Copper in Human Health and Disease: Insights from Inherited Disorders

Abstract

In recent years, our understanding of copper metabolism in humans has advanced considerably, driven in large part by insights from genetic disorders. Studies of Menkes disease, Wilson disease, MEDNIK and KIDAR syndromes, and most recently CTR1 deficiency, have illuminated the fundamental principles of copper acquisition, intracellular distribution, and systemic elimination. These discoveries revealed not only the canonical roles of CTR1, ATP7A, and ATP7B, but also uncovered auxiliary pathways of copper uptake, novel chaperone and organelle-specific distribution mechanisms, and the importance of trafficking adaptors in maintaining copper balance. Beyond its classical enzymatic functions, copper has emerged as a dynamic regulator of cell signaling, autophagy, metabolism, and immune surveillance, with mitochondrial dysfunction and cuproptosis representing key pathogenic outcomes of copper imbalance. The expanding view of copper as both a nutrient and a signaling ion highlights the complexity of its physiological regulation. In this review, we summarize the current knowledge of human copper homeostasis, focusing on how lessons from inherited disorders continue to redefine our understanding of copper physiology and inform therapeutic approaches.

Introduction

Systemic Copper Homeostasis: Acquisition, Distribution, and Elimination

Copper is an essential micronutrient required for proper metabolic function and normal tissue development. This versatile metal acts as a cofactor for numerous vital enzymes, thereby supporting a wide range of cellular processes. It drives energy metabolism as the central metal ion of cytochrome c oxidase and provides antioxidant protection as a component of Cu/Zn superoxide dismutase. In addition, it facilitates dopamine biosynthesis by serving as a cofactor for dopamine β-hydroxylase. Copper is also indispensable for connective tissue development, as it is a critical cofactor for lysyl oxidase, the enzyme responsible for collagen cross-linking in the extracellular matrix (1–3). Beyond its classical role as an enzymatic cofactor, copper has recently been attributed with important regulatory functions in diverse signaling networks, including those governing cell proliferation, autophagy, lipolysis, and immune surveillance (4–7).

Although essential, copper is toxic in excess and therefore must be maintained within a narrow optimal range to prevent both deficiency and overload. In mammals, copper homeostasis is characterized by remarkably stable tissue and fluid concentrations. Across most mammalian species (with some exceptions, such as dogs), copper levels remain consistent: approximately 5 µg/g in the liver and heart, 4–5 µg/g in the brain, and ~1 µg/g in muscle (8). As an essential trace element, copper must be obtained from the diet (Figure 1), with typical daily intakes ranging from 2–4 mg but occasionally reaching up to 10 mg (9). A healthy 70-kg adult human body contains roughly 100 mg of copper, primarily localized to the liver, brain, and kidneys. Mammals also display an efficient capacity to normalize copper excess; for instance, mouse organs and fluids rapidly return to baseline copper levels even after a threefold increase in total body copper (10). The liver plays a central role in this process, as it mediates biliary excretion of excess copper, which is widely regarded as the primary regulatory pathway for systemic copper balance (2, 3).

Figure 1. Copper homeostasis in human body.

Copper (Cu) absorption occurs within the body through the small intestine, where it is transported into enterocytes via the apical copper transporter CTR1. From there, Cu is exported across the basolateral membrane into the portal circulation by ATP7A, a process that requires ATP7A to traffic from the trans-Golgi network (TGN) to the plasma membrane (blue arrow). In Menkes disease, loss of ATP7A activity prevents Cu efflux, leading to its retention within enterocytes and systemic Cu deficiency (green dash bar). Under normal conditions, most absorbed Cu is delivered to the liver through portal circulation. In the liver hepatocytes internalize Cu and ATP7B incorporates it into newly synthesized ceruloplasmin within the TGN, establishing ceruloplasmin (CP) as the major Cu carrier in the blood (green arrows). When Cu levels rise, ATP7B relocates to excretory sites (orange arrows), including the apical canalicular membrane and associated vesicles, to promote biliary Cu elimination. In Wilson disease, pathogenic variants in ATP7B impair either its enzymatic activity or trafficking, thereby blocking Cu incorporation into ceruloplasmin and its secretion into bile (red bars). Consequently, Cu accumulates in hepatocytes, driving progressive hepatic toxicity.

Because both copper deficiency and overload are detrimental, mammals have evolved finely tuned mechanisms to regulate copper acquisition, distribution, utilization, and elimination. These processes depend on the expression, activity, and copper-dependent trafficking of specialized transporters (2, 11). Dietary copper is absorbed mainly in the small intestine, particularly in the duodenum (Figure 1). Several studies suggest that copper uptake in enterocytes requires the activity of the high-affinity copper transporter 1 (CTR1) at the apical surface of the cells (12, 13), although other mechanisms have also been proposed (14, 15).

Once inside the enterocyte, copper must traverse the basolateral membrane to enter the bloodstream. This export is mediated by the copper-transporting ATPase ATP7A, which traffics from the trans-Golgi network (TGN) to the basolateral membrane to release copper into the portal circulation (2, 11, 16, 17) (Figure 1, blue arrow). Following intestinal absorption, most copper is delivered to the liver, where polarized hepatocytes orchestrate systemic copper distribution and detoxification. Uptake of circulating copper occurs mainly through CTR1. Copper import through CTR1 and its subsequent intracellular distribution are facilitated by the cytosolic tripeptide glutathione [γ-Glu-Cys-Gly(GSH)] (18). Within hepatocytes, imported copper is either stored in GSH- or metallothionein-bound form, or delivered to specific targets via copper chaperones (3, 11, 16). One such chaperone, ATOX1, transfers copper to the P-type ATPase ATP7B (19, 20), which plays a pivotal role in systemic copper homeostasis. In addition to ATOX1, both GSH and glutaredoxin1 have been shown to contribute to copper transfer to ATP7B (21). Under basal conditions, ATP7B resides in the TGN, where it transfers copper into the secretory pathway for incorporation into newly synthesized ceruloplasmin, the major circulating copper-binding protein (11, 17, 22, 23). Secretion of copper-loaded ceruloplasmin into the bloodstream supplies peripheral tissues and organs (23), where copper supports essential processes such as mitochondrial respiration, neurotransmitter synthesis, tyrosine metabolism, redox balance, and extracellular matrix remodeling. When hepatocellular copper levels rise, ATP7B relocalizes from the Golgi to endo-lysosomal compartments and ultimately to the apical canalicular membrane (Figure 1, orange arrows), where it facilitates copper excretion into bile (24, 25). In this way, ATP7B enables the liver to distribute copper to the body and eliminate excess via biliary secretion and fecal excretion.

Inherited Disorders of Copper Metabolism

The importance of physiological mechanisms regulating copper balance is underscored by severe genetic disorders that result in either systemic copper deficiency or toxic overload (26, 27).

Copper deficiency is most prominently associated with Menkes disease, an X-linked disorder caused by mutations in ATP7A. Loss of ATP7A function leads to entrapment of dietary copper in the intestine and impaired delivery to peripheral tissues and the brain. Consequently, patients exhibit profound developmental and neurological deficits. Additional symptoms arise from impaired activity of copper-dependent enzymes, including connective tissue abnormalities, hypopigmentation, and vascular defects such as twisted blood vessels (26–28). A significant proportion of affected individuals die in early childhood. Beyond classic Menkes disease, certain ATP7A mutations give rise to milder phenotypes, such as distal motor neuron myelopathy and occipital horn syndrome (28). Usually, milder phenotypes are associated with residual ATP7A activity (28–30). For example, in occipital horn syndrome, splice-site mutations result in a small but measurable amount (3–5%) of correctly spliced, wild-type ATP7A transcript, compared with less than 0.5% in fibroblasts from Menkes disease patients missing the same exon in ATP7A (30). Thus, the mild occipital horn syndrome phenotype arises from residual expression of functional ATP7A, sufficient to maintain limited copper transport activity. These results highlight the complexity of correlating specific ATP7A mutations with clinical outcomes and underscore the difficulty of predicting disease severity solely based on genotype.

