Mammalian copper homeostasis: physiological roles and molecular mechanisms

Abstract

In the past decade, evidence for the numerous roles of copper (Cu) in mammalian physiology has grown exponentially. The discoveries of Cu involvement in cell signaling, autophagy, cell motility, differentiation, and regulated cell death (cuproptosis) have markedly extended the list of already known functions of Cu, such as a cofactor of essential metabolic enzymes, a protein structural component, and a regulator of protein trafficking. Novel and unexpected functions of Cu transporting proteins and enzymes have been identified, and new disorders of Cu homeostasis have been described. Significant progress has been made in the mechanistic studies of two classic disorders of Cu metabolism, Menkes disease and Wilson's disease, which paved the way for novel approaches to their treatment. The discovery of cuproptosis and the role of Cu in cell metastatic growth have markedly increased interest in targeting Cu homeostatic pathways to treat cancer. In this review, we summarize the established concepts in the field of mammalian Cu physiology and discuss how new discoveries of the past decade expand and modify these concepts. The roles of Cu in brain metabolism and in cell functional speciation and a recently discovered regulated cell death have attracted significant attention and are highlighted in this review.

Keywords: ATP7A; ATP7B; SLC31A1; brain; cuproptosis.

Graphical abstract

FIGURE 1.

The multifaceted role of copper (Cu) in cell physiology. The cartoon illustrates (in blue) the role of Cu as an enzyme cofactor and corresponding physiological processes. In yellow: the role of Cu as a regulator of protein structure and function, which is mediated through direct binding to specific targets. In gray: the Cu-dependent processes for which importance of Cu is firmly established, but the mechanisms of Cu action are not yet fully understood. DBH, dopamine-β-hydroxylase; LOX, lysyl oxidase; MEMO1, mediator of cell motility 1; MUC2, mucin 2; PAM, peptidyl alpha-amidating monooxygenase; PDE3, phosphodiesterase 3.

FIGURE 2.

Copper (Cu) is absorbed from the dietary sources by the small intestine and transported to tissues, with the liver being the major recipient of incoming Cu. The liver has the highest Cu concentration (3.47 µg/g wet tissue). In the liver, Cu is incorporated into ceruloplasmin, which is then secreted into the bloodstream. Excess Cu is exported into bile and removed with feces. The brain also has high Cu content (3.32 µg/g wet tissue). In other tissues, Cu plays a crucial role in angiogenesis, cardiac development, hematopoiesis, inflammatory response, and myogenesis. Image created with BioRender.com, with permission.

FIGURE 3.

Cartoon depicting the cross-sectional view of copper (Cu) transporter CTR1 (left) and the experimental structure of the NH₂ terminally truncated CTR1 (PDB 6M98; right). Ctr1 is a homotrimer. The extracellular domain of CTR1 contains clusters of Met (orange) and His (purple) residues that concentrate Cu and guide it toward the selectivity filter. The translocation pathway for Cu (green circles) is formed by the transmembrane domain 2 (TM2) of each monomer. Each TM2 contains an invariant MxxM motif (red circles) that is essential for activity. Cu binds to the first triad of Met residues (red circles at top) and then is handed to the second triad (red circles at bottom) before entering the intramembrane cavity. The COOH-terminal His-Cys-His motif (purple and pink circles) may regulate Cu release, transfer Cu to Cu chaperones (CCS and Atox1), and interact with VEGFR2 (210). Image created with BioRender.com, with permission.

FIGURE 4.

Copper (Cu) transporter CTR1 (SLC31A1) localization and traffic in a generic nonpolarized cell. In the vast majority of cells, CTR1 is located in the basolateral membrane and transfers Cu from Cu carriers in the blood into the cytosol. Intracellular Cu elevation triggers CTR1 endocytosis to early endosomes. Upon Cu depletion, CTR1 binds the retromer and returns to the plasma membrane. Upon prolonged Cu elevation, endocytosed CTR1 is degraded. Image created with BioRender.com, with permission.

FIGURE 5.

Copper (Cu)-dependent localization of Cu transporters in the small intestine. Cu (red circles) and Cu transporters (CTRs) are distributed unequally in the villi and the crypts. CTR1 and ATP7A, which are required for dietary Cu uptake, are enriched in the villi. Their expression in the crypt is low. ATP7B and Cu show an opposite pattern: highly enriched in the crypt and relatively low in the villus. The regulation of CTRs is also distinct. At steady state, the enteric CTR1 is targeted predominantly to the basolateral membrane. Cu depletion causes CTR1 trafficking from the plasma membrane to vesicles located in the vicinity of the apical membrane. ATP7A provides the major route for dietary Cu to enter the bloodstream from enterocytes. At steady state, ATP7A cycles between the trans-Golgi network (TGN) and endocytic vesicles, where it sequesters Cu for further export via vesical fusion. ATP7B is present predominantly in vesicles, although TGN localization is also observed. Upon Cu elevation, the abundance of ATP7B increases and the localization is uniformly vesicular. Image created with BioRender.com, with permission.

