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    Topic review

    Molecular Mechanisms of Copper Homeostasis

    Subjects: Molecular Biology
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    Copper (Cu) is a trace element necessary for the growth and development of all living organisms and is the third most abundant trace element in the body after iron and zinc. Copper is an essential for maintaining life processes in all living cells, because several copper-dependent enzymes play an important role in kay physiological processes as cellular  respiration, oxygen–radicals scavenging, iron transport and neurotransmitter synthesis. However, copper excess is highly toxic because copper ions are potent generator of free radicals what leads to damage of proteins, lipids and nucleic acids. To avoid these harmful reactions living organisms developed complex mechanisms for maintaining the balance between essential and toxic copper levels. In the cell copper homeostasis is controlled by a network of copper-binding proteins and transporters. Copper uptake is mediated by membrane transporters CTR1 and CTR2 proteins, in the cytoplasm it is bound to unique group of metallochaperones (ATOX1, CCS, COX17) and transported to different cell compartments where it is linked to recipient proteins. Cu-transporting ATPases (ATP7A and ATP7B) are responsible for copper transfer into the Golgi apparatus, where the copper is added to the active sites of enzymes and also it is directed on the path of excess cellular copper removal to prevent its toxicity. Activity of copper-dependent ferroxidases, ceruloplasmin (Cp) and its homologue hephaestin (Heph) are necessary for normal iron metabolism. 

    1. Introduction

    Copper (Cu) is a trace element necessary for the growth and development of all living organisms and is the third most abundant trace element in the body after iron and zinc. In an organism, this reactive element Copper (Cu) is an essential micronutrient and plays a role in several fundamental processes that are critical for the normal growth and development of all living organisms.  In humans, copper is the third most abundant trace element in the body after iron and zinc[1], however, the organism of adult men contains only about 100 mg of Cu.  Due to its ability to accept and donate electrons this reactive element can exist in two oxidation states: as a reduced, cuprous (Cu+) and as an oxidized, cupric (Cu2+) ion. The extensive range of redox potentials of copper and its capacity to participate in one-electron transfer reactions determine the biological activity and function of this microelement. Owing to its redox properties, copper is an integral part of enzymes, where the metal works as a prosthetic group to facilitate the transfer of electrons from one molecule to another. Copper can coordinate with several electron donor ligands and in the proteins is often ligated to amino acids like cysteine, methionine, and histidine. Also, the cyclic amino acids tyrosine, phenylalanine, and tryptophan have significant copper-binding capacities[2]. Copper is a cofactor for more than 30 enzymes, and many of them Cu-dependent enzymes catalyze reactions used in fundamental metabolic processes including respiration (cytochrome c oxidase), detoxication of oxygen free radicals (superoxide dismutases: SOD1 and SOD3), connective tissue formation (lysyl oxidase), neurotransmitter synthesis (dopamine ß-hydroxylase and peptidylglycine-amidating monooxygenase, PAM), pigment production (tyrosinase), and iron metabolism (ceruloplasmin and hephaestin). Copper also takes part in the processes of myelination and regulation of the circadian rhythms, and is necessary for angiogenesis and coagulation. Copper deficiency leads to the impaired function of these enzymes and in consequence to disturbances in the key metabolic processes. Conversely, by means of the same redox activity, copper can generate an extremely harmful hydroxyl radical (·OH) through the Fenton reaction, which may cause oxidative damage to proteins and nucleic acids, lipid peroxidation, and enzyme inhibition. For this reason, excess copper is highly toxic to organisms. To deal with this dual nature of copper, tightly regulated molecular mechanisms have evolved and operate in organisms to ensure its bioavailability and to reduce its toxicity. The balance between essential and toxic copper concentrations at both cellular and systemic levels is maintained by a complex network of proteins involved in the regulation of copper uptake, transport, utilization, storage, and excretion. Cellular copper uptake, transport, and utilization are coordinated by a closely integrated network of three groups of proteins. First, copper transporters belonging to the CTR/SLC31 (copper transporter) family, proteins that are involved in copper uptake and intracellular distribution. Second, metallochaperones ATOX1, COX17, and CCS, cytosolic copper transporters, which bind Cu ions and deliver them to the specific cellular organelles, and third, P-type Cu-transporting ATP-ases (ATP7A and ATP7B), localized in the trans-Golgi network (TGN). In the Golgi apparatus, ATP7A and ATP7B proteins transfer copper into the lumen of the secretory pathway where this metal is incorporated into the active sites of the Cu-dependent enzymes. Both ATP7A and ATP7B are also involved in the ATP-dependent transport of Cu ions across the plasma or intracellular membranes [3][4].

