The Crossroads between Host Copper Metabolism and Influenza Infection.

Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.

Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b.

OBJECTIVE: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Activity and Trafficking of Copper-Transporting ATPases in Tumor Development and Defense against Platinum-Based Drugs.

Membrane trafficking pathways emanating from the Golgi regulate a wide range of cellular processes. One of these is the maintenance of copper (Cu) homeostasis operated by the Golgi-localized Cu-transporting ATPases ATP7A and ATP7B. At the Golgi, these proteins supply Cu to newly synthesized enzymes which use this metal as a cofactor to catalyze a number of vitally important biochemical reactions. However, in response to elevated Cu, the Golgi exports ATP7A/B to post-Golgi sites where they promote sequestration and efflux of excess Cu to limit its potential toxicity. Growing tumors actively consume Cu and employ ATP7A/B to regulate the availability of this metal for oncogenic enzymes such as LOX and LOX-like proteins, which confer higher invasiveness to malignant cells. Furthermore, ATP7A/B activity and trafficking allow tumor cells to detoxify platinum (Pt)-based drugs (like cisplatin), which are used for the chemotherapy of different solid tumors. Despite these noted activities of ATP7A/B that favor oncogenic processes, the mechanisms that regulate the expression and trafficking of Cu ATPases in malignant cells are far from being completely understood. This review summarizes current data on the role of ATP7A/B in the regulation of Cu and Pt metabolism in malignant cells and outlines questions and challenges that should be addressed to understand how ATP7A and ATP7B trafficking mechanisms might be targeted to counteract tumor development.

Altered zinc balance in the Atp7b-/- mouse reveals a mechanism of copper toxicity in Wilson disease.

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.

In Atp7b-/- Mice Modeling Wilson's Disease Liver Repopulation With Bone Marrow-Derived Myofibroblasts or Inflammatory Cells and Not Hepatocytes Is Deleterious.

In Wilson's disease, Atp7b mutations impair copper excretion with liver or brain damage. Healthy transplanted hepatocytes repopulate the liver, excrete copper, and reverse hepatic damage in animal models of Wilson's disease. In Fah-/- mice with tyrosinemia and α-1 antitrypsin mutant mice, liver disease is resolved by expansions of healthy hepatocytes derived from transplanted healthy bone marrow stem cells. This potential of stem cells has not been defined for Wilson's disease. In diseased Atp7b-/- mice, we reconstituted bone marrow with donor cells expressing green fluorescent protein reporter from healthy transgenic mice. Mature hepatocytes originating from donor bone marrow were identified by immunostaining for green fluorescence protein and bile canalicular marker, dipeptidylpeptidase-4. Mesenchymal and inflammatory cell markers were used for other cells from donor bone marrow cells. Gene expression, liver tests, and tissues were analyzed for outcomes in Atp7b-/- mice. After bone marrow transplantation in Atp7b-/- mice, donor-derived hepatocytes containing bile canaliculi appeared within weeks. Despite this maturity, donor-derived hepatocytes neither divided nor expanded. The liver of Atp7b-/- mice was not repopulated by donor-derived hepatocytes: Atp7b mRNA remained undetectable; liver tests, copper content, and fibrosis actually worsened. Restriction of proliferation in hepatocytes accompanied oxidative DNA damage. By contrast, donor-derived mesenchymal and inflammatory cells extensively proliferated. These contributed to fibrogenesis through greater expression of inflammatory cytokines. In Wilson's disease, donor bone marrow-derived cells underwent different fates: hepatocytes failed to proliferate; inflammatory cells proliferated to worsen disease outcomes. This will help guide stem cell therapies for conditions with proinflammatory or profibrogenic microenvironments.

Exome sequencing of an adolescent with nonalcoholic fatty liver disease identifies a clinically actionable case of Wilson disease.

Diagnostic whole-exome sequencing has proven highly successful in a range of rare diseases, particularly early-onset genetic conditions. In more common conditions, however, exome sequencing for diagnostic purposes remains the exception. Here we describe a patient initially diagnosed with a common, complex liver disease, nonalcoholic fatty liver disease (NAFLD), who was determined to have Wilson disease (WD) upon research-related exome sequencing. The patient presented as a 14.5-yr-old adolescent with chronically elevated aminotransferases, normal ceruloplasmin, and histologic examination consistent with NAFLD with advanced fibrosis. He was enrolled in a large longitudinal study of patients with NAFLD and was found to have WD by exome sequencing performed 4 yr later. This new diagnosis, confirmed clinically by 24 h urine copper quantification, led to a change in the therapy from lifestyle counseling to directed treatment with d-penicillamine, a copper chelating agent. In this case, the likelihood of making the correct diagnosis and thereby choosing the appropriate treatment was increased by exome sequencing and careful interpretation. This example illustrates the utility of exome sequencing diagnostically in more common conditions not currently considered as targets for genome-wide evaluation and adds to a growing body of evidence that patients diagnosed with more common conditions often in fact have rarer genetically determined syndromes that have escaped clinical detection.

Pathogenesis of Wilson disease.

Wilson disease is an autosomal-recessive disorder originating from a genetic defect in the copper-transporting ATPase ATP7B that is required for biliary copper secretion and loading of ceruloplasmin with copper. Impaired ATP7B function in Wilson disease results in excessive accumulation of copper in liver, brain, and other tissues. Toxic copper deposits may induce oxidative stress, modify expression of genes, directly inhibit proteins, and impair mitochondrial function, leading to hepatic, neuropsychiatric, renal, musculoskeletal, and other symptoms. Hepatocyte dysfunction initially manifests as steatosis and later may progress to other hepatic phenotypes such as acute liver failure, hepatitis, and fibrosis. In the brain, copper accumulates in astrocytes, leading to impairment of the blood-brain barrier and consequent damage to neurons and oligodendrocytes. Basal ganglia and brainstem are the brain regions with highest susceptibility to copper toxicity and their lesions lead to various combinations of movement and psychiatric disorders. This chapter will give an overview of the essential requirement of copper for biologic processes and the molecular mechanisms employed by cells to maintain their copper levels in a proper range. We will specify the physiologic functions of ATP7B and the consequences of its dysfunction and summarize the current knowledge on the pathogenesis of liver and neuropsychiatric disease. Finally, we will describe the consequences of copper overload in Wilson disease in other tissues.