FIGURE 6.

Copper (Cu)-distribution pathways within a generalized cell. Cu enters cells predominantly via the Cu transporter SLC31A1 (CTR1) and binds to cytosolic Cu chaperones (ATOX1, CCS, COX17, and COX19) and possibly other soluble proteins. CCS delivers Cu to cytosolic SOD1 via heterodimerization. ATOX1 exchanges Cu with the NH₂-terminal domain of the Cu transporters ATP7A and ATP7B, which transfer Cu to Cu-dependent enzymes in the lumen of trans-Golgi network (TGN). When Cu is elevated, ATP7A and ATP7B move out of TGN to vesicles where they sequester excess Cu and eventually export it out of cells. ATOX1 also traffics to the nucleus in a TRAF4-facilitated fashion. ATOX1 can exchange Cu with mediator of cell motility 1 (Memo1), which binds to microtubules. Cox17 and Cox19 may be involved in Cu transfer to mitochondria but are not essential for this process. SLC25A3 is located in the inner membrane of mitochondria and maintains Cu content in the matrix. Mitochondria Cu chaperones are located in the inner membrane and facilitate Cu incorporation into cytochrome c oxidase (COX). Metallothioneins are induced in response to Cu elevation and bind Cu with high affinity to prevent toxicity (created with BioRender). Cu-accepting proteins in each of 3 intracellular Cu distribution pathways have specific structural features that facilitate Cu exchange with respective chaperones. Recent studies in yeast suggest that this classic model is somewhat simplistic and that a significant number of Cu-binding proteins exists in a cytosol forming an exchangeable “Cu pool” (220). Whether these proteins receive Cu from a chaperone, the Cu-glutathione complex, or other carriers is not yet clear.

FIGURE 7.

Human SOD1 in a complex with its copper (Cu) chaperone (CCS) in the elongated (top) and compact (bottom) conformations. CCS (yellow) has 3 domains. The similarity between the CCS domain 1 and ATOX1 (blue) is illustrated by the overlay of their structures. Domain 2 of CCS is structurally similar to SOD1 (purple), with which it heterodimerizes. Domain 3 carries cysteines that facilitate the formation of the disulfide bond in SOD1. In the elongated state (top; PDB 6FON), domain 1 of CCS can accept Cu from different sources (GSH, CTR1, and/or ATOX1). Following Cu binding to domain 1, CCS adopts compact information (bottom; PDB 6FP6). This brings domain 1 and domain 3 to the vicinity of the Cu entry site and leads to the insertion of Cu into SOD1, which has a zinc (Zn) atom already bound (256).

FIGURE 8.

Schematic of chaperone-mediated Cu (red circles) delivery to cytochrome c oxidase (COX). Double red circles in COX are a CuA site and a single circle is a CuB site. Cu-L, low-molecular-weight Cu ligand in the mitochondria matrix. Image created with BioRender.com, with permission.

FIGURE 9.

Current view of the structure and mechanism of Cu⁺¹-transporting P-type ATPases. A: structure of a truncated ATP7B (PDB accession 7SI3; Ref. 294); for other conformers and the detailed discussion of structural changes during the Cu-transport cycle, see Ref. . B: cartoon illustrates the main steps of ATP-driven Cu transport: binding of ATP and Cu stabilizes the E1 conformation of the transporter allowing the ATP hydrolysis and a transient phosphorylation (P) of the invariant Asp residue within the P domain. N, N domain. Hydrolysis of this energy-rich acyl-phosphate bond is facilitated by the A domain and triggers the conformational transition resulting in the release of Cu at the opposite site of the membrane. The transporter then returns to the state that can bind Cu and ATP from the cytosol.

FIGURE 10.