    2. An Outline of Systemic Copper Metabolism in Mammals

    In mammals during gestation copper is transported from the mother circulation to the foetus throughout the placenta and copper uptake is enhanced in the last part of gestation [Linder 2014]. Mammalian foetuses accumulate stores of copper in the liver, and this indispensable element is utilised by a rapidly grown young organism during the time of lactation [5][6].  In adult mammals, copper is predominantly absorbed from a diet in the duodenum and small intestine and is transported to the blood from enterocytes. In adult humans, the average daily intake of copper ranges from 0.8 to 3 mg. In the blood, absorbed copper is bound to albumin, histidine, or glutathione, and is then transported via the portal vein into the liver, a central organ that maintains copper homeostasis. In hepatocytes, copper is bound to apo-ceruloplasmin (CP) and in the form of holo-ceruloplasmin complex is again excreted to the circulatory system, and with blood redistributed to other organs and tissues. Ceruloplasmin CP is a main copper carrier in the blood, transporting this microelement to various organs and tissues. Approximately 90% of serum copper occurs in the complex with CP, while the remaining 10% is bound to albumin or amino acids. The liver is also the site of copper storage in this organ  excess copper can be stored in the form of Cu-metallothionein complex and metallothionein is the dominant copper-binding protein in the fetal liver [7][8]. Copper, when delivered to various organs, is utilized in metabolic processes by Cu-containing dependent enzymes. Although various tissues differ in copper requirements, the set of proteins regulating copper distribution within the cells is thought to be the same in all tissues. The liver also takes part in removing copper from the body because On the other hand, excess cytosolic copper in hepatocytes is expelled into the bile and removed from the organism in feces and this is the main route of copper extraction [9][6]. Only 2% of copper is removed from the body by the kidneys in urine.

    3. Cellular Copper Uptake, Transport, and Utilization Are Orchestrated by a Closely Integrated Network of Proteins  Orchestration of cellular copper metabolism by Closely Integrated Network of Proteins

    The protein-mediated distribution of copper from the uptake system (CTR1) to the export system (Cu-ATPases) is a unique feature of copper transport compared with the transport of other ions such as sodium or calcium [3]. Over the last three decades, a remarkable increase has been made in our understanding of the molecular mechanisms of copper homeostasis, as well as the structure and function of Cu-containing proteins. A real breakthrough was the discovery in 1993 of ATP7A and ATP7B proteins: Cu-transporting ATPases. Currently, we know that cellular copper homeostasis requires a series of copper importers, carriers, chaperones, recipient proteins, and exporters to achieve the essential level of this biometal and prevent its toxicity (summarized in Figure 1). High binding affinities of the cellular machinery prevent the release of Cu+ into the various cellular compartments, resulting in the effective absence of free Cu+. In consequence, cellular and systemic Cu+ movement appears to be mediated via ligand exchange upon protein-protein interaction [10]. Cellular copper uptake, transport, and utilization are coordinated by a closely integrated network of three groups of proteins.

    Figure 1. Copper metabolism in polarized mammalian cells. Cupric ions (Cu2+) are reduced to cuprous ions (Cu+) by metalloreductase metaloreductase and can then be transported into the cell by the transmembrane transporters CTR1 or CTR2. In the cytosol, Cu+ ions can be reversibly bound by metallothionein (MT) and stored in the Cu-MT complex. In the cytoplasm Cu+ ions are bound by cytosolic metallochaperones: copper chaperone for superoxide dismutase 1 (CCS), COX17, and ATOX1, which deliver copper to various proteins such as superoxide dismutase 1 (SOD1), cytochrome c oxidase (CCO), ATP7A, and ATP7B. As a result, copper is delivered to different cellular compartments such as the cytoplasmic reticulum, mitochondria, or the Golgi apparatus. The ATP7A and ATP7B proteins located in the trans-Golgi network (TGN) incorporate Cu+ ions into the apoenzymes. ATP7B can transfer copper to apo-ceruloplasmin (CP) to form holo-ceruloplasmin. Then copper in the complex with ceruloplasmin can be transported to the blood vessels. Cu-transporting ATPases can remove the excess copper outside the cell: ATP7A across the basolateral membrane and ATP7B across the apical membrane of polarized cells. During the copper overload, Cu-transporting ATPases bind copper ions and are transported in vesicles to the plasma membrane by exocytosis. Then these ATPases can remove the excess copper outside the cell: ATP7A across the basolateral membrane and ATP7B across the apical membrane of polarized cells. Subsequently, they return to the TGN membrane by retrograde transport.
    This figure was made using free available illustrations from under the Creative Commons Attribution 3.0 Unported License.

