Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation.
ABSTRACT: Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(?hep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(?hep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(?hep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(?hep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(?hep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(?hep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(?hep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.
Project description:COMMD1 (previously known as MURR1) belongs to a novel family of proteins termed the copper metabolism gene MURR1 domain (COMMD) family. The 10 COMMD family members are well conserved between vertebrates, but the functions of most of the COMMD proteins are unknown. We recently established that COMMD1 is associated with the hepatic copper overload disorder copper toxicosis in Bedlington terriers. Recent in vitro studies indicate that COMMD1 has multiple functions, including sodium transport and NF-kappaB signaling. To elucidate the function of Commd1 in vivo, we generated homozygous Commd1 null (Commd1(-/-)) mice. Commd1(-/-) embryos died in utero between 9.5 and 10.5 days postcoitum (dpc), their development was generally retarded, and placenta vascularization was absent. Microarray analysis identified transcriptional upregulation of hypoxia-inducible factor 1 (HIF-1) target genes in 9.5-dpc Commd1(-/-) embryos compared to normal embryos, a feature that was associated with increased Hif-1alpha stability. Consistent with these observations, COMMD1 physically associates with HIF-1alpha and inhibits HIF-1alpha stability and HIF-1 transactivation in vitro. Thus, this study identifies COMMD1 as a novel regulator of HIF-1 activity and shows that Commd1 deficiency in mice leads to embryonic lethality associated with dysregulated placenta vascularization.
Project description:Hereditary forms of copper toxicosis exist in man and dogs. In man, Wilson's disease is the best studied disorder of copper overload, resulting from mutations in the gene coding for the copper transporter ATP7B. Forms of copper toxicosis for which no causal gene is known yet are recognized as well, often in young children. Although advances have been made in unraveling the genetic background of disorders of copper metabolism in man, many questions regarding disease mechanisms and copper homeostasis remain unanswered. Genetic studies in the Bedlington terrier, a dog breed affected with copper toxicosis, identified COMMD1, a gene that was previously unknown to be involved in copper metabolism. Besides the Bedlington terrier, a number of other dog breeds suffer from hereditary copper toxicosis and show similar phenotypes to humans with copper storage disorders. Unlike the heterogeneity of most human populations, the genetic structure within a purebred dog population is homogeneous, which is advantageous for unraveling the molecular genetics of complex diseases. This article reviews the work that has been done on the Bedlington terrier, summarizes what was learned from studies into COMMD1 function, describes hereditary copper toxicosis phenotypes in other dog breeds, and discusses the opportunities for genome-wide association studies on copper toxicosis in the dog to contribute to the understanding of mammalian copper metabolism and copper metabolism disorders in man.
Project description:Wilson disease (WD) results from accumulation of copper and caused due to mutations in ATP7B, a copper transporting ATPase. Besides regular hepatic and neurological symptoms, WD patients occasionally manifest atypical symptoms due to unknown cause. To understand the molecular etiology of atypical WD manifestations, we screened COMMD1, a gene implicated in canine copper toxicosis, in 109 WD patients including those with atypical symptoms. In a patient showing apoptotic symptoms and high urinary copper surpassing normal WD levels, we identified a novel, putative mutation in COMMD1. Two other changes were also identified in the gene. We have examined genotype-phenotype correlation between the detected changes and the atypical presentation of the WD patient.
Project description:ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.
Project description:The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in ATP7A and ATP7B contributes to the biological understanding of protein function, with relevance for future development of therapy.
Project description:Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.
Project description:Copper-responsive intracellular ATP7B trafficking is crucial for maintaining the copper balance in mammalian hepatocytes and thus copper levels in organs. The copper metabolism domain-containing protein 1 (COMMD1) binds both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], whereas COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that COMMD1 is localized to endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking has not yet been defined. In this study, experiments using quantitative colocalization analysis reveal that COMMD1 modulates copper-responsive ATP7B trafficking through recruitment to PtdIns(4,5)P2 Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and the trans-Golgi network (TGN) in high copper conditions, although excess expression of COMMD1 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to inhibit PtdIns(4,5)P2 binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-mediated function for COMMD1 is proposed that is consistent with decreased cellular copper export as a result of disruption of the ATP7B trafficking itinerary and early endosome accumulation when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as COMMD1 abundance could both be important in maintenance of specific membrane protein trafficking pathways.
Project description:Wilson disease is a genetic disorder of copper metabolism. Impaired biliary excretion results in a gradual accumulation of copper, which leads to severe disease. The specific gene defect lies in the Wilson disease protein, ATP7B, a copper-transporting ATPase that is highly active in hepatocytes. The two major functions of ATP7B in the liver are the copper loading of ceruloplasmin in the Golgi apparatus, and the excretion of excess copper into the bile. In response to elevated copper levels, ATP7B shows a unique intracellular trafficking pattern that is required for copper excretion from the Golgi apparatus into dispersed vesicles. We analyzed the translocation of ATP7B by both confocal microscopy and RNA interference, testing current models that suggest the involvement of Murr1/COMMD1 and Rab7 in this pathway. We found that although the ATP7B translocation is conserved among nonhepatic cell lines, there is no co-localization with Murr1/COMMD1 or the Rab marker proteins of the endolysosomal system. Consistent with this finding, the translocation of ATP7B was not impaired by the depletion of either Murr1/COMMD1 or Rab7, or by a dominant-negative Rab7 mutant. In conclusion, our data suggest that the translocation of ATP7B takes place independently of Rab7-regulated endosomal traffic events. Murr1/COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson disease protein.
Project description:The transcription factor NF-?B plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-?B activation may protect tissues from stress, a prolonged NF-?B activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-?B activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-?B transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD.
Project description:Copper-transporter ATP7B maintains copper homeostasis in the human cells and delivers copper to the biosynthetic pathways for incorporation into the newly synthesized copper-containing proteins. ATP7B is a target of several hundred mutations that lead to Wilson disease, a chronic copper toxicosis. ATP7B contains a chain of six cytosolic metal-binding domains (MBDs), the first four of which (MBD1-4) are believed to be regulatory, and the last two (MBD5-6) are required for enzyme activity. We report the NMR structure of MBD1, the last unsolved metal-binding domain of ATP7B. The structure reveals the disruptive mechanism of G85V mutation, one of the very few disease causing missense mutations in the MBD1-4 region of ATP7B.