Expression and copper binding properties of the N-terminal domain of copper P-type ATPases of African trypanosomes.

Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.

Lipid and energy metabolism in Wilson disease.

Copper accumulation and deficiency are reciprocally connected to lipid metabolism. In Wilson disease (WD), which is caused by a genetic loss of function of the copper-transporting P-type ATPase beta, copper accumulates mainly in the liver and lipid metabolism is dysregulated. The underlying mechanisms linking copper and lipid metabolism in WD are not clear. Copper may impair metabolic machinery by direct binding to protein and lipid structures or by generating reactive oxygen species with consequent damage to cellular organelles vital to energy metabolism. In the liver, copper overload results in mitochondrial impairment, down-regulation of lipid metabolism, and the development of steatosis with an etiology not fully elucidated. Little is known regarding the effect of copper overload on extrahepatic energy homeostasis. This review aims to discuss alterations in hepatic energy metabolism associated with WD, highlights potential mechanisms involved in the development of hepatic and systemic dysregulation of lipid metabolism, and reviews current knowledge on the effects of copper overload on extrahepatic energy metabolism.

A Gene Therapy Approach to Improve Copper Metabolism and Prevent Liver Damage in a Mouse Model of Wilson Disease.

Wilson disease (WD), an autosomal recessive disease caused by mutations in a copper-transporting P-type ATPase (Atp7b), causes severe liver damage. This disease is currently treated with the lifelong use of copper chelation therapy, which has side effects and does not fix copper metabolism. Here, we thoroughly characterized a mouse model of WD, the toxic milk mouse, and used the model to test a gene therapy approach for treating WD. WD mice accumulated copper in the liver from birth; severe copper accumulation and concurrent liver disease were evident by 2 months of age. Intravenously administering an adeno-associated viral (AAV) 8 vector expressing a codon-optimized version of the human ATP7B transgene into 2-month-old WD mice significantly decreased liver copper levels compared with age-matched, uninjected, WD mice. We also observed a significant dose-dependent decrease in liver disease. Male mice injected with 1011 genome copies of AAV8 vector showed only mild histopathological findings with a complete lack of liver fibrosis. Therefore, we conclude that administering gene therapy at the early stages of disease onset is a promising approach for reducing liver damage and correcting copper metabolism in WD.

Copper Delivery to Chloroplast Proteins and its Regulation.

Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.