting beta (ATP7B) mutations and results in copper toxicity in liver and brain. Although the defective gene was identified in 1993, the specific mechanisms underlying copper toxicity and the remarkable phenotypic diversity of the disease are still poorly understood. Animal models harboring defects in the ATP7B homolog have helped to reveal new insights into pathomechanisms of WD.
Four rodent models with ATP7B gene defects have been described – the Long–Evans Cinnamon (LEC) rat, inbred mouse models (toxic milk (tx), the Jackson Laboratory toxic milk (tx-j)), and the genetically engineered ATP7B^–/– (knockout) mouse – all of which develop liver disease to different extents. Copper accumulation in parts of the brain accompanied by some neurologic involvement was revealed in LEC rats and tx/tx-j mice, but the pathology is less severe than human neurologic WD. Several dogs show hepatic copper toxicity resembling WD; however, brain involvement has not been observed and the underlying genetic defect is different.
These models are of great value for examination of copper distribution and metabolism, gene expression, and investigation of liver and brain pathology. The availability of disease models is essential for therapeutic interventions such as drug, gene, and cell therapy. Findings made by animal studies may facilitate the development of specific therapies to ameliorate WD progression.
Recent advance in the molecular genetics of Wilson disease and hereditary hemochromatosis
2016, European Journal of Medical Genetics
Metabolic liver diseases such as Wilson disease (WD) and hereditary hemochromatosis (HH) possess complicated pathogenesis and typical hereditary characteristics with the hallmarks of a deficiency in metal metabolism. Mutations in genes encoding ATPase, Cu + transporting, beta polypeptide (ATP7B) and hemochromatosis (HFE) or several non-HFE genes are considered to be causative for WD and HH, respectively. Although the identification of novel mutations in ATP7B for WD and HFE or the non-HFE genes for HH has increased, especially with the application of whole genome sequencing technology in recent years, the biological function of the identified mutations, as well as genotype–phenotype correlations remain to be explored. Further analysis of the causative gene mutation would be critical to clarify the mechanisms underlying specific disease phenotypes. In this review, we therefore summarize the recent advances in the molecular genetics of WD and HH including the updated mutation spectrums and the correlation between genotype and phenotype, with an emphasis on biological functional studies of the individual mutations identified in WD and HH. The weakness of the current functional studies and analysis for the clinical association of the individual mutation was also discussed. These works are essential for the understanding of the association between genotypes and phenotypes of these inherited metabolic liver diseases.

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Regular ArticleFunctional Studies on the Wilson Copper P-Type ATPase and Toxic Milk Mouse Mutant

Abstract

Biochem. Biophys. Res. Commun.

FEBS Lett.

Gastroenterology

Genomics

Genomics

J. Biol. Chem.

FEBS Lett.

Anal. Biochem.

J. Immunol. Methods

Biochem. Biophys. Res. Commun.

Am. J. Hum. Genet.

J. Biol. Chem.

Regular Article
Functional Studies on the Wilson Copper P-Type ATPase and Toxic Milk Mouse Mutant