More recently, another inherited disorder associated with severe copper deficiency has been identified, caused by mutations in the principal copper influx transporter CTR1 (SLC31A1) (31, 32). A homozygous missense variant in CTR1 (p.Arg95His) was reported in infants with profound neurodevelopmental impairment, including intractable seizures, severe developmental delay, and rapid neurodegeneration characterized by cerebral and cerebellar atrophy exceeding that typically observed in untreated Menkes disease (31). Functional studies demonstrated reduced intracellular copper levels and impaired mitochondrial function in patient-derived samples, highlighting the essential role of CTR1-mediated copper uptake in sustaining mitochondrial energy metabolism. Transcriptomic analyses of human fetal brain and cerebral organoids further revealed high CTR1 expression in excitatory neurons and radial glial cells, suggesting that these populations are particularly vulnerable to disrupted copper influx (31). The overlapping phenotypes of CTR1 deficiency and Menkes disease emphasize the critical importance of adequate copper supply for human development and survival.

Copper overload also has devastating consequences, most prominently manifested in Wilson disease. This autosomal recessive disorder is caused by mutations in ATP7B, which is predominantly expressed in the liver (33–35). Loss of ATP7B function prevents hepatocytes from transporting copper into the Golgi and secretory pathway or excreting it into bile (11, 22, 33). As a result, copper accumulates in the liver, leading to severe hepatotoxicity. Hepatocellular injury and increased circulating copper subsequently promote copper deposition in extrahepatic tissues, particularly the brain(33–35). Most patients present with hepatic manifestations, including hepatitis, fibrosis, and cirrhosis, which can culminate in acute liver failure. A substantial proportion of patients also exhibit neurological symptoms, such as tremor, dystonia, parkinsonism, rigidity, and bradykinesia (34, 35). The cellular mechanisms underlying copper toxicity include oxidative damage, disruption of mitochondrial Fe–S clusters, and cuproptosis (33, 36, 37). If untreated, the disease is fatal; survival depends on timely copper-chelation therapy or, in chelator-resistant cases, liver transplantation (33–35).

Aceruloplasminemia is another inherited disorder of copper metabolism, caused by mutations in the ceruloplasmin gene. Ceruloplasmin is a copper-containing ferroxidase synthesized primarily in the liver. After translation, the protein undergoes a maturation process involving the ATP7B-mediated incorporation of copper atoms (38), which are essential for its proper folding, stability, and enzymatic activity (39–41). Impaired copper loading or structural defects in the protein result in nonfunctional ceruloplasmin that is rapidly degraded (40, 41). The absence of active ceruloplasmin leads to decreased serum copper levels and disrupts iron metabolism, causing iron accumulation in tissues, particularly in the brain, liver, and pancreas, causing neurodegeneration, diabetes, and retinal degeneration (42).

Finally, significant abnormalities of copper metabolism have been reported in MEDNIK and KIDAR syndromes (43–45). These disorders are not caused by mutations in copper transporters themselves, but rather by mutations in genes encoding components of the adaptor protein complex 1 (AP-1), which regulates the sorting and trafficking of ATP7A and ATP7B. Indeed, defects in ATP7A/B trafficking and localization have been observed upon AP-1 dysfunction (see below). This highlights that copper homeostasis depends not only on the transporters themselves, but also on the cellular machinery that regulates their localization, trafficking, and function.

Copper Uptake Into The Cells

CTR1: The Core High-Affinity Pathway

Cellular copper levels are precisely regulated by specialized protein transport systems that adapt to both copper-deficient and copper-rich conditions. Copper enters cells primarily via the high-affinity copper transporter SLC31A1, commonly known as CTR1 (Figure 2). The essential role of CTR1 in copper uptake is highlighted by the embryonic lethality observed in mice lacking this transporter (46, 47) and by the severe neurological manifestations reported in humans carrying CTR1 mutations (31, 32).

Figure 2. Copper uptake, distribution and export cellular pathways.

The figure schematically shows main copper (Cu) transport pathways within the cell. Cu transport across cellular membranes is shown by black arrows. Other thin solid lines indicate transport through endocytic or biosynthetic pathways. Thick gray arrow shows sequestration by metallothioneins (MTs). Thick pink arrow indicates Cu-dependent activation of proliferation. See main text for details.

However, the role of CTR1 in regulating intestinal copper absorption remains controversial. Dietary copper absorption occurs predominantly in the duodenum, where Ctr1 has been detected at the apical membrane of enterocytes, suggesting its possible involvement in copper uptake from the gut lumen (13). This role is further supported by studies using tissue-specific knockout mice, in which conditional deletion of Ctr1 in the intestinal epithelium significantly impairs systemic copper uptake, leading to marked copper deficiency in peripheral tissues (12). Notably, this genetic ablation does not prevent copper accumulation within enterocytes, indicating that CTR1 is not solely responsible for apical copper uptake and suggesting the involvement of additional, possibly compensatory, transport mechanisms at the luminal surface.

Other studies suggest that apical membrane localization of CTR1 is largely restricted to neonatal mice whereas in adults, CTR1 exhibits a predominantly intracellular or even basolateral localization (15, 48). These findings suggest that CTR1 physiological role is not in direct luminal uptake but rather in importing copper from the bloodstream, implying that intestinal copper absorption requires an additional, yet-unidentified apical Cu²⁺ reduction and uptake pathway.

Structurally, CTR1 functions as a homotrimeric membrane complex, forming a central pore through which copper ions are translocated. Each subunit includes an extracellular N-terminal region containing with a total of 57 potential copper-binding residues in the trimer, suggesting that this extracellular domain might be able to capture copper from the environment and to deliver it directly toward the transmembrane transport channel. Furthermore, this trimeric conformation is important for the reduction of Cu²⁺ to Cu⁺(49) establishing CTR1 as the principal high-affinity copper influx transporter in mammalian cells.

Recent study elucidated the mechanism by which CTR1 interacts with both Cu²⁺ and Cu⁺ species (50). It showed that histidine–methionine clusters within the extracellular N-terminal domain transiently coordinate Cu²⁺ ions and promote their reduction to Cu⁺ before translocation through the channel. Methionine residues are strategically positioned around the entrance to the Cu translocation channel in CTR1 and are thought to function as a selectivity gate for Cu⁺. While CTR1 does not transport Cu²⁺ directly, it can take up Ag⁺, a monovalent metal ion similar in size and electronic configuration to Cu⁺, which competes for uptake and inhibits copper entry (50). This specificity suggests a finely tuned selectivity filter that favors monovalent copper ions. The process of Cu²⁺ reduction to Cu⁺ not only facilitates metal uptake but also triggers copper-dependent endocytosis of CTR1, serving as an autoregulatory mechanism that adjusts transporter availability according to extracellular copper levels (50).

It has been proposed that members of the STEAP family of metalloreductases assist in converting extracellular Cu²⁺ to Cu⁺, thereby enabling CTR1-mediated uptake (51). However, no definitive structural or biochemical interaction between CTR1 and STEAP proteins has been demonstrated (51). Thus, CTR1 functions as both a redox enzyme and a transporter, integrating copper reduction and uptake within a single molecular complex.

Under physiological conditions, copper is not present in a free, unbound state—either in circulation or inside the cell. Instead, it is tightly associated with proteins and small ligands. Thus, CTR1 likely operates under a local concentration gradient created by its methionine- and histidine-rich extracellular domain, which acts as a binding site to concentrate Cu⁺ at the membrane surface (52).

Once in the cytosol, Cu⁺ is buffered by GSH and transferred to specific high-affinity protein chaperones that mediate its delivery to various intracellular destinations (18, 53). These chaperones outcompete GSH for Cu⁺ binding, forming a dynamic pool of exchangeable copper (18, 53).

CTR1 function is also regulated at the levels of both expression and localization. Acute exposure of cells to excess copper triggers endocytic internalization of CTR1, thereby reducing its presence at the plasma membrane and limiting further copper uptake that could compromise intracellular copper balance (54). In Wilson disease, which is characterized by abnormal intracellular copper accumulation, hepatocytes downregulate CTR1 expression as an adaptive mechanism to mitigate continued copper buildup and associated toxicity (55, 56).