Copper (Cu)-handling machinery of neurons and astrocytes. Astrocytes play an important role in brain Cu homeostasis, as they are highly effective in storing and releasing Cu. Cu enters the brain through blood capillary of the blood-brain barrier and is taken up by astrocytes via the Cu transporter CTR1. Metallothionein MT3 and GSH are abundant in astrocytes and facilitate Cu buffering and storage. ATP7B may be involved in Cu transfer to the GPI-anchored ceruloplasmin, which is relatively abundant in astrocytes (8). ATP7A exports Cu, which is taken up by neighboring neurons via CTR1. Different neurons express ATP7A and ATP7B at different ratios (317). ATP7A and ATP7B are involved in the biosynthesis of Cu-dependent enzymes, removal of excess Cu, and sequestering Cu in vesicles for Cu storage or signaling. Cu-dependent enzymes, like dopamine-β-hydroxylase (DBH), are synthesized in specific types of neurons, where they produce neuromodulators that are packaged in secretory granules and then released in response to signaling events (CCS, Cu chaperone for SOD1; DA, dopamine; NE, norepinephrine). In response to changes in Cu levels, ATP7B traffics toward the dendrites (somatodendritic polarity). ATP7A is present in the cell body and traffics toward the axons in response to Cu elevation; ATP7A is also located at the synapse where it releases Cu in response to NMDA receptor signaling (139). Cu-binding protein PrPc (prion) is highly expressed at the synaptic cleft and is endocytosed in the presence of high Cu. Image created with BioRender.com, with permission.

FIGURE 11.

Copper (Cu) transporters in the brain barriers. Left: blood blood-brain barrier is composed of endothelial cells of the capillaries surrounded by astrocytic end feet, neurons, and pericytes. The endothelial cells express the Cu transporters CTR1, ATP7A, and ATP7B. The CTR1 retrieves Cu from the blood and ATP7A exports Cu toward brain parenchyma through the abluminal surface. Astrocytes import Cu through CTR1 and export Cu through ATP7A. Right: epithelial cells of choroid plexus (ChPl). The basolateral membrane of ChPl epithelia faces the blood capillaries, whereas the apical membrane is exposed to the cerebral spinal fluid. Both ATP7A and ATP7B are expressed in ChPl epithelia. Upon Cu elevation in ChPl, ATP7A traffics toward the apical surface allowing Cu entry into the cerebral spinal fluid/brain parenchyma, whereas ATP7B may export Cu through the basolateral membrane. CTR1 was detected at the apical surface, and its presence at the basolateral membrane needs to be examined further. Image created with BioRender.com, with permission.

FIGURE 12.

Potential roles of blood blood-brain barrier (BBB) and blood-cerebral spinal fluid (CSF) barrier in the entry of copper (Cu) into the brain. Cu (red circles) is taken from the blood predominantly by the Cu transporter CTR1 located in BBB (blue cylinder) and then exported out of BBB cells into the interstitial fluid (ISF) by ATP7A (green cylinder). Some Cu is taken by the parenchyma cells (via CTR1), while excess Cu enters the epithelium of the choroid plexus via CTR1 for storage. ATP7B (gray cylinder) may store Cu in vesicles and/or export Cu back into circulation via basolateral membrane. ATP7A transports to the CSF. Cu entering the brain parenchyma from CSF is stored in Cu storage vesicles in the subventricular zone (SVZ).

FIGURE 13.

Structure of elesclomol in a complex with copper (Cu).

FIGURE 14.

Molecular events in ATP7B^-/- hepatocytes leading to Wilson’s disease pathology. In the absence of ATP7B function, copper (Cu) accumulates in the cytosol (where it is sequestered by metallothioneins MT1/MT2), in mitochondria (where it inhibits mytochondria function), and in the nucleus causing changes in RNA splicing and LXR/RXR activity. Later, when Cu-binding capacity of MT1/2 is saturated, GSH:GSSG ratio decreases, leading to increased oxidated stress further augmented by low selenium (Se) levels. This causes upregulation of NRF2, further decrease in nuclear receptor activity (FXR/RXR) and mitochondria deterioration, and then relocation of transcription factor EB in the nucleus and activation of autophagocytosis. Image created with BioRender.com, with permission.

FIGURE 15.

Mechanisms driving copper (Cu)-regulated cell death. A: levels of Cu are dictated by the natural Cu flux regulated by specific Cu importers/exporters and the efficiency and abundance of specific Cu ionophores. Availability and reactivity of Cu are regulated by mechanisms of Cu release from its protein/metabolite/ionophore bound state and its redox regulation dictating its reactivity and binding partners. Intracellular localization of Cu in specific organelles will dictate the downstream targets of Cu and the resulting mechanisms of toxicity. B: targets of Cu in the cell as previously characterized include the targeting of signaling cascades (such as Cu-binding kinases); mitochondrial Cu accumulation will result in targeting of lipoylated and Fe-S cluster-containing proteins. Cu directly affects protein homeostasis by directly engaging with the ubiquitin-proteasome system machinery and/or by facilitating the aggregation of proteins. Cu can also directly target metabolism and redox-regulating enzymes promoting metabolic and redox stress, cell adhesion, and histone biology. Image created with BioRender.com, with permission.