No abstract

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-␥ 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 hostpathogen interface.

The Menkes P‐type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper‐translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P‐type ATPase has a role in copper efflux has come from studies using copper‐resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper‐resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady‐state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady‐state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.

Copper uptake at the plasma membrane and subsequent delivery to copper-dependent enzymes is essential for many cellular processes, including mitochondrial oxidative phosphorylation, free radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. However, intracellular levels of this nutrient must be controlled because it is potentially toxic in excess concentrations. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. In this study, we demonstrate that levels of the hCtr1 protein at the plasma membrane of HEK293 cells were reduced when cells were exposed to elevated copper. This decrease in surface hCtr1 levels was associated with an increased rate of endocytosis, and low micromolar concentrations of copper were sufficient to stimulate this process. Inhibitors of clathrindependent endocytosis prevented the trafficking of hCtr1 from the plasma membrane, and newly internalized hCtr1 and transferrin were co-localized. Significantly, elevated copper concentrations also resulted in the degradation of the hCtr1 protein. Our findings suggest that hCtr1-mediated copper uptake into mammalian cells is regulated by a post-translational mechanism involving copper-stimulated endocytosis and degradation of the transporter.

The trace element copper is indispensable for all aerobic life forms. Its ability to cycle between two oxidation states, Cu 1؉ and Cu 2؉ , 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.