Finally, CTR1 appears to play a role in inter-organ communication that regulates systemic copper supply. Kim and colleagues (57) described crosstalk between cardiac, intestinal, and hepatic copper homeostasis, showing that tissue-specific deletion of CTR1 in the heart or intestine disrupts systemic copper balance and accelerates copper mobilization from intestinal and hepatic stores. Although the molecular mediators of this heart–gut–liver communication remain unclear, these findings highlight that CTR1 contributes not only to cellular copper uptake but also to inter-organ copper signaling, reinforcing its role as a central regulator of systemic copper physiology.

DMT1: A Copper Entry Route Linked to Iron Metabolism

The divalent metal transporter 1 (DMT1, also known as SLC11A2), a broadly selective transporter that mediates uptake of multiple transition metals including iron and copper, has also been proposed to contribute to copper uptake (Figure 2). DMT1 can translocate Cu²⁺ across the apical membrane, although copper imported via this route requires subsequent intracellular reduction before incorporation into cuproenzymes (58). Some reports also suggest that DMT1 may transport Cu⁺ as well (59).

Functional studies have investigated potential functional interactions between DMT1 and CTR1. For example, in mice subjected to iron deficiency, DMT1 expression was upregulated at the brush-border membrane, coinciding with increased intestinal absorption of both iron and copper (59). In human endothelial and tumor cells, suppression of either CTR1 or DMT1 alone partially inhibited copper uptake, whereas simultaneous knockdown of both transporters almost completely abolished copper import (60, 61). Our findings further indicate that DMT1 supports copper accumulation in ATP7B-deficient HepG2 cells, despite significantly reduced CTR1 expression in this cell line (56). Together, these observations suggest that DMT1-mediated copper import may serve as an auxiliary or compensatory mechanism when CTR1 activity is impaired (56, 60, 61).

Non-Canonical Mechanisms of Copper Import

ZnT1: A Dual Zinc-Copper Transporter and Gatekeeper of Cuproptosis

Apart from the well-known copper transporters CTR1 and DMT1, additional transporters have recently emerged as potential mediators of copper import (Figure 2). A notable example is zinc transporter 1 (ZnT1), which has been shown to mediate Cu²⁺ entry into cells. CRISPR-based knockout and functional studies demonstrated that ZnT1 is essential for initiating cuproptosis, thereby expanding the repertoire of known copper transporters (62). Using cryo-electron microscopy (cryo-EM), the study provided evidence that ZnT1 functions as a dual Zn²⁺ and Cu²⁺ transporter. It possesses a unique inter-subunit disulfide bond, which is crucial for stabilizing the outward-open conformation of its protomers and facilitating efficient Cu²⁺ transport.

ZnT1 appears to act as a compensatory pathway when CTR1, another major copper transporter, is impaired, suggesting functional redundancy and a dynamic interplay between zinc and copper metabolism (62). This transporter has also been proposed as a critical modulator of hepatic copper loading and redistribution, where ZnT1 is highly expressed at the basolateral membrane. This function may be particularly relevant under conditions of pathological copper accumulation, such as Wilson disease.

Further research indicates that ZnT1 contributes to cuproptosis through regulation of the MTF1–MT1X axis. Specifically, ZnT1 knockout increases intracellular zinc levels, which in turn activates the MTF1 transcription factor and induces expression of Metallothionein 1X (MT1X). MT1X can buffer excess copper in mitochondria, thereby conferring resistance to cuproptosis (63). On the other hand, ZnT1 did not emerge in a genome-wide screen for targets that reduce copper toxicity in Wilson disease (56), suggesting that its involvement in copper import may be modulated by specific physiological, pathological, or cell-specific contexts.

LAT1/SLC7A5 and Cu-Histidine Complex Transport

A novel and clinically significant pathway for copper uptake is mediated by the L-type amino acid transporter 1 (LAT1, SLC7A5), which is traditionally known for transporting large neutral amino acids. Recent studies have demonstrated that LAT1 can transport copper when complexed with histidine (Cu–His₂) (64). This mechanism is particularly relevant in therapeutic contexts such as Menkes disease, where Cu–His₂ is administered to bypass defective endogenous copper uptake pathways. LAT1-mediated transport of Cu–His₂ complexes provides an alternative route for cells to acquire bioavailable copper, potentially circumventing the need for reduction and direct uptake via transporters like CTR1. Although the quantitative contribution of LAT1 to overall copper uptake across different tissues remains to be fully elucidated, its role highlights a promising target for novel copper supplementation strategies.

CD44/Hyaluronic Acid-Mediated Endocytosis

Beyond transporter-based systems, receptor-mediated mechanisms of copper uptake are emerging. Copper complexed with hyaluronic acid can be internalized via the CD44 receptor through endocytosis, bypassing classical copper transporters (65). This pathway is particularly intriguing in cancer biology, as CD44 is a marker of cancer stem cells and is often upregulated in tumors with dysregulated copper metabolism. However, hyaluronic acid also suppresses copper efflux machinery, raising the question of whether the observed increase in intracellular copper reflects truly enhanced uptake or reduced export (65). This mechanism may represent a cell-type-specific adaptation relevant to actively proliferating or stem-like cells.

Endocytic Copper Uptake via Prion Protein

The cellular prion protein (PrP) contains N-terminal octapeptide repeats capable of binding multiple Cu²⁺ ions with high affinity and mediating their internalization via endocytosis (66). Binding of copper induces conformational changes in PrP (67) that favor its uptake through both clathrin-dependent and clathrin-independent pathways (68, 69). Notably, disruption of the copper-binding domains markedly impairs PrP endocytosis (56).

The role of PrP as an endocytic copper receptor has been recognized for decades (66, 68, 69). However, its physiological and pathological significance in copper uptake remained unclear until recently. A study from our laboratory identified PrP as a critical regulator of hepatic copper uptake and a novel driver of copper-induced toxicity in Wilson disease (56). In ATP7B-deficient hepatocytes, impaired copper export capacity triggers transcriptional upregulation of PRNP, leading to enhanced PrP-mediated copper endocytosis and severe intracellular copper overload (56). This creates a self-amplifying loop in which elevated copper levels activate the metal-responsive transcription factor MTF1 (56, 70), which further induces PRNP expression, exacerbating copper accumulation and hepatotoxicity. As noted above, in this setting CTR1 expression is downregulated, suggesting that PrP-driven copper endocytosis supplants the canonical high-affinity Cu⁺ uptake pathway as the dominant copper entry route in Wilson disease hepatocytes (56).

Genetic suppression of PrP in Atp7b⁻/⁻ mice markedly reduces hepatic copper content, alleviates mitochondrial damage, limits fibrosis, and improves survival, identifying PrP as a promising therapeutic target to complement conventional chelation therapy (56). Moreover, PrP suppression in these mice was associated with a significant reduction in cholangiocarcinoma formation, underscoring its impact on copper-related tumorigenesis (56).

Mechanistically, PrP-mediated copper internalization appears to converge with established copper-handling pathways. Progressive acidification along the endocytic route reduces PrP’s copper-binding affinity (67), releasing copper ions for subsequent reduction by STEAP metalloreductases and translocation across the endosomal membrane via DMT1 (59, 71). Consistent with this mechanism, both STEAP proteins and DMT1 have been shown to potentiate copper toxicity in ATP7B-deficient cells (56).

In sum, recent studies reveal an expanding array of cellular mechanisms driving copper import. These mechanisms are no longer limited to the well-known CTR1- and DMT1-mediated pathways, but also include novel transmembrane transporters and endocytic copper receptors. Under normal conditions, CTR1 likely serves as the principal high-affinity copper importer. However, in scenarios where additional copper is required for physiological needs or CTR1 function is impaired—due to developmental, pathological, or cell-type-specific contexts—alternative pathways can become more prominent. Elevated expression of copper transporters and endocytic receptors operating in lower-affinity pathways can still sustain significant copper influx circumventing CTR1. Collectively, the diversity and abundance of these uptake routes confer cellular plasticity, enabling precise regulation of copper import tailored to specific physiological and pathological conditions.

