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.     

Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance

The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.     

Copper-dependent interaction of glutaredoxin with the N termini of the copper-ATPases (ATP7A and ATP7B) defective in Menkes and Wilson diseases

The P-type ATPases affected in Menkes and Wilson diseases, ATP7A and ATP7B, respectively, are key copper transporters that regulate copper homeostasis. The N termini of these proteins are critical in regulating their function and activity, and contain six copper-binding motifs MxCxxC. In this study, we describe the identification of glutaredoxin (GRX1) as an interacting partner of both ATP7A and ATP7B, confirmed by yeast two-hybrid technology and by co-immunoprecipitation from mammalian cells. The interaction required the presence of copper and intact metal-binding motifs. In addition, the interaction was related to the number of metal-binding domains available. GRX1 catalyses the reduction of disulphide bridges and reverses the glutathionylation of proteins to regulate and/or protect protein activity. We propose that GRX1 is essential for ATPase function and catalyses either the reduction of intramolecular disulphide bonds or the deglutathionylation of the cysteine residues within the CxxC motifs to facilitate copper-binding for subsequent transport.     

Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B

ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.     

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.     

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.     

Glutaredoxin1 protects neuronal cells from copper-induced toxicity

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P_1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.     

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.     

The multi-layered regulation of copper translocating P-type ATPases

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.     

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.     

Independent Evolution of Heavy Metal-Associated Domains in Copper Chaperones and Copper-Transporting ATPases

Copper chaperones are small cytoplasmic proteins that bind intracellular copper (Cu) and deliver it to Cu-dependent enzymes such as cytochrome oxidase, superoxide dismutase, and amine oxidase. Copper chaperones are similar in sequence and structure to the Cu-binding heavy metal-associated (HMA) domains of Cu-transporting ATPases (Cu-ATPases), and the genes for copper chaperones and Cu-ATPases are often located in the same operon. Phylogenetic analysis shows that Cu chaperones and HMA domains of Cu-ATPases represent ancient and distinct lineages that have evolved largely independently since their initial separation. Copper chaperone–Cu-ATPase operons appear to have evolved independently in different prokaryotic lineages, probably due to a strong selective pressure for coexpression of these genes. Received: 14 December 2000 / Accepted: 9 May 2001     

Genes regulating copper metabolism

The metabolism of Cu is intimately linked with its nutrition. From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands. Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly being understood. Cloning and sequencing of the genes for Menkes disease and Wilson disease has shown that membrane-bound enzymes analogous to Cu-ATPases in prokaryotes are equally important to Cu transport and homeostasis in mammalian cells. The primary structure of the mammalian Cu-ATPases has been deduced from cDNAs from tissues and organs. It now appears that mammalian Cu-ATPase have tissue and developmental specificity. In this review, we will focus on the Cu-ATPase that has been identified with Menkes disease. An emphasis will be placed on the existence of multiple forms of the ATPase and some indication as to how the different isoforms befit their role in the normal physiology of copper, specifically transmembrane transport and maintenance of a favorable internal cellular environment.     

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of approximately 30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 +/- 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A(4)-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A(4)-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX.     

NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells

Cu is an essential cofactor of cellular proteins but is toxic in its free state. The hepatic Cu-ATPase ATP7B has two functions in Cu homeostasis: it loads Cu+ onto newly synthesized apoceruloplasmin in the secretory pathway, thereby activating the plasma protein; and it participates in the excretion of excess Cu+ into the bile. To carry out these two functions, the membrane protein responds to changes in intracellular Cu levels by cycling between the Golgi and apical region. We used polarized hepatic WIF-B cells and high-resolution confocal microscopy to map the itinerary of endogenous and exogenous ATP7B under different Cu conditions. In Cu-depleted cells, ATP7B resided in a post-trans-Golgi network compartment that also contained syntaxin 6, whereas in Cu-loaded cells, the protein relocated to unique vesicles very near to the apical plasma membrane as well as the membrane itself. To determine the role of ATP7B's cytoplasmic NH2 terminus in regulating its intracellular movements, we generated seven mutations/deletions in this large [approximately 650 amino acid (AA)] domain and analyzed the Cu-dependent behavior of the mutant ATP7B proteins in WIF-B cells. Truncation of the ATP7B NH2 terminus up to the fifth copper-binding domain (CBD5) yielded an active ATPase that was insensitive to cellular Cu levels and constitutively trafficked to the opposite (basolateral) plasma membrane domain. Fusion of the NH2-terminal 63 AA of ATP7B to the truncated protein restored both its Cu responsiveness and correct intracellular targeting. These results indicate that important targeting information is contained in this relatively short sequence, which is absent from the related CuATPase, ATP7A.     

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.     

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.     

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).     

Structural organization of human Cu-transporting ATPases: learning from building blocks

Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B play an essential role in human physiological function. Their primary function is to deliver copper to the secretory pathway and export excess copper from the cell for removal or further utilization. Cells employ Cu-ATPases in numerous physiological processes that include the biosynthesis of copper-dependent enzymes, lactation, and response to hypoxia. Biochemical studies of human Cu-ATPases and their orthologs have demonstrated that Cu-ATPases share many common structural and mechanistic characteristics with other members of the P-type ATPase family. Nevertheless, the Cu-ATPases have a unique coordinate environment for their ligands, copper and ATP, and additional domains that are required for sophisticated regulation of their intracellular localization and activity. Here, we review recent progress that has been made in understanding the structure of Cu-ATPases from the analysis of their individual domains and orthologs from microorganisms, and speculate about the implications of these findings for the function and regulation of human copper pumps.     

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.