Copper Toxicity Is Not Just Oxidative Damage: Zinc Systems and Insight from Wilson Disease.
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ABSTRACT:
SUBMITTER: Barber RG
PROVIDER: S-EPMC8003939 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Wilson disease (WD) is an autosomal recessive disorder caused by mutation in the ATP7B gene that affects copper transport in the body. ATP7B mutation damages copper transporter function, ultimately resulting in excessive copper accumulation and subsequent toxicity in both the liver and brain. Mechanisms of copper toxicity, however, are not well defined. The Atp7b-/- mouse model is well-characterized and presents a hepatic phenotype consistent with WD. In this study, we found that the untreated Atp7b-/- mice accumulate approximately 2-fold excess hepatic zinc compared to the wild type. We used targeted transcriptomics and proteomics to analyze the molecular events associated with zinc and copper accumulation in the Atp7b-/- mouse liver. Altered gene expression of Zip5 and ZnT1 zinc transporters indicated a transcriptional homeostatic response, while increased copper/zinc ratios associated with high levels of metallothioneins 1 and 2, indicated altered Zn availability in cells. These data suggest that copper toxicity in Wilson disease includes effects on zinc-dependent proteins. Transcriptional network analysis of RNA-seq data reveals an interconnected network of transcriptional activators with over-representation of zinc-dependent and zinc-responsive transcription factors. In the context of previous research, these observations support the hypothesis that mechanisms of copper toxicity include disruption of intracellular zinc distribution in liver cells. The translational significance of this work lies in oral zinc supplementation in treatment for WD, which is thought to mediate protective effects through the induction of metallothionein synthesis in the intestine. This work indicates broader impacts of altered zinc-copper balance in WD, including global transcriptional responses and altered zinc balance in the liver.
| S-EPMC6310031 | biostudies-literature
Project description:To find new genes that might be targeted for reduction of Copper (Cu) toxicity in Wilson disease we employed genome-wide shRNA screening in ATP7B-KO HepG2 cells, a bona fide WD cell model. Cells were screened using the Dharmacon™ Decode™ Pooled Lentiviral shRNA Screening Library targeting 18205 human genes (Dharmacon #RHS6083). The Lentiviral RNAi screening library is made of 10 pools of 9570 GIPZ short hairpin RNAs (shRNAs) packaged into high-titer lentiviral particles. Genomic DNA was isolated from the cells and read counts for each shRNAs were obtained by deep sequencing to determine the abundance of each shRNA in the Cu-treated cells compared to controls. This analysis allowed the identification of genes whose downregulation promoted survival of ATP7B-KO cells upon Cu overload. To exclude that some shRNAs would have been responsible for ATP7B-KO survival by suppressing generic proapoptotic genes, we run the same screening with the proapoptotic drug vinblastine instead of CuCl2. Genes, whose suppression allowed cell to resist Cu but not vinblastine were considered as Cu-specific hits and through this approach we identified PrP as a suitable target for reduction of Cu toxicity in WD. Indeed, we found that Cu accumulation in ATP7B-KO cells stimulates expression of PrP, which brings to toxic Cu overload while PrP suppression significantly reduces Cu accumulation and toxicity in ATP7B-deficient cells. In sum our data highlight an important regulatory role of PrP in copper metabolism and as a therapeutic tool for Wilson disease.
2024-12-09 | GSE260454 | GEO
Project description:To find new genes that might be targeted for reduction of Copper (Cu) toxicity in Wilson disease we employed genome-wide shRNA screening in ATP7B-KO HepG2 cells, a bona fide WD cell model. Cells were screened using the Dharmacon™ Decode™ Pooled Lentiviral shRNA Screening Library targeting 18205 human genes (Dharmacon #RHS6083). The Lentiviral RNAi screening library is made of 10 pools of 9570 GIPZ short hairpin RNAs (shRNAs) packaged into high-titer lentiviral particles. Genomic DNA was isolated from the cells and read counts for each shRNAs were obtained by deep sequencing to determine the abundance of each shRNA in the Cu-treated cells compared to controls. This analysis allowed the identification of genes whose downregulation promoted survival of ATP7B-KO cells upon Cu overload. To exclude that some shRNAs would have been responsible for ATP7B-KO survival by suppressing generic proapoptotic genes, we run the same screening with the proapoptotic drug vinblastine instead of CuCl2. Genes, whose suppression allowed cell to resist Cu but not vinblastine were considered as Cu-specific hits and through this approach we identified PrP as a suitable target for reduction of Cu toxicity in WD. Indeed, we found that Cu accumulation in ATP7B-KO cells stimulates expression of PrP, which brings to toxic Cu overload while PrP suppression significantly reduces Cu accumulation and toxicity in ATP7B-deficient cells. In sum our data highlight an important regulatory role of PrP in copper metabolism and as a therapeutic tool for Wilson disease.