Intracellular Copper Distribution

Upon entry via membrane transporters such as CTR1 or DMT1, copper must be distributed from the entry sites to various intracellular destinations, including cytosolic proteins as well as more distant compartments such as mitochondria and organelles of the biosynthetic pathway. Copper distribution to these destinations represents a logistical challenge (Figure 2).

First, owing to its redox activity, copper is potentially toxic and therefore must be tightly regulated within the cell. As a result, free ionic copper is virtually absent from the cytosol (72). Instead, copper is managed by a molecular network that mitigates its potential toxicity. One of the key components of this network is GSH. The small size of GSH allows it to bind copper within the CTR1 cavity, thereby preventing uncontrolled copper release into the cytosol (18). Due to the relatively low affinity of Cu⁺ binding to GSH (73), GSH-bound copper constitutes a labile pool that can be readily exchanged with other copper-binding proteins to ensure safe intracellular delivery or storage (1, 3).

Second, the physical separation between membrane importers and copper-dependent enzymes localized in organelles precludes simple protein–protein handoffs. In this context, chaperone proteins—such as ATOX1, CCS, and COX17—play critical roles in intracellular copper trafficking. ATOX1 ferries copper to the Golgi (19, 20), CCS delivers copper to SOD1 (74, 75) and to certain signaling molecules such as MEK (76), and COX17 transports copper to the mitochondria (77).

However, the mechanisms by which these chaperones navigate the cytoplasmic space remain incompletely understood. While passive diffusion can be considered a baseline mechanism, the spatial separation between copper entry points and utilization sites suggests that additional layers of regulation are required for vectorial transport. In this context, copper-binding sites have been reported in molecular motors such as dynactin (78), which may enable motor-mediated transport of copper-bound cargoes—including proteins, their complexes, or even organelles—over long distances along cytoskeletal highways such as microtubules.

Another possible mechanism for copper transport between compartments involves inter-organelle contact sites. Several organelles have been shown to establish physical contacts with one another (79), and such interactions could facilitate targeted copper delivery—particularly to high-demand compartments such as the mitochondria and Golgi apparatus (see below).

Copper in the Mitochondria

Mitochondria are among the primary consumers of intracellular copper due to the metal’s essential role in cytochrome c oxidase (COX), the terminal complex of the electron transport chain (80, 81). COX subunit IV (COX-IV), located deep within the mitochondrial inner membrane, has high affinity for copper and represents a major sink for intracellular copper (80–82). Accordingly, mitochondrial copper levels must be tightly regulated: both deficiency and overload are deleterious.

In Menkes disease, copper fails to reach the mitochondria of target tissues—including the brain—due to systemic deficiency and intracellular misdistribution(83). This leads to COX dysfunction and mitochondrial abnormalities characterized by reduced ATP production, further leading to hypotonia and neurodevelopmental delay (84). The neurological symptoms of Menkes—seizures, spasticity, and neurodegeneration—are at least partly due to defective mitochondrial respiration in the central nervous system, where COX activity is critical for maintaining neuronal energy metabolism(83). Mitochondrial pathology has been observed in brain biopsies from Menkes patients and in ATP7A-deficient mouse models (84). Mitochondrial dysfunction in a mouse model of Menkes disease can be counteracted by the copper ionophore elesclomol, which delivers copper to mitochondria (85).

Copper overload - most dramatically seen in Wilson disease - is also catastrophic for mitochondrial function (Figure 3 A, B). Due to the presence of numerous high-affinity copper-binding proteins and redox centers, mitochondria often act as early intracellular accumulators of copper during overload (86–88). Copper-induced mitochondrial damage in Wilson disease includes: swelling of the cristae and intermembrane space (see examples in Figure 3B), collapse of mitochondrial membrane potential, release of pro-apoptotic factors such as cytochrome c, increased reactive oxygen species (ROS) production (55, 86–88). In extreme cases, excess copper induces cuproptosis, a recently characterized form of regulated cell death in which copper binds directly to lipoylated components of the tricarboxylic acid (TCA) cycle, leading to aggregation of these enzymes, destabilization of Fe–S cluster proteins, and proteotoxic stress (37).

Figure 3. Impact of copper accumulation on mitochondria in animal model of Wilson disease.

Electron microscopy images of liver tissue from Atp7b+/- and Atp7b-/- mice are shown. Normal mitochondria are present in the liver of Atp7b+/- animals (A, white arrows). Mice lacking ATP7B exhibit abnormal mitochondria (B, black arrowheads). Additionally, mitophagy is activated in Atp7b-/- mice (B, black arrowheads). Scale bar: 600 nm.

Given the critical need for balanced copper delivery, precise mechanisms must exist to guide copper from the influx sites at the plasma membrane or endocytic compartments to the mitochondrial inner membrane. Yet, this pathway remains incompletely resolved.

Once in the cytosol, copper is believed to be handed off to the mitochondrial chaperone COX17, which shuttles copper into the intermembrane space (77) (Figure 2; green dash line). From there, it is transferred to Sco1, Sco2, and Cox11, which metallate the copper-binding subunits of COX (80). However, there is currently no known direct interaction between COX17 and copper importers like CTR1 or DMT1, leaving unclear how COX17 acquires newly imported copper from the cytosolic copper pool.

Further complexity arises in the delivery of copper across the inner mitochondrial membrane into the matrix. A key transporter implicated here is SLC25A3 (Figure 2), originally identified as a phosphate carrier but recently shown to mediate copper import into the matrix for COX metallation (89).

Another candidate is SLC25A37 (mitoferrin-1), traditionally recognized for iron import but possibly promiscuous under pathological conditions. In genome-wide shRNA screen, SLC25A37 was identified as a genetic modifier of copper toxicity in Wilson disease models (56), suggesting a previously unappreciated role in copper transport within mitochondria under stress conditions. These findings imply that SLC25A37 may contribute to mitochondrial copper overload in disease states, either by directly transporting copper or by facilitating redox interactions that enhance copper entry. Whether SLC25A37 is physiologically relevant for normal copper homeostasis or mainly involved under stress conditions remains an open question.

The Secretory Pathway

The secretory pathway is a major intracellular route for copper utilization, primarily serving a biosynthetic function—delivering copper to enzymes synthesized and processed within the TGN. These copper-dependent enzymes are involved in key physiological processes, including extracellular matrix remodeling, pigmentation, iron homeostasis, and neurodevelopment (2, 3). Copper delivery to this compartment is mediated by ATP7A and ATP7B (2, 3, 11), which receive copper from the cytosolic chaperone ATOX1 (19, 20) (Figure 2, dash blue arrows). ATOX1, in turn, is loaded by the plasma membrane copper importer CTR1 (90, 91).

Although ATP7A and ATP7B also contribute to copper homeostasis—relocating to post-Golgi compartments and the plasma membrane to export excess copper under high-copper conditions (Figure 2, blue arrows) —their role in biosynthetic copper delivery within the secretory pathway is particularly critical (2, 11, 17, 22). In this context, they incorporate copper into enzymes during their maturation in the Golgi apparatus. Failure of this function leads to a broad spectrum of biochemical and systemic abnormalities.

In Menkes disease, mutations in ATP7A severely impair copper delivery to the secretory pathway. The resulting absence of copper loading into key enzymes has devastating effects across multiple tissues (2, 11, 26). Enzymes most critically affected by ATP7A loss include peptidylglycine α-amidating monooxygenase (PAM), β-hydroxylase (DBH), tyrosinase, and lysyl oxidase (LOX) (92–95). Their dysfunction contributes respectively to neurodevelopmental delay, autonomic dysregulation, hypopigmentation, and connective tissue abnormalities like vascular fragility and joint laxity (28, 84).

By contrast, Wilson disease results from mutations in ATP7B, with the main biosynthetic defect involving impaired copper incorporation into ceruloplasmin, a multi-copper ferroxidase synthesized in hepatocytes and secreted into plasma (23). Under normal conditions, ATP7B loads copper into apoceruloplasmin within the TGN, stabilizing the protein and enabling its enzymatic activity. In the absence of functional ATP7B, ceruloplasmin is unstable and rapidly degraded, leading to markedly reduced serum ceruloplasmin levels (23, 33). The loss of ceruloplasmin’s ferroxidase activity disrupts iron homeostasis by impairing Fe²⁺ oxidation and transferrin loading, contributing to hepatic iron accumulation alongside copper and thus promoting oxidative stress and liver damage (23, 33). Furthermore, the failure to mobilize copper into the bloodstream exacerbates hepatic copper retention and toxicity.

