Cellular Copper Homeostasis

The underlying mechanisms of the physiological regulation of copper homeostasis are now becoming understood as a result of the advances in the molecular and cellular studies of the genes and proteins involved. An overriding principle that lies behind the types of mechanism that have been

Cu(I

Cu(I

Fig. 2. Copper transport in a nonpolarized human cell such as a fibroblast. Copper enters the cell via the plasma membrane transporter, hCTR1, Cu(II) is thought to be reduced to Cu(I) by membrane reductases. As soon as copper enters the cell, it is distributed between the various copper chaperones, hCOX17, CCS, and ATOX 1, that carry the ion variously to the mitochondrion, Cu/Zn superoxide dismutase, or to ATP7A in the trans-Golgi network (TGN). ATP7A pumps copper into the TGN vesicles, where it is incorporated into secreted cuproenzymes, such as lysyl oxidase. ATP7A is constitutively recycling between the TGN and the plasma membrane, and the if copper levels in the cell increase, more ATP7A is found on the plasma membrane, allowing efflux of the metal ion. Cu(I) is also found on glutathione, and if the ATP7A-mediated efflux is insufficient to maintain low cytoplasmic copper levels, metallothioneins (MTs) are induced and bind the excess copper.

Fig. 2. Copper transport in a nonpolarized human cell such as a fibroblast. Copper enters the cell via the plasma membrane transporter, hCTR1, Cu(II) is thought to be reduced to Cu(I) by membrane reductases. As soon as copper enters the cell, it is distributed between the various copper chaperones, hCOX17, CCS, and ATOX 1, that carry the ion variously to the mitochondrion, Cu/Zn superoxide dismutase, or to ATP7A in the trans-Golgi network (TGN). ATP7A pumps copper into the TGN vesicles, where it is incorporated into secreted cuproenzymes, such as lysyl oxidase. ATP7A is constitutively recycling between the TGN and the plasma membrane, and the if copper levels in the cell increase, more ATP7A is found on the plasma membrane, allowing efflux of the metal ion. Cu(I) is also found on glutathione, and if the ATP7A-mediated efflux is insufficient to maintain low cytoplasmic copper levels, metallothioneins (MTs) are induced and bind the excess copper.

evolved to handle copper is the dual nature of copper in biological systems: Copper is both an essential cofactor for various Cu-dependent enzymes and a potential pro-oxidant. To function normally and to protect itself from potential damage a cell must be able to sense and respond to changes in intracellular copper levels. Different mechanisms will be activated in response to deficiency or excess as cells adopt an acquisition or detoxification mode depending on their copper status. Whole-body copper homeostasis depends on these mechanisms, which are modified in cells such as the intestinal enterocyte (acquisition) or the hepatocyte (detoxification). These modifications are discussed in more detail in Section 2.3. (see Figs. 3 and 4).

Much of our understanding of human cellular copper homeostasis has been gleaned from studies in yeast. Such comparisons have revealed a remarkable conservation of the molecules required for cellular copper homeostasis, but some important differences have been discovered. Figure 2 summarizes the current state of knowledge of copper homeostasis in a human cells such as a fibroblasts that require copper, but are not involved in the physiological regulation of whole-body copper status. Copper entry into the cell across the plasma membrane is mediated by the protein hCTR1 (16). The human protein was identified following the discovery of the equivalent yeast protein Ctr1p encoded by the gene CTR1. In Saccharomyces cerevisiae, CTR1 was discovered, paradoxically as a gene essential, not for copper transport but for iron transport (17). This discovery provided a clear link between copper and iron transport that had been known for years but was not well understood. In S. cerevisiae, Cu is required for Fet3p, a multicopper oxidase related to the mammalian ceruloplasmin. Fet3p catalyzes the oxidation of Fe2+ to Fe3+, allowing entry of iron into the cell by a ferric transporter. The yeast Ctr1p is a 406-amino-acid protein with 3 putative transmembrane domains and has the motif M-X-X-M repeated 11 times in the extracellular (periplasmic) amino terminal domain of the protein, these motifs presumably bind copper from the extracellular environment. The human ortholog is considerably smaller, consisting of 190 amino acids, and includes a methionine and histi-dine-rich N-terminal domain reminiscent of Ctrlp. hCTRl also is thought to have three transmembrane domains but lacks much of the cytoplasmic region found in the yeast protein. This gene is expressed in all tissues so far examined, with liver, heart, and pancreas showing the highest levels of expression and the brain and muscle having the lowest levels. In yeast, Ctrlp specifically transports Cu(I); thus, a copper reductase is required on the plasma membrane to reduce the Cu(II) present in the oxidative extracellular environment. These reductases act on both Fe(III) and Cu(II), are known in yeast, and are products of the FRE genes (seven are known) (18). It is unclear whether the transport of Cu(I) occurs by direct coupling of the reductases and Ctrl at the plasma membrane. A reductase that uses NADH has been described in rat liver cells and may be required for mammalian plasma membrane copper transport (19).

