Michael J. Petris

A Role for the ATP7A Copper-transporting ATPase in Macrophage Bactericidal Activity

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, suggesting a role for ATP7A-dependent copper transport in the bactericidal activity of macrophages. Significantly, a copper-sensitive mutant of Escherichia coli lacking the CopA copper-transporting ATPase was hypersensitive to killing by RAW264.7 macrophages, and this phenotype was dependent on ATP7A expression. Collectively, these data suggest that copper-transporting ATPases, CopA and ATP7A, in both bacteria and macrophage are unique determinants of bacteria survival and identify an unexpected role for copper at the host-pathogen interface.

Copper Homeostasis at the Host-Pathogen Interface

The trace element copper is indispensable for all aerobic life forms. Its ability to cycle between two oxidation states, Cu1+ and Cu2+, has been harnessed by a wide array of metalloenzymes that catalyze electron transfer reactions. The metabolic needs for copper are sustained by a complex series of transporters and carrier proteins that regulate its intracellular accumulation and distribution in both pathogenic microbes and their animal hosts. However, copper is also potentially toxic due in part to its ability to generate reactive oxygen species. Recent studies suggest that the macrophage phagosome accumulates copper during bacterial infection, which may constitute an important mechanism of killing. Bacterial countermeasures include the up-regulation of copper export and detoxification genes during infection, which studies suggest are important determinants of virulence. In this minireview, we summarize recent developments that suggest an emerging role for copper as an unexpected component in determining the outcome of host-pathogen interactions.

The Mammalian Zip5 Protein Is a Zinc Transporter That Localizes to the Basolateral Surface of Polarized Cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.

A Histidine-rich Cluster Mediates the Ubiquitination and Degradation of the Human Zinc Transporter, hZIP4, and Protects against Zinc Cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.

Advances in the Understanding of Mammalian Copper Transporters

Copper (Cu) is an essential micronutrient. Its ability to exist in 2 oxidation states (Cu(1+) and Cu(2+)) allows it to function as an enzymatic cofactor in hydrolytic, electron transfer, and oxygen utilization reactions. Cu transporters CTR1, ATP7A, and ATP7B play key roles in ensuring that adequate Cu is available for Cu-requiring processes and the prevention of excess Cu accumulation within cells. Two diseases of Cu metabolism, Menkes disease and Wilson disease, which are caused by mutations in ATP7A and ATP7B, respectively, exemplify the critical importance of regulating Cu balance in humans. Herein, we review recent studies of the biochemical and cell biological characteristics of CTR1, ATP7A, and ATP7B, as well as emerging roles for Cu in new areas of physiology.

The SLC31 (Ctr) copper transporter family

Copper is essential for many copper-dependent processes, including mitochondrial oxidative phosphorylation, free-radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. The identification of proteins for high affinity copper uptake and export has greatly expanded our understanding of cellular copper homeostasis. Copper export in human cells is mediated by the ATP7A and ATP7B P-type ATPases, which are, respectively, affected in the genetic disorders of copper metabolism, Menkes disease and Wilson disease. A different class of transporter known as the SLC31 or Ctr family of proteins mediates cellular copper uptake. These high-affinity copper transporters exist in all eukaryotes and their discovery has provided new insights into how cells acquire and regulate this essential nutrient. The following is a brief overview of the SLC31 copper transporter family with a focus on the human hCtr1 protein.

Copper transport into the secretory pathway is regulated by oxygen in macrophages.

Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A.

Altered microglial copper homeostasis in a mouse model of Alzheimer’s disease

J. Neurochem. (2010) 114, 1630–1638.

P2Y2 nucleotide receptor-mediated responses in brain cells.

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimers disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the Gq protein-coupled P2Y2 nucleotide receptor subtype (P2Y2R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y2R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y2R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y2R activation increases astrocyte migration. P2Y2R activation by UTP increases the expression in astrocytes of αVβ3/5 integrins that bind directly to the P2Y2R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y2R, an interaction required for Go and G12 protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y2R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y2Rs in neurons. Data with rPCNs suggest that P2Y2R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y2Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y2Rs in glial cells can promote neuroprotective responses, suggesting that P2Y2Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.

A copper treatable Menkes disease mutation associated with defective trafficking of a functional Menkes copper ATPase

Copper dependency in humans is most dramatically illustrated in Menkes disease, an X linked recessive copper deficiency disorder that is generally lethal in early childhood.1,2 Menkes disease is caused by mutations in a transmembrane copper transporting P type ATPase, MNK (or ATP7A), which is expressed in virtually all non-hepatic tissues.3–5 Studies using cultured cells suggest that MNK is located in the trans-Golgi network (TGN), where it transports copper to copper dependent enzymes synthesised within secretory compartments.6–8 In addition to this biosynthetic role, MNK functions in the efflux of excess copper from cells via a process of copper stimulated trafficking to the plasma membrane.6,9 Copper export via MNK from intestinal enterocytes is essential for supplying the blood with dietary copper. Similarly, MNK mediated copper export from the capillary endothelium of the blood brain barrier is thought to supply copper to the central nervous system. In Menkes patients, these processes are defective resulting in a range of symptoms attributable to deficiencies in copper dependent metabolism. These include neurological degeneration, mental retardation, seizures, arterial and bone abnormalities, hypothermia, and hypopigmentation.2 Classical Menkes disease rapidly progresses and is generally lethal during early childhood, although milder variants of the disease exist.10,11 The treatment of Menkes disease involves parenteral injections of copper-histidine, which in the most successful cases reduces neurological defects and prolongs life expectancy.2,12,13 This copper replacement therapy bypasses the intestinal blockage of dietary copper absorption and increases circulating copper levels. However, to prevent the onset of neurological symptoms in Menkes patients, copper must be delivered across the endothelial cells of the blood brain barrier to supply copper to the central nervous system. Within the central nervous system, copper transport into secretory compartments of neurones and other cells to supply copper …