Together, these disorders highlight the essential biosynthetic roles of ATP7A and ATP7B in delivering copper to enzymes within the Golgi. Their dysfunction not only alters copper distribution but also disrupts key metabolic pathways, demonstrating the broader physiological consequences of impaired copper incorporation in the secretory pathway.

In contrast to the Golgi apparatus, whether and how another major compartment of the biosynthetic pathway—the endoplasmic reticulum (ER)—receives copper remains unclear, as does its potential role in maintaining copper homeostasis. It is known that ER quality control machinery is involved in the retention and degradation of various ATP7B mutants, including the most common H1069Q and R778L variants (96, 97). ER-mediated degradation of these misfolded mutants leads to copper accumulation and toxicity in Wilson disease (96, 97). Interestingly, the ER-retained G875R mutant of ATP7B is able to escape from the ER and traffic to the TGN in copper-treated cells (98). This indicates that copper facilitates the proper folding of this ATP7B variant in the ER. However, under physiological conditions, knowledge about ER-resident proteins that utilize copper is very limited.

Recent studies suggest that sulfatase-modifying factor 1 (SUMF1), also known as formylglycine-generating enzyme, uses copper as a cofactor (99, 100). (Figure 2 violet arrow) SUMF1 is a key ER-resident protein responsible for the post-translational activation of sulfatases by oxidizing a specific cysteine (or serine) residue to Cα-formylglycine (FGly), a modification essential for the enzymatic activity of lysosomal sulfatases. Mutations in SUMF1 cause multiple sulfatase deficiency (MSD), a severe lysosomal storage disorder (101). Two MSD-causing mutations, N259I and A279V, completely abolish SUMF1 activity (102) and are located near copper-binding cysteines at positions 269 and 274 in the enzyme’s active site (103), strongly suggesting that copper is critical for SUMF1 function. However, how ER-localized SUMF1 acquires copper remains unknown (Figure 2, dashed violet arrow).

One hypothesis is that SUMF1 transiently passes through the Golgi, where it may acquire copper from ATP7A or ATP7B. SUMF1 has been shown to travel from the ER to the Golgi, but it is subsequently retrieved back to the ER through a mechanism that involves recognition of its N-terminal RDEL motif (104). However, MSD-like features have not been observed in patients with Menkes or Wilson disease, suggesting that copper delivery to SUMF1 is likely not mediated by ATP7A or ATP7B in the Golgi, but rather by a yet-to-be-identified ER-localized transporter. Therefore, elucidating the molecular mechanisms of copper delivery to SUMF1 could provide critical insights into how copper is supplied to the ER and the importance of this process in the pathophysiology of MSD.

Copper in the endo-lysosomal and autophagic compartments

The organelles of the endo-lysosomal system represent an important hub for copper sorting and storage (105). Copper may enter this system through endocytosis when bound to transporters such as CTR1, or receptors such as PrP or CD44 (54, 56, 65, 66). How far endocytosed copper penetrates along the endocytic route remains unclear. It appears that PrP may carry copper into late endosomal compartments (56), where the acidic pH promotes its dissociation from the receptor (Figure 2, purple arrows).

Another major pathway of copper entry into the endo-lysosomal system involves transmembrane transport by ATP7B. In hepatocytes exposed to excess copper, ATP7B traffics to endo-lysosomal organelles, where it sequesters copper into their lumen (24) (Figure 2, blue arrows). This mechanism is likely not limited to hepatic tissue, as ATP7B-positive vacuolar organelles have also been identified in the intestine, where they appear to contribute to copper storage (106).

A key unresolved question is whether copper in endo-lysosomal compartments is merely stored or also delivered to specific recipient proteins to support their function. It is not clear whether lysosomes contain dedicated copper-sequestering proteins, analogous to metallothioneins. Among lysosomal enzymes, acid sphingomyelinase requires copper as a cofactor (107). Notably, analyses of lysosomes in ATP7B-deficient cells have revealed major alterations, including accumulation of undigested material and reduced degradative capacity, possibly due to dysfunction of enzymes such as acid sphingomyelinase (55). This phenotype resembles a mild lysosomal storage disorder and underscores the role of ATP7B-mediated copper supply in sustaining lysosomal function in the liver. Why lysosomal dysfunction remains relatively mild in the absence of ATP7B-mediated copper delivery is not fully understood. One possible explanation is that the transporter SLC46A3 can provide an alternative source of lysosomal copper (108), at least partially compensating for the loss of ATP7B. SLC46A3 may also serve as a primary lysosomal copper supplier in tissues and cell types where ATP7B is expressed at low levels.

Copper may also reach the lysosomal compartment via autophagy (Figure 2, teal arrows). The autophagic pathway is particularly relevant under conditions of copper overload, as it protects cells from copper-mediated toxicity by sequestering damaged components (55). Indeed, autophagy is activated in Wilson disease as a consequence of ATP7B deficiency and copper accumulation, which primarily damages mitochondria (86–88). Sequestration of such compromised mitochondria into autophagosomes (Figure 3B, arrowhead) protects the cell from the release of pro-apoptotic factors. Autophagosomes may also capture other cellular components aberrantly bound to copper (55). Subsequent fusion of these autophagosomes with lysosomes delivers copper for sequestration in the lysosomal compartment (Figure 2, teal arrows). Elevated lysosomal copper levels have been documented in both cellular and animal models of Wilson disease (55). Autophagy of copper-containing material may also occur under physiological conditions, as copper-bound metallothioneins sequestered by autophagy have been detected in lysosomes (109). However, whether a specific copper-directed autophagic pathway (“cuprophagy”), analogous to ferritin-specific autophagy (“ferritinophagy”), exists remains unclear and requires further investigation.

How copper activates autophagy is an intriguing question. Our studies in a Wilson disease cell model indicate that elevated copper stimulates nuclear translocation of TFEB, the master transcriptional regulator of autophagy genes, thereby inducing their transactivation(55). This is accompanied by reduced mTOR activity, which normally inhibits TFEB via phosphorylation (55). An additional copper-dependent mechanism involves ULK1 and ULK2, key kinases driving autophagy initiation (Figure 2 teal arrows). An elegant study from Brady’s lab demonstrated that copper can directly bind to ULK1/2 and allosterically activate them, thereby, stimulating biogenesis of the autophagic structures (5).

Thus, the endo-lysosomal and autophagic pathways not only function as key hubs for copper storage, detoxification, and distribution, but are also themselves regulated by copper, underscoring a reciprocal relationship that is critical for cellular homeostasis and highly relevant in disorders such as Wilson disease.

Copper Transport to the Nucleus

The physiological roles of copper within the nucleus remain incompletely understood, yet emerging evidence suggests that this compartment participates in copper-dependent regulatory processes. Some nuclear proteins have been identified as potential copper-binding targets. For example, the histone H3-H4 tetramer has been proposed to possess copper reductase activity, implicating chromatin components in nuclear copper handling (110). Moreover, nuclear accumulation of copper has been observed in hepatic cells of Atp7b^-/- mice, where it correlates with marked alterations in gene expression profiles (111). These findings indicate that perturbations in nuclear copper homeostasis may have a direct impact on transcriptional regulation.

The delivery of copper to the nucleus has been primarily attributed to the copper chaperone ATOX1, which is classically known for shuttling Cu(I) to the ATP7A and ATP7B transporters in the secretory pathway (see above). Beyond this canonical role, ATOX1 has been increasingly recognized as a potential regulator of gene transcription. Several studies have documented its nuclear localization (112, 113). It was proposed that ATOX1 might act as a transcription factor (112), although in vitro assays failed to demonstrate direct binding of ATOX1 to DNA (113).

