Role of Glutaredoxin1 and Glutathione in Regulating the Activity of the Copper-transporting P-type ATPases,ATP7A and ATP7B

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.     

Copper-dependent interaction of dynactin subunit p62 with the N terminus of ATP7B but not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.     

Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins

Copper plays an essential role in human physiology and is indispensable for normal growth and development. Enzymes that are involved in connective tissue formation, neurotransmitter biosynthesis, iron transport, and others essential physiological processes require copper as a cofactor to mediate their reactions. The biosynthetic incorporation of copper into these enzymes takes places within the secretory pathway and is critically dependent on the activity of copper-transporting ATPases ATP7A or ATP7B. In addition, ATP7A and ATP7B regulate intracellular copper concentration by removing excess copper from the cell. These two transporters belong to the family of P₁-type ATPases, share significant sequence similarity, utilize the same general mechanism for their function, and show partial colocalization in some cells. However, the distinct biochemical characteristics and dissimilar trafficking properties of ATP7A and ATP7B in cells, in which they are co-expressed, indicate that specific functions of these two copper-transporting ATPases are not identical. Immuno-detection studies in cells and tissues have begun to suggest specific roles for ATP7A and ATP7B. These experiments also revealed technical challenges associated with quantitative detection of copper-transporting ATPases in tissues, as illustrated here by comparing the results of ATP7A and ATP7B immunodetection in mouse cerebellum. This work was supported by the National Institute of Health grants PO1 GM 067166–01 and DK R01 DK071865 to S.L.     

Interactions between Copper-binding Sites Determine the Redox Status and Conformation of the Regulatory N-terminal Domain of ATP7B

Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins.     

Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.     

At sixes and sevens: cryptic domain in the metal binding chain of the human copper transporter ATP7A

ATP7A and ATP7B are structurally similar but functionally distinct active copper transporters that regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. Both proteins have a chain of six cytosolic metal-binding domains (MBDs) believed to be involved in the copper-dependent regulation of the activity and intracellular localization of these enzymes. Although all the MBDs are quite similar in structure, their spacing differs markedly between ATP7A and ATP7B. We show by NMR that the long polypeptide between MBD1 and MBD2 of ATP7A forms an additional seventh metastable domain, which we called HMA1A (heavy metal associated domain 1A). The structure of HMA1A resembles the MBDs but contains no copper-binding site. The HMA1A domain, which is unique to ATP7A, may modulate regulatory interactions between MBD1–3, contributing to the distinct functional properties of ATP7A and ATP7B.     

Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.     

A mutation in the ATP7B copper transporter causes reduced dopamine beta-hydroxylase and norepinephrine in mouse adrenal

The copper-transporting ATPases Atp7A and Atp7B play a major role in controlling intracellular copper levels. In addition, they are believed to deliver copper to the copper-requiring proteins destined for the secretory vesicles. One cuproprotein, dopamine -hydroxylase (DBH) functions in the biosynthesis of norepinephrine and epinephrine, neurohormones in endocrine and nervous tissue. To evaluate the consequences of loss of Atp7B on the function of DBH, the level of proteins in adrenal gland were compared between normal mice and mice containing a null mutation in the ATP7B gene. The levels of DBH, as well as another vesicular protein, chromogranin A, are reduced in the ATP7B–/– mice. In addition to the lower level of enzyme, the products of DBH catalytic activity, norepinephrine and epinephrine, are also decreased. Although these changes are a consequence of ATP7B gene function, Atp7B mRNA is not normally expressed in the adrenal gland. Instead, Atp7A mRNA is present. The levels of copper and DBH RNA within adrenals of the ATP7B–/– mice are not different from the wild type. The results of these experiments suggest that copper-requiring enzymes are affected by a loss of ATP7B even in tissue not normally expressing this protein. Therefore the multisystemic effects observed in Wilson disease, the human disorder characterized by mutation in ATP7B, may be a secondary consequence of the major accumulation of copper in liver.     

Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B

The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.     

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H₂O₂ in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H₂O₂ levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H₂O₂ is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).     

Cell-Specific Trafficking Suggests a new role for Renal ATP7B in the Intracellular Copper Storage

Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2–3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.     