The potential pro-oxidant toxicity of copper necessitates that the Cu(I) ion be complexed at all times [copper is thought to be in the Cu(I) state in the cytoplasm]. It has been known for some time that intracellular copper is associated with glutathione (GSH) (20) and, after exposure of cells to excess copper, the small metal binding proteins metallothioneins (MTs) are induced (21). More recently, experiments involving yeast mutants have uncovered a number of cytoplasmic copper carriers termed copper chaperones that accept and deliver copper to specific locations in the cell [reviewed in by Harrison et al. (22)]. Individual copper chaperones appear to be responsible for the specificity of delivery of copper to the different compartments of the cell, including the cytoplasm, the trans-Golgi network (TGN), and other less understood vesicles. The mechanisms whereby these chaperones are themselves loaded with copper and what determines the choice between the different chaperones once the copper enters the cell are unknown. Presumably, the regulation of the concentration of these chaperones is part of cellular copper homeostasis, but little is known about this process in mammalian cells.

The Cox17p chaperone in yeast has a role in delivering copper to mitochondria for incorporation into cytochrome oxidase (23). Cox17p is a 69-amino-acid polypeptide present in the cytosol and the intermembrane space of the mitochondrion (24). Inactivation of the COX17 gene in yeast results in loss of cytochrome oxidase function as a result of a failure in the assembly of a functional multisubunit complex, leading to respiratory deficiency. The putative Cu-binding sites on Cox17p are tandem cysteine residues and two Cu(I) ions are bound in a binuclear cluster (25). The human equivalent COX17 has been identified by complementation of the cytochrome oxidase deficiency in a yeast strain with a null mutation in the COX17 gene (26). Little is known about the physiological role of the COX17 protein in mammalian cells, but a study of the expression patterns in rodent tissue show expression in a wide range of tissues, as expected from its role as a copper chaperone to cytochrome oxidase.

The copper chaperone for SOD1 (CCS, also known as LYS7 in yeast) transports copper to copper/ zinc superoxide dismutase (SOD) and was identified by complementation of yeast mutant, which was SOD deficient but had an intact SOD gene (27). In yeast, CCS is a 249-amino-acid protein that includes a single Cu-binding domain (MTCXXC) in the N-terminal region and the C-terminal half contains a domain with homology to SOD1. The human protein hCCS is 274 amino acids in length has 28% amino acid identity with the yeast protein (27). An interaction with CCS and Cu/Zn SOD has been demonstrated and the localization of the two proteins in cells is identical, with a diffuse cytoplasmic and nuclear distribution (28).

Another copper chaperone, Atx1 (antioxidant 1), was first identified as a suppressor of oxidative damage in yeast cells lacking SOD1 (29). Atx1 is a 73-amino-acid cytosolic polypeptide with a singe amino terminal MTCXXC copper-binding motif. Atx1 binds one Cu(I) atom per polypeptide involving the thiol ligands of the two cysteines (30). The Cu-Atx1 complex is proposed to move to the trans-Golgi network (TGN) where it interacts with the N-terminal MTCXXC residues of the yeast Menkes ortholog Ccc2 protein and copper is transferred by a series of two- or three-coordinate Cu-bridged intermediates (30). ATOX1 (formerly HAH1), the human ortholog of Atx1, has a 47% amino acid identity with Atx1 and contains an MTCXC domain (31). ATOX1 is believed to transport copper from CTR1 to the Menkes and Wilson proteins in the TGN (31).

The Menkes disease protein (ATP7A) and Wilson protein (ATP7B) are highly related copper-transporting P-type ATPases that have both biosynthetic and protective roles in cellular copper homeostasis in human cells under different copper conditions. During normal copper exposure, the Cu-ATPases are located in the TGN of the cell, where the metal is incorporated into various copper-dependent enzymes such as lysyl oxidase in fibroblasts (mediated by ATP7A as shown in Fig. 2) or ceruloplasmin in hepatocytes (mediated by ATP7B; see Fig. 4). If the copper levels in the cell start to rise, perhaps when the capacity of the copper chaperones is exceeded, a remarkable mechanism is activated that enables the cell to reduce its intracellular copper concentrations. This mechanism involves the Cu-induced trafficking of ATP7A and ATP7B to the plasma membrane of the cell (as shown in Fig. 2A for ATP7A) (32) or to a vesicular compartment (ATP7B; see Fig. 4A) (33). In these locations, the excess copper can be effluxed from the cell or sequestered into a vesicle for later efflux. Because of its central role in copper homeostasis, the mechanism of copper-induced trafficking is of considerable interest and will be discussed in more detail in Section 2.3. Induction of the genes encoding the cysteine-rich metallothioneins (MTs), which avidly bind intracellular copper ions, provides a last line of defense against copper toxicity (21). Presumably, this induction occurs when the capacity of the ATPases to efflux copper is insufficient to reduce the intracellular concentrations to a safe level.

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