A yeast two-hybrid screen for ATOX1 interactors revealed that sveral binding partners are proteins associated with DNA or RNA (114), indicating that ATOX1 may influence gene expression through protein–protein interactions. Interestingly, several of these ATOX1-interacting proteins contain CXXC motifs, suggesting that their interaction with ATOX1 is likely to be copper-mediated (114). Overexpression of nuclear-targeted ATOX1 leads to widespread alterations in transcript abundance (115), supporting a role for ATOX1 in modulating transcriptional activity. Collectively, these observations point to a model in which copper, delivered by ATOX1, participates in nuclear transcriptional regulation through metal-dependent protein networks.

Copper Supply to Cytosolic Proteins and Signaling Networks

Cytosol is the first cellular environment encountered by copper once it is imported across the plasma membrane or released from the endo-lysosomal system. From there, copper can either be redistributed to other intracellular compartments (see above) or retained in the cytosol. The cytosolic fates of copper are diverse. First, copper can be targeted to specific proteins - such as the antioxidant enzyme superoxide dismutase 1 (SOD1) - to support their catalytic activity (74, 75) (Figure 2, dashed orange arrows) Second, surplus copper can be sequestered by metallothioneins (Figure 2, solid grey arrow) or complexed with GSH, thereby buffering potentially toxic levels of the metal (3, 116). Finally, emerging evidence suggests that copper itself may act as a signaling cue, directly modulating the activity of critical intracellular pathways (4–6, 117). While the first two modes of cytosolic copper handling have been extensively characterized (3, 116), the signaling roles of copper are only beginning to be elucidated (4–6, 117).

MAP kinase signaling, proliferation, and stress response

Recent work has uncovered a direct requirement for copper in the regulation of the MAPK/ERK signaling cascade (Figure 2 pink arrows). MEK1 and MEK2, the dual-specificity kinases that activate ERK1/2, bind copper, which stimulates their catalytic activity (4, 76). Depletion of intracellular copper impairs MEK–ERK signaling, leading to reduced ERK phosphorylation and downstream transcriptional activity, thereby blunting proliferative responses to growth factor stimulation. Conversely, increasing copper availability enhances ERK pathway activity, linking cellular copper status to mitogenic signaling and cell proliferation (4, 76).

Copper also modulates stress-responsive MAPK pathways. Findings from our laboratory revealed that pathological copper accumulation, as occurs in hepatocyte-like cells carrying ATP7B mutations, aberrantly activates the stress-responsive kinases p38 and JNK, contributing to oxidative stress, mitochondrial dysfunction, and hepatocellular injury (118, 119). Importantly, pharmacological or genetic interventions that lower cytosolic copper restore MAPK activity to baseline, highlighting that copper overload is a proximal trigger of stress signaling (118, 119). Together, these studies position copper as a critical modulator of MAPK signaling, linking physiological copper availability to proliferative cues while implicating copper overload in stress kinase activation and Wilson disease pathogenesis.

Autophagy-related signaling

Beyond its role in proliferation, copper regulates signaling networks controlling autophagy through multiple mechanisms (Figure 2 teal arrows). ULK1 and ULK2, the serine/threonine kinases orchestrating autophagy initiation (120), require copper binding for their activation. Under conditions of copper deficiency, ULK1/2 activity is attenuated, leading to impaired autophagic flux and reduced cellular capacity to adapt to stress or nutrient deprivation. Restoration of copper levels reinstates ULK1/2 activity and promotes autophagy induction (5).

Copper also modulates autophagy via the mTOR–TFEB axis. Under nutrient-rich conditions, mTOR phosphorylates TFEB, a master regulator of the autophagy–lysosomal gene network, retaining it in the cytoplasm and preventing autophagy induction (121). In ATP7B-deficient cells, pathological copper accumulation promotes TFEB nuclear translocation and activation of its transcriptional program, in part through copper-dependent inhibition of mTOR (55). As a result, autophagy is upregulated and functions as a protective mechanism in Wilson disease, enabling ATP7B-deficient hepatocytes to counteract copper-induced toxicity (55). Notably, high-calorie diets, which sustain mTOR activity and suppress autophagy (121), exacerbate disease phenotypes in Wilson disease animal models (122), indicating that diet and lifestyle may critically influence copper-dependent physiological and pathogenic mechanisms. This copper dependence adds an additional layer of regulation to the autophagy pathway, positioning copper as a key factor in balancing cell survival, stress adaptation, and metabolic homeostasis.

Phosphodiesterase regulation and cAMP signaling

Chris Chang’s laboratory demonstrated a direct role of copper in cyclic nucleotide signaling through inhibition of phosphodiesterase 3B (PDE3B) (6). Using mouse models of altered copper homeostasis, pharmacological modulation of copper levels, and imaging studies in 3T3-L1 adipocytes, they showed that copper elevation suppresses PDE3B activity, leading to increased intracellular cAMP concentrations and enhanced lipolysis. Biochemical analyses identified a conserved cysteine residue within a PDE3-specific loop as critical for copper-dependent inhibition (6). These findings highlight copper as an endogenous regulator of second messenger signaling, extending its biological role beyond enzyme cofactors to include dynamic control of metabolic processes.

PD-L1 regulation and immune evasion

Copper also regulates immune checkpoint molecules. Intracellular copper availability influences the expression of programmed death-ligand 1 (PD-L1), a key mediator of tumor immune evasion (7). Mechanistically, elevated copper promotes the activation of redox-sensitive signaling cascades, including STAT3 and NF-κB, both known transcriptional drivers of PD-L1 expression. Increased copper availability upregulates PD-L1 at the cell surface, enabling tumor cells to suppress cytotoxic T-cell activity, whereas copper chelation or reduced copper uptake diminishes PD-L1 expression and restores anti-tumor immune responses (7). These findings highlight a novel signaling role for copper in shaping the tumor–immune interface and suggest that targeting copper homeostasis could synergize with immune checkpoint blockade therapies.

Although these findings underscore the novel role of copper as an important signaling molecule, many questions remain. Until recently, only a few signaling components—such as MEK1/2, ULK1/2, and PDE3B—have been shown to bind copper directly (4–6). Future efforts should focus on identifying additional copper-dependent signaling proteins and elucidating the mechanisms by which copper regulates them, whether allosterically or via substrate-binding sites. Another critical question concerns the delivery of copper to cytosolic signaling proteins—that is, which chaperones shuttle copper from entry sites to specific targets. For example, CCS transfers copper to MEK1 (76), whereas the carriers responsible for delivering copper to ULK1/2 or PDE3B remain unknown.

Together, these studies establish copper as a versatile intracellular regulator, modulating kinase signaling, autophagy, second messenger pathways, and immune checkpoints. By integrating copper availability into multiple cellular signaling networks, cells can coordinate growth, metabolism, stress responses, and immune interactions, highlighting the centrality of copper in both physiological and pathological contexts.

Copper in Inter-Organelle Communication

Copper trafficking between organelles can occur via direct or indirect mechanisms. Indirect mechanisms involve one organelle influencing copper availability to others. A clear example is the Golgi apparatus, where ATP7A and ATP7B utilize copper for incorporation into newly synthesized proteins or facilitate its excretion/sequestration. In Wilson disease, loss of ATP7B compromises the Golgi ability to transport copper into the secretory pathway and out of the cell. Consequently, copper accumulation in the cell disrupts the homeostasis of other organelles, including mitochondria, lysosomes, and autophagosomes (55, 86, 88). Similarly, loss of ATP7A function in Golgi has been reported to affect redox balance in mitochondria (123). Another example involves mitochondrial proteins such as SCO1, which can transduce redox signals to the Golgi by regulating the mitochondria-to-cytosol partitioning of COX19 (124). Through this mechanism, mitochondria can influence the activity of ATP7A, the primary Golgi copper-transporting protein.

Direct inter-organelle copper exchange has not yet been definitively demonstrated. However, a growing body of evidence suggests that ion exchange may occur at so-called contact sites between different organelles (e.g., ER–mitochondria or ER–Golgi) (79). These sites represent areas where the distance between contacting organelles is minimal, and their membranes are tethered by specific protein complexes (79). Considering that ions such as Ca²⁺ can be transported through these contact sites, it cannot be ruled out that copper ions might be transferred between organelles in a similar manner.