2024-12-09 | GSE277865 | GEO
Project description:ATP7A and ATP7B, two homologous copper-transporting P1B-type ATPases, play crucial roles in cellular copper homeostasis, and mutations cause Menkes and Wilson diseases, respectively. ATP7A/B contains a P-type ATPase core consisting of a membrane transport domain and three cytoplasmic domains, the A, P, and N domains, and a unique amino terminus comprising six consecutive metal-binding domains. Here, we present a cryo-electron microscopy structure of frog ATP7B in a copper-free state. Interacting with both the A and P domains, the metal-binding domains are poised to exert copper-dependent regulation of ATP hydrolysis coupled to transmembrane copper transport. A ring of negatively charged residues lines the cytoplasmic copper entrance that is presumably gated by a conserved basic residue sitting at the center. Within the membrane, a network of copper-coordinating ligands delineates a stepwise copper transport pathway. This work provides the first glimpse into the structure and function of ATP7 proteins and facilitates understanding of disease mechanisms and development of rational therapies.
| S-EPMC8896786 | biostudies-literature
Project description:We evaluate Wilson disease (WD) treatment with zinc acetate (U.S. Food and Drug Administration approved) and alternative zinc salts. Studies examining zinc therapy in WD are few, and data on alternative zinc salts are limited. We describe one of the largest recent studies of zinc therapy in WD. First, we conducted a single-center retrospective review of 59 patients with WD (age 6-88 years, 32 female patients) treated with zinc (50-150 mg) for 0.8 to 52 years (median, 26 years); most were on prior chelation therapy (n = 39). Second, we developed a survey to explore patients' zinc therapy experience. Primary endpoints were alamine aminotransferase (ALT) and urine copper excretion (µg/24 hours). Urine copper was categorized as low <25 μg (possible overtreatment), target 25-100 μg, or elevated >100 μg (possible noncompliance or treatment failure). The target range was reached in 81% of patients on zinc acetate, 73% on zinc gluconate, and 57% on alternative zinc. Low urine copper was not associated with a high ALT. ALT was normal in 77% of patients with target urine copper but only in 16% with urine copper >100 µg. ALT elevations were not significantly different between zinc salts (Kruskal-Wallis, P = 0.26). Our survey demonstrated the mean age of starting zinc was 26.8 years (3.5-65 years); most were treated with zinc acetate (45%) and zinc gluconate (42%). Before zinc treatment, 45% of patients were symptomatic; the majority of patients (80%) were asymptomatic on zinc. Gastrointestinal side effects were the predominant reason for changing zinc salts (38%), but most reported no side effects on current zinc therapy (67%). Conclusion: Effective treatment with zinc is possible in many patients with WD. The potential for treatment failure suggests close monitoring and consideration of alternative treatments are paramount for those without both a normal serum ALT and appropriate urine copper excretion.
| S-EPMC6671772 | biostudies-literature
Project description:Copper accumulation in tissues due to a biallelic pathogenic mutation of the gene: ATP7B results in a clinical phenotype known as Wilson disease (WD). Aberrations in copper homeostasis can create favourable conditions for superoxide-yielding redox cycling and oxidative tissue damage. Drugs used in WD treatment aim to remove accumulated copper and normalise the free copper concentration in the blood. In the current study the effect of decoppering treatment on copper metabolism and systemic antioxidant capacity parameters was analyzed. Treatment naïve WD patients (TNWD) (n = 33), those treated with anti-copper drugs (TWD) (n = 99), and healthy controls (n = 99) were studied. Both TNWD and TWD patients characterised with decreased copper metabolism parameters, as well as decreased total antioxidant potential (AOP), glutathione (GSH) level, activity of catalase, glutathione peroxidase (GPx), and S-transferase glutathione, compared to controls. TWD patients had significantly lower copper metabolism parameters, higher total AOP and higher levels of GSH than TWD individuals; however, no difference was observed between these two patient groups with respect to the rest of the antioxidant capacity parameters. Patients who had undergone treatment with D-penicillamine or zinc sulphate did not differ with respect to copper metabolism or antioxidant capacity parameters, with the exception of GPx that was lower in D-penicillamine treated individuals. These data suggest that anti-copper treatment affects copper metabolism as well as improves, but does not normalize, natural antioxidant capacity in patients with WD. We propose to undertake studies aimed to evaluate the usefulness of antioxidants as well as selenium as a supplemental therapy in WD.