Biochemical basis of regulation of human copper-transporting ATPases

Copper is essential for cell metabolism as a cofactor of key metabolic enzymes. The biosynthetic incorporation of copper into secreted and plasma membrane-bound proteins requires activity of the copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B. The Cu-ATPases also export excess copper from the cell and thus critically contribute to the homeostatic control of copper. The trafficking of Cu-ATPases from the trans-Golgi network to endocytic vesicles in response to various signals allows for the balance between the biosynthetic and copper exporting functions of these transporters. Although significant progress has been made towards understanding the biochemical characteristics of human Cu-ATPase, the mechanisms that control their function and intracellular localization remain poorly understood. In this review, we summarize current information on structural features and functional properties of ATP7A and ATP7B. We also describe sequence motifs unique for each Cu-ATPase and speculate about their role in regulating ATP7A and ATP7B activity and trafficking.     

Functional characterization of new mutations in Wilson disease gene (ATP7B) using the yeast model

The Wilson disease gene, a copper transporting ATPase (Atp7b), is responsible for the sequestration of Cu into secretory vesicles, and this function is exhibited by the orthologous Ccc2p in the yeast. In this study, we aimed to characterize clinically relevant new mutations of human ATP7B (p.T788I, p.V1036I and p.R1038G-fsX83) in yeast lacking the CCC2 gene. Expression of human wild type ATP7B gene in ccc2Δ mutant yeast restored the growth deficiency and copper transport activity; however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. Our data support that p.T788I, p.V1036I and p.R1038G-fsX83 mutations cause functional deficiency in ATP7B functions and suggest that these residues are important for normal ATP7B function.     

Mechanism of tumor resistance to cisplatin mediated by the copper transporter ATP7B

The Wilson disease protein (ATP7B) is a copper-transporting ATPase that is responsible for regulating copper homeostasis in human tissues. ATP7B is associated with cancer resistance to cisplatin, one of the most widely used anticancer drugs. This minireview discusses the possible mechanisms of tumor resistance to cisplatin mediated by ATP7B. Cisplatin binds to the N-terminal cytosolic domain of ATP7B, which contains multiple copper-binding sites. Active platinum efflux catalyzed by ATP7B is unlikely to significantly contribute to cisplatin resistance in vivo. Transient platinum sequestration in the metal-binding domain followed by transfer to an acceptor protein or a low molecular weight compound is proposed as an alternative mechanism of cisplatin detoxification in the cell.     

Hepatic copper-transporting ATPase ATP7B: function and inactivation at the molecular and cellular level

Copper-transporting ATPase ATP7B (Wilson disease protein) is a member of the P-type ATPase family with characteristic domain structure and distinct ATP-binding site. ATP7B plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The dual function of ATP7B in hepatocytes is coupled with copper-dependent intracellular relocalization of the transporter. The final destination of ATP7B in hepatocytes during the copper-induced trafficking process is still under debate. We show the results of immunocytochemistry experiments in polarized HepG2 cells that support the model in which elevated copper induces trafficking of ATP7B to sub-apical vesicles, and transiently to the canalicular membrane. In Atp7b ^-/- mice, an animal model of Wilson disease, both copper delivery to the trans-Golgi network and copper export into the bile are disrupted despite large accumulation of copper in the cytosol. We review the biochemical and physiological changes associated with Atp7b inactivation in mouse liver and discuss the pleiotropic consequences of the common Wilson disease mutation, His1069Gln.     

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.     

ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.     

Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hepatocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular trafficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mutation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the constitutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.     

An atomic-level investigation of the disease-causing A629P mutant of the Menkes protein, ATP7A

Menkes disease is a fatal disease that can be induced by various mutations in the ATP7A gene, leading to unpaired uptake of dietary copper. The ATP7A gene encodes a copper(I)-translocating ATPase. Here the disease-causing A629P mutation, which occurs in the last of the six copper(I)-binding soluble domains of the ATPase (hereafter MNK6), was investigated. To understand why this apparently minor amino acid replacement is pathogenic, the solution structures and dynamics on various time-scales of wild-type and A629P-MNK6 were determined both in the apo- and copper(I)-loaded forms. The interaction in vitro with the physiological ATP7A copper(I)-donor (HAH1) was additionally studied. The A629P mutation makes the protein beta-sheet more solvent accessible, possibly resulting in an enhanced susceptibility of ATP7A to proteolytic cleavage and/or in reduced capability of copper(I)-translocation. A small reduction of the affinity for copper(I) is also observed. Both effects could concur to pathogenicity.