Recent studies indicate that close contacts between the Golgi and lysosomes in the perinuclear region may play an important role in intracellular copper handling (125). Other potentially relevant contacts include those between endo-lysosomal organelles and mitochondria. It is tempting to speculate that when mitochondria require copper, it could be rapidly mobilized from lysosomes at these contact sites.

These sites may also contribute to the propagation of copper toxicity in the context of Wilson disease. In ATP7B-deficient cells, we observed that PrP promotes copper toxicity by accelerating its endocytosis and subsequent DMT1-mediated transport across the membranes of endo-lysosomal organelles (56). However, the mechanism by which copper is delivered to mitochondria—the primary sites of toxicity—remains unclear. One hypothesis is that copper is transferred via contact sites between endo-lysosomal organelles and mitochondria.

Understanding the role of direct inter-organelle copper exchange will require investigating how copper itself influences the formation and regulation of these contact sites under both physiological and pathological conditions.

Copper Excretion

Copper excretion plays a fundamental role in maintaining cellular and systemic copper balance. By eliminating excess intracellular copper, excretory mechanisms protect cells from copper-mediated toxicity. This process is also critical for preserving metal homeostasis, as tightly controlled intracellular copper levels are required for the proper function of numerous cuproenzymes. Moreover, efficient copper excretion prevents pathological accumulation in vital organs such as the liver and brain, thereby reducing the risk of tissue damage observed in copper-overload disorders such as Wilson disease (1–3).

Within the cell, the Golgi apparatus plays a central role in copper excretion, with the TGN serving as a crucial trafficking hub that coordinates intracellular copper distribution (16). The TGN regulates both the incorporation of copper into essential cuproenzymes and the export of excess copper, thereby linking intracellular copper handling to physiological functions as well as to the pathogenesis of genetic diseases. Two key regulators, ATP7A and ATP7B, mediate the delivery of copper ions into the Golgi lumen for biosynthetic needs, but when intracellular copper levels rise, they relocalize away from the TGN (16, 17) (Figure 2 blue arrows). This dynamic trafficking enables cells to export excess copper or sequester it within intracellular organelles for detoxification, underscoring the Golgi’s pivotal role in maintaining copper homeostasis.

ATP7B as a main driver of systemic copper excretion

At the systemic level, the liver plays a central role in copper excretion by channeling excess copper into the bile. A key element of this process is the ability of ATP7B to traffic from the Golgi apparatus where it normally resides (Figure 4A) to specialized copper excretion sites in hepatocytes (2, 11, 17, 22). Under conditions of moderate copper elevation, ATP7B relocalizes to endo-lysosomal organelles (Figure 4B), where it facilitates copper sequestration by actively transporting the metal into the lysosomal lumen (24). Its positioning on the outer lysosomal membrane allows efficient copper import from the cytosol into the lysosome interior (24), while the acidic pH of endo-lysosomal compartments enhances ATP7B’s transport activity (126). Subsequently, when intracellular copper levels decline, endo-lysosomal organelles can release copper back to the cytosol through different transporters, including CTR1, CTR2, and DMT1 (127–129), thereby resupplying the cell with copper required for essential metabolic processes.

Figure 4. Copper-mediated relocalization of ATP7B.

Distribution of endogenous ATP7B in HepG₂ cells is shown. A. Under low copper conditions, ATP7B localizes to the TGN (white arrows). B. Under high copper conditions, ATP7B redistributes to lysosomes, highlighted in green false colour (white arrows). C. Additionally, increased copper levels promote ATP7B delivery to the canalicular membrane, highlighted in blue false colour (black arrows). Scale bar, 750 nm.

If intracellular copper concentrations continue to rise and threaten cellular integrity, ATP7B-positive lysosomes migrate toward the apical (biliary/canalicular) domain of the hepatocyte surface (24), where excess copper is eliminated through two complementary mechanisms. First, lysosomal exocytosis releases copper stored in lysosomes directly into the canalicular space (24). Second, lysosomal fusion delivers ATP7B to the apical membrane of hepatocytes, enabling the direct transport of copper from the cytosol into the bile (24). Together, these mechanisms ensure the efficient removal of toxic copper from hepatic cells and safeguard systemic copper homeostasis.

Copper-dependent interaction of ATP7B with p62 (DNCT4), a subunit of the dynactin microtubule motor complex, appears to serve as a trigger for lysosomal exocytosis and subsequent copper excretion into the bile. p62 contains a copper-binding domain and associates with ATP7B in the presence of copper (78), thereby linking ATP7B-positive lysosomes to microtubules and promoting their movement toward the microtubule minus ends, which are oriented toward the canalicular surface of hepatocytes (24). Consistently, genetic suppression of p62 markedly reduces copper-dependent lysosomal exocytosis and the delivery of ATP7B to the canalicular surface in polarized hepatic cells (24).

The regulated trafficking of ATP7B from the TGN to the plasma membrane in response to copper in hepatocytes resembles another stimulus-dependent pathway: the trafficking of secretory granules in neurons and endocrine or exocrine cells (130). In both cases, cargo release occurs only upon specific physiological stimuli—copper elevation for ATP7B, and signals such as calcium influx or hormonal cues for secretory granules. Both pathways rely on tightly regulated sorting, cytoskeletal transport, and controlled fusion with the plasma membrane. This resemblance suggests that, although serving distinct biological purposes—copper detoxification in the case of ATP7B and protein secretion for granules—these trafficking routes may utilize shared regulatory modules and likely originate from an evolutionarily conserved mechanism.

Road map of ATP7B trafficking to copper excretion sites

In the past, whether ATP7B traffics through lysosomes and whether it reaches the plasma membrane has been a matter of debate. Early studies in hepatic WIF-B cells suggested that, upon copper elevation, ATP7B relocates directly to the apical membrane without transiting through an endo-lysosomal intermediate (131). By contrast, studies in another human hepatic cell line, Huh7, concluded that copper does not alter ATP7B localization, which remained confined to late endosomes and lysosomes undergoing exocytosis to eliminate excess copper (132, 133). Our own immunofluorescence and electron microscopy studies in HepG2 cells demonstrated that ATP7B can be detected both in lysosomes and at the apical membrane of hepatocytes, particularly under conditions of elevated copper stimulation (24). Subsequently, other intermediate endosomal stations for ATP7B trafficking were proposed (134, 135). This apparent discrepancy among in vitro models such as WIF-B, Huh7, and HepG2 cells likely reflects differences in cellular context, including the degree of polarization, complexity of regulatory cues, and steady-state copper levels (25). Importantly, in vivo findings in mice provide compelling evidence that copper stimulates ATP7B trafficking from the TGN to lysosomes and that subsequent lysosomal exocytosis promotes both biliary copper excretion and delivery of ATP7B to the canalicular surface of hepatocytes (24).

This finding supports a model in which ATP7B dynamically traffics to the apical membrane, complementing lysosomal exocytosis to maximize copper export efficiency (24, 105). Although plasma membrane localization of ATP7B may be transient or less abundant than its lysosomal pool, even brief presence at the canalicular surface becomes critical under conditions of high copper load, enabling hepatocytes to respond rapidly to toxic copper accumulation. This illustrates how the hepatocytes have evolved a finely tuned sensing mechanism that allows ATP7B to adjust dynamically to intracellular copper levels. By modulating its localization and activity according to cellular copper status, ATP7B ensures precise regulation—sequestering copper in lysosomes when levels are moderate and facilitating rapid export at the plasma membrane when levels become toxic (24, 105).