| S-EPMC3905172 | biostudies-literature
Project description:Background & aimsWilson disease (WD) is caused by accumulation of copper primarily in the liver and brain. During maintenance therapy of WD with D-penicillamine, current guidelines recommend on-treatment ranges of urinary copper excretion (UCE) of 200-500 μg/24 h and serum non-ceruloplasmin-bound copper (NCC) of 50-150 μg/L. We compared NCC (measured by two novel assays) and UCE from patients with clinically stable WD on D-penicillamine therapy with these recommendations.MethodsThis is a secondary analysis of data from the Chelate trial (NCT03539952) that enrolled physician-selected patients with clinically stable WD on D-penicillamine maintenance therapy (at an unaltered dose for at least 4 months). We analyzed laboratory samples from the first screening visit, prior to interventions. NCC was measured by either protein speciation (NCC-Sp) using anion exchange high-performance liquid chromatography protein speciation followed by copper determination with inductively coupled plasma mass spectroscopy or as exchangeable copper (NCC-Ex). NCC-Sp was also analyzed in healthy controls (n = 75).ResultsIn 76 patients with WD with 21.3±14.3 average treatment-years, NCC-Sp (mean±SD: 56.6±26.2 μg/L) and NCC-Ex (mean±SD: 57.9±24.7 μg/L) were within the 50-150 μg/L target in 61% and 54% of patients, respectively. In addition, 36% and 31%, respectively, were even below the normal ranges (NCC-Sp: 46-213 μg/L, NCC-Ex: 41-71 μg/L). NCC-Ex positively correlated with NCC-Sp (r2 = 0.66, p <0.001) but with systematic deviation. UCE was outside the 200-500 μg/24 h target range in 58%. Only 14/69 (20%) fulfilled both the NCC-Sp and UCE targets. Clinical or biochemical signs of copper deficiency were not detected.ConclusionClinically stable patients with WD on maintenance D-penicillamine therapy frequently have lower NCC-Sp or higher UCE than current recommendations without signs of overtreatment. Further studies are warranted to identify appropriate target ranges of NCC-Sp, NCC-Ex and UCE in treated WD.Impact and implicationsChelator treatment of patients with Wilson disease (WD) is currently guided by measurements of non-ceruloplasmin-bound copper (NCC) and 24 h urinary copper excretion (UCE) but validation is limited. In 76 adults with ≈21 years history of treated WD and clinically stable disease on D-penicillamine therapy, NCC was commonly found to be below normal values and recommended target ranges whether measured by protein speciation (NCC-Sp) or as exchangeable copper (NCC-Ex), while UCE values were above the recommended target range in 49%. Common wisdom would suggest overtreatment in these cases, but no clinical or biochemical signs of copper deficiency were observed. Exploratory analysis of liver enzymes suggested that NCC below levels seen in controls may be beneficial, while the relation to UCE was less clear. The data calls for critical re-evaluation of target ranges for treatment of WD, specific for drug and laboratory methodology.Clinical trial number(NCT03539952).
| S-EPMC11321293 | biostudies-literature
Project description:Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.
| S-EPMC4070386 | biostudies-literature
Project description:Wilson's disease (WD) is an autosomal recessive disorder, resulting from variations in ATP7B gene. Clinical heterogeneity, including neuropsychiatric and hepatic manifestations over a large range of age groups make diagnosis difficult. Most of WD patients suffer severe disabilities and even die. So, overall goal of proposed study is the genetic and clinical characterization of Wilson's disease cases from Pakistani population. Clinical data was collected, and patients were investigated for variations in selected ATP7B exons using PCR based Sanger sequencing. Pathogenic effect predictions for detected variants were carried out using PROVEAN, MutationTaster2, and HSF software's. Clinical heterogeneity was observed in patients including reduced serum ceruloplasmin, signs of chronic liver damage and raised 24 h urinary copper excretion. Mean age of onset was 11.3 years. Kayser-Fleischer rings were present in 75% of cases. About 82.5% patients belonged to inbred families. Patients having neurological disorder were above 12 years of age. Total ten variants in analyzed region of ATP7B gene, including a reported variation (p. L227Yfs*35) were found in patients. The study also identified 4 putative novel synonymous variants (c.251A>C, c.15T>A, c.6T>C, c.238C>T) and 5 reported polymorphisms (c.83C>A, c.39_40insCGGCG, p.V456L, c.39_40insCGCCG and c.1544-53A>C). Reliable understanding of clinical presentations and genotype-phenotype correlation provide insight to function and structure of ATP7B and may assist in disease prognosis and family counseling. The study revealed clinical presentation of Pakistani WD cases and identification of sequence variants in screened region of ATP7B.
| S-EPMC9239485 | biostudies-literature
Project description:Screening for genes that promote copper toxicity in a cell model of Wilson disease
| PRJNA1081796 | ENA