    3.1. Copper transport to the cells

    The first group comprises copper transporters belonging to the CTR/SLC31 (copper transporter) family: CTR1 and CTR2 proteins belonging to the CTR/SLC31 (copper transporter) family that are involved in the copper uptake and intracellular distribution. In mammalian cells, the copper import is primarily mediated by the high-affinity copper membrane transporter CTR1 (more than 80% of intracellular copper import is CTR1-dependent). In humans, the SLC31A1 protein, widely known as CTR1, is encoded by the SLC31A1 (solute carrier family 31 member 1) gene (gene ID: 1317) and in mice by Slc31a1 gene (gene ID:20529). Human CTR1 is ubiquitously expressed with the highest expression in the liver, kidneys, small intestine, ovaries, testes, and heart. CTR1 is composed of three transmembrane domains: an extracellular N-terminal domain with characteristic Mets motif rich in methionine and histidine, a large intracellular loop, and a short intracellular C domain. CTR1 transports copper in the form of Cu+ ions in an ATP-independent manner using conserved methionine residues located in the N-terminal domain. Two-dimensional electron microscopy studies demonstrated that CTR1 forms a pore for the transport of Cu+ ions across membranes by the formation of homotrimer within the cell membrane. The pore is lined at its extracellular side by a ring of six methionine residues that are believed to provide an initial coordinating site for the permeating Cu. The intracellular  C-terminus provides a putative Cu-binding site at the exit of the pore in the cytosol. Numerous studies showed that the intracellular localization of CTR1 protein is dependent on copper status. Under basal copper concentration, CTR1 is found in both locations, i.e. in the plasma membrane and in the cytoplasm. Under low copper conditions, CTR1 is mainly localized in the plasma membrane. High copper levels induce CTR1 endocytosis and translocation, in vesicles, to the cytoplasm. Such a mechanism probably prevents the excessive import of copper into the cells, and thus counteracts intracellular copper overload and toxicity. Results of the studies performed on mice showed that expression of CTR1 protein, encoded by the Slc31a1 gene (gene ID: 20529), is required for normal embryonic development. Expression of the Slc31a1 gene in mouse embryos starts at day 7 (E7) and Slc31a1-/- knockout mice die in the midgestation at day 9 (E9) [11]. Slc31a1-/- embryos at the day E7.5 exhibited dramatic size reduction compared to the control fetus. Morphological analysis of Slc31a1-/- embryos revealed severe developmental impairments. For example, their neural ectoderm and mesoderm cell layers were poorly developed. Heterozygous Slc31a1+/- mice developed normally but in those mice copper concentration in the brain and spleen was reduced by around 50%. Similarly, the activity of copper-dependent enzyme SOD1 (encoded by superoxide dismutase 1, soluble gene, Sod1, gene ID: 20655) was also reduced [11][12].

    In contrast to CTR1, CTR2, a second cellular copper transporter, encoded in humans by SLC31A2 (solute carrier family 31 member 2) gene (gene ID: 1318) and in mice by the Slc31a2 gene (gene ID: 20530), exhibits exclusively intracellular localization. CTR2 is localized in the membranes of the vacuoles, vesicles, endosomes, and lysosomes of mammalian cells. CTR2 is characterized by a low affinity for Cu ions, probably because this protein lacks the Met, His rich motif in the N-terminus domain. The major role of CTR2 is to facilitate copper release from degraded cuproenzymes in lysosomes and to transport it back into the cytosol for reutilization. Similarly to the SLC31A1/Slc31a1, expression of the SLC31A2/Slc31a2 was found in all tissues and organs with high abundance in liver, kidney, and testis [13][14].

    3.2. Intracellular copper transport and distribution

    The second group comprises metallochaperones, cytosolic copper transporters that bind Cu ions and deliver them to the cellular organelles to prevent possible participation of Cu ions in the Fenton reaction. These metallochaperones include among others an antioxidant 1 protein (ATOX1), which binds Cu+ ions and transports them from the Golgi apparatus to ATP7A and ATP7B proteins. ATOX1-ATP7A and ATOX1-ATP7B interactions are essential for intracellular copper translocation and trafficking because ATOX1 also regulates the catalytical activities of both ATPases.

    3.2.1. ATOX1 – metallochaperone, transcription factor, and antioxidant protein

    Antioxidant 1 copper chaperone protein (ATOX1) is a cytosolic protein, which transports Cu from the membrane-bound CTR1 to the Cu-ATPases – ATP7A and ATP7B located in the membrane of the trans-Golgi network (TGN) and stimulates their catalytic activity. Human ATOX1 is a small (consists of 68 amino-acids) and soluble protein that is ubiquitously expressed and is encoded by the ATOX1 gene (gene ID: 475),  which is localized on chromosome 5 (in mice Atox1 gene (gene ID: 11927) is localized on chromosome 11) [15][16]. The large-scale analysis of the human transcriptome showed the highest expression of ATOX1 in the adult kidney, liver, and spleen. ATOX1 protein contains a single metal-binding site (MBS) with a characteristic MetxCysxxCys motif.  In the MBS copper ion is bound by sulfur atoms from two cysteines, thus one molecule of ATOX1 can bind and transfer one Cu+ ion [16]. Among the different roles that ATOX1 plays, the most important is copper transfer to the Cu transporting ATPases and there are several data that indicate that ATOX1 delivers Cu to metal-binding domains (MBD) of ATP7A and ATP7B [17][18]. The MetxCysxxxCys copper-binding sequence of ATOX1 has significant homology and similar folding to the N-terminal metal-binding domain of Cu-ATPases and interacts with them in a copper-dependent manner [16][19]. Thus, Cu delivered to the TGN is transferred into the lumen, via the ATP7A and ATP7B action, where it is incorporated into the Cu-dependent enzymes. Inactivation of ATOX1 inhibits the maturation of secretion of cuproenzymes as well as copper export from cells [16][20].  It is known, that ATOX1 regulates the catalytic activity of ATP7B because ATOX1-Cu transfer stimulates phosphorylation of this protein [15]. ATOX1 also can play a role as a Cu-ATPase inhibitor because at low cellular copper concentration apo-form of ATOX1 can remove copper from 4-5 MBDs of Cu-ATPases and therefore down-regulate enzyme activity at about 50% [3].  Cellular functions of ATOX1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of gene transcription, and antioxidant defense [16]. ATOX1 protein contains nuclear localization signal (NLS) at the C-terminal domain, and in the nucleus plays acts as a transcription factor (TF) regulating genes expression [21][22]. Direct copper-dependent interactions of ATOX1 to the promoter region of cyclin D1 gene (Ccnd1) was demonstrated [21]. Under a high concentration of copper ions inside the cell, ATOX1 also stimulates the expression of the SOD3 (superoxide dismutase 3, extracellular) gene. SOD3 protein is the major extracellular antioxidant enzyme protecting against O2• by catalyzing the dismutation of two superoxide radicals into hydrogen peroxide and oxygen. Results obtained by Itoh et al (2009) revealed that copper induces binding of ATOX1 to the ATOX1-responsive element of mouse Sod3 promoter (gene ID: 20657), which is indispensable for the copper-dependent SOD3 transcription [23]. ATOX1 plays an essential role in inflammatory neovascularization and wound healing as a metallochaperone delivers copper ions through the CTR1-Atox1-ATP7A pathway to the active centre of lysyl oxidase (LOX), the enzyme which is involved in the process of angiogenesis [24][25][26].   

    ATOX1 protein participates in placental copper transport. Experiments on mice with knockout of the Atox1 gene (Atox1-/- mice) showed that most of the copper is trapped in the placenta in ATOX1 deficient mice. In the embryonic cells, such as syncytiotrophoblasts and capillary endothelium cells, in the absence of functional ATOX1 chaperone, copper is not delivered to ATP7A and ATP7B proteins, which are responsible for copper transfer between mother and foetus [27][28]. Most of the homozygous Atox1-/- pups died just after birth only around 45% survived the critical period, however, they also died subsequently before weaning. The phenotype of homozygous Atox-/- mice was very close to that observed in copper-deprived animals. Those Atox1-/- mice exhibited growth retardation, central nervous system deformations, skin laxity, hypothermia, and hypopigmentation, they also suffered from peripartum hemorrhaging. Some of the pups had severe congenital eye defects (microphthalmia). On a postnatal day 2 (day P2), tested knockout mice showed lower copper concentration in the liver and in the brain (around 50% compared to the wild type littermates). Also, the activity of the copper-dependent enzymes such as cytochrome c oxidase in the brain and tyrosinase in the skin cells was lower. Heterozygous mice Atox1+/- were indistinguishable from their wild-type littermates [27]

    3.2.2. CCS - metallochaperone mediated SOD1 maturation

    Copper chaperone for superoxide dismutase 1 (CCS) is a ubiquitously expressed protein with a molecular weight of 35 kDa, which forms a 70 kDa homodimer. CCS (copper chaperone for superoxide dismutase) gene (gene ID: 9973) is localized on chromosome 11 in humans and mouse Ccs (gene ID: 12460) on chromosome 19. In the cells, CCS is localized in the cytosol and intermembrane space of mitochondria [29][30][31]. As a  metallochaperone, CCS binds Cu+ and is responsible for delivering copper to Cu,Zn-superoxide dismutase (SOD1) ensuring conversion of apo-SOD1 to holo-SOD1 [29][32].  CCS protein consists of three domains that carry out separate functions of copper-binding, trafficking, and docking to adaptor SOD1 protein. N-terminal domain I is an ATOX1-like domain, and it includes MetxCysxxCys motif in the loop responsible for binding copper [32][33][34]. Domain II and III are required for interaction with and activation of SOD1. Central and largest domain II contains a region of close homology with SOD1 (in humans it is close to 50%) and it is responsible for the formation of heterodimeric CCS-SOD1 intermediate and docking CCS to the SOD1  during protein metallation. On the C-terminus, there is the smallest domain III but this structure contains the most conserved motif CysxCys across whole CCS protein. It is responsible for binding copper to apo-SOD1 by catalyzing the formation of disulfide bonds between copper and protein [34][32], and any mutation in this region causes the CCS dysfunction. CCS plays a crucial role not only in SOD1 metallation but also is necessary for SOD1 activation and maturation [35][31]. SOD1 maturation from monomer to a functional enzyme requires zinc-binding, copper acquisition, the formation of an intramolecular disulfide bond between cysteine (Cys) 57 and Cys 146, and homodimerization [30][34]. In the absence of metal cofactors and upon reduction of the disulfide bonds, the SOD1 dimer is destabilized and exists as an inactive monomer. Only when both metal ions are present and a disulfide bond is formed, the active, dimeric form of SOD1 is stabilized [30][30][35]. CCS protein expression is very sensitive to copper level, and even a marginal decrease in copper level caused increased CCS protein production and thus, CCS is proposed as a marker of copper deficiency [36][37]. Conversely, the concentration of CCS protein is reduced in conditions of cellular copper excess, presumably due to increased proteasomal degradation of CCS [36][38]. Ccs-/- knockout mice with the deletion of exons 1 and 2 were viable and exhibited normal amounts but the abnormal activity of CCS protein in comparison to the wild type littermates. Ccs-/- mice demonstrated a similar phenotype to Sod1 knockout mice, because in Ccs-/- mice copper ions are not delivered to the SOD1 enzyme. In Ccs-/- mice the activity of SOD1 protein was lowered by 15% in the brain, kidneys, and spinal cord and by 30% in the liver, even if copper absorption in hepatocytes was normal. Ccs-/- females had problems with fertility and the structure of their ovaries was disturbed. Histological analysis of Ccs-/- female ovary revealed abnormally formed follicles instead of corpus luteum [35].

    3.2.3. COX17 – metallochaperone essential for Cu-mitochondrial pathway

    COX17 is a small (8 kDa) hydrophilic protein located both in the cytoplasm and in the mitochondrial intramembrane space (IMS). This small metallochaperone contains 6 conserved cysteine residues and thus bonds and transports Cu+ ions from CTR1 membrane protein and delivers copper to CCO through next metallochaperones directly associated with metalation of CCO [39][40