The trafficking of ATP7B remains a central focus in copper metabolism research. Recent findings have identified a subpopulation of lysosomes flanking the TGN, termed the TGN-proximal lysosomal compartment, as an intermediate station in ATP7B distribution. ATP7B was detected in this compartment even under basal copper conditions, suggesting that TGN-proximal lysosomes play a specific role in ATP7B function and copper homeostasis (125). One plausible hypothesis is that they may serve as early sequestration sites when intracellular copper levels rise, as their lower pH favors the copper-transporting activity of ATP7B (125). The close proximity of these lysosomes to the TGN would permit rapid ATP7B exchange between compartments, enabling the protein to switch efficiently between a copper-sequestering, homeostatic role in lysosomes and a biosynthetic role in the TGN, such as ceruloplasmin metalation. This exchange may be facilitated by transient contact zones between TGN membranes and TGN-proximal lysosomes, where ATP7B might be transferred through kiss-and-run mechanisms that allow selective protein exchange without full membrane fusion. Importantly, TGN-proximal lysosomes containing ATP7B are found exclusively in hepatic cells (125), but not in other ATP7B-expressing cell types, suggesting that hepatocytes have evolved this specialized subcompartment to accommodate their unique need for rapid switching between biosynthetic and homeostatic copper handling.

Extra-hepatic cells primarily rely on ATP7A to excrete copper (136, 137). When intracellular copper levels increase, post-Golgi membrane carriers mediate the trafficking of ATP7A to the plasma membrane, where it facilitates copper export to prevent copper-induced toxicity. This trafficking is dynamic and reversible; once copper levels return to baseline, ATP7A is retrieved to the Golgi apparatus (138). The regulation of ATP7A post-Golgi trafficking involves interactions with cytoskeletal elements, clathrin, and clathrin adaptor proteins (139, 140).

Disruption of cellular copper excretion mechanisms in genetic disorders

The ability of ATP7A and ATP7B to traffic from the Golgi to copper excretion or sequestration sites is a critical function for maintaining copper homeostasis. The importance of this function is highlighted by the severe, often fatal, genetic disorders caused by mutations in these genes. In the case of ATP7A mutations, which underlie Menkes disease, enterocytes fail to export dietary copper across the basolateral membrane—a process normally mediated by ATP7A when intracellular copper levels rise (2, 11, 17, 26, 28). Consequently, dietary copper remains trapped in the intestinal cells, leading to systemic copper deficiency.

In Wilson disease, loss-of-function mutations in ATP7B impair the excretion of copper into bile, disrupting a critical pathway for systemic copper elimination. Consequently, copper progressively accumulates in hepatocytes, eventually exceeding the liver’s storage capacity and spilling over into extrahepatic tissues, particularly the brain and kidneys. This toxic accumulation leads to chronic liver injury and neuropsychiatric manifestations (33–35).

Hepatic copper accumulation, indicative of impaired systemic copper excretion, has also been documented in MEDNIK and KIDAR syndromes (43, 45). These disorders are caused by mutations in the AP1S1 (MEDNIK) and AP1B1 (KIDAR) genes, which encode the σ1A and β1 subunits of the clathrin adaptor protein complex 1 (AP-1), respectively (43, 44, 141). AP-1 localizes at the TGN/endosome interface and recognizes specific motifs in the cytoplasmic tails of physiologically important transmembrane proteins, including ATP7A/B, Man6PR, transferrin receptor, E-cadherin, desmoplakin, and Na⁺/K⁺-ATPase in both epithelial and neuronal cells (142–145). By directing the sorting and trafficking of these proteins, AP-1 plays a crucial role in maintaining intracellular protein distribution, cellular polarity, and homeostasis (142, 143).

The pronounced impairment of copper metabolism observed in MEDNIK and KIDAR patients underscores the importance of AP-1 in trafficking copper ATPases and, consequently, in regulating copper flux toward the secretory pathway and the extracellular space (43–45). However, the precise role of AP-1 in ATP7A/B trafficking remains highly controversial. While several studies have implicated AP-1 in anterograde, copper-dependent trafficking from the TGN to the plasma membrane (45, 139, 140), others have highlighted its critical role in retrograde trafficking of ATP7A/B from endosomes back to the TGN (145, 146). Recent study in polarized MDCK cells reveal that AP-1 critically regulates the trafficking polarity of ATP7A and ATP7B (147). Under basal copper, ATP7A and ATP7B occupy distinct TGN subdomains. Upon copper elevation, ATP7A traffics from the TGN to the basolateral membrane of these cells, while ATP7B moves via recycling and apical sorting endosomes to the apical surface. Loss of AP-1 strongly inhibits retrograde transport of both transporters back to the Golgi and disrupts basolateral targeting of ATP7A. Isoform-specific analyses indicate that ubiquitous AP-1A ensures TGN retention and directional trafficking of both proteins, whereas epithelial-specific AP-1B, present in MDCK cells, mediates copper-independent apical delivery of ATP7B (147). This study underscores a specific role of different AP-1 subunits indicating that their tissue-specific expression matters for ATP7A/B trafficking and copper balance. In this context lack of detectable expression of key AP-1B subunit µ1B leaves open the possibility that trafficking mechanisms of ATP7B in hepatocytes may differ (148). Thus, further investigation is required to clarify the role of MEDNIK- and KIDAR-related AP-1 subunits in copper homeostasis, as studies using patient-derived cells have not yet provided definitive answers.

Clinically, MEDNIK and KIDAR syndromes are very similar and present with a combination of features characteristic of both Menkes disease (caused by ATP7A dysfunction) and Wilson disease (linked to ATP7B dysfunction), including neurodegeneration, liver defects, and abnormal copper and ceruloplasmin levels (43–45). Studies in fibroblasts from MEDNIK patients revealed that mutations in AP1S1 disrupt the proper localization and function of ATP7A. In particular, loss of AP1S1 function causes ATP7A to mislocalize from the Golgi to peripheral compartments even under basal copper conditions (43). Trapping in these compartments appears to reduce ATP7A’s capacity to transport copper to the secretory pathway and/or the extracellular space. Hepatic copper accumulation in MEDNIK patients (43) suggests that AP1S1 deficiency may have a similar impact on ATP7B trafficking and its ability to promote biliary copper excretion. However, analysis of hepatocyte-like cells derived from a MEDNIK patient did not reveal significant impairment of ATP7B trafficking, even though these cells exhibited copper overload consistent with liver pathology (149).

It was expected that similar ATP7A/B phenotypes would be observed in KIDAR patients, given the clinical resemblance between KIDAR and MEDNIK syndromes and the fact that both are caused by dysfunction of the same AP-1 protein complex. However, fibroblasts from AP1B1-deficient KIDAR patients retained ATP7A in the Golgi (45). Similar Golgi retention was observed for ATP7B in AP1B1-silenced HEK293 cells of kidney origin (45), leading to the conclusion that AP1B1 disruption causes copper accumulation by preventing ATP7A/B trafficking from the Golgi to copper excretion sites. Notably, ATP7B trafficking has not yet been examined in physiologically relevant hepatic cells from KIDAR patients.

The observation that AP1S1 and AP1B1 disruptions produce distinct trafficking and localization phenotypes for the same copper transporter further complicates our understanding of AP-1’s role in ATP7A/B trafficking and, consequently, in the maintenance of copper homeostasis. We propose that a detailed cell biology analysis of the compartmentalization of endogenously expressed ATP7A/B in physiologically relevant cell types is needed to elucidate how AP1S1 or AP1B1 mutations affect their trafficking. Furthermore, these studies should be complemented by an analysis of subcellular copper pools to clarify how AP-1 regulates copper excretion and overall copper homeostasis.

Concluding Remarks

The expanding view of copper biology has reshaped our understanding of this trace element from a static enzymatic cofactor into a dynamic regulator of physiology, influencing development, energy metabolism, connective tissue integrity, neural function, and immune surveillance. Yet, many questions remain unresolved. How do canonical and non-canonical uptake routes integrate to match copper supply with tissue-specific demands, and how are these circuits rewired across development, aging, and disease? What molecular machinery governs inter-organelle transfer—particularly to mitochondria, lysosomes, and the ER—and how does this scale to whole-body physiology? Which chaperones deliver copper to signaling proteins, and how does this contribute to systemic regulation of stress responses, autophagy, and immunity? Addressing these questions will require bridging structural and cellular insights with integrative physiology, spanning advanced models, multi-omics, and human studies. Such efforts promise not only to define how copper supports resilience at both cellular and organismal levels but also to unlock therapeutic strategies for disorders of copper imbalance and for conditions where copper-driven signaling is pathologically exploited, such as cancer and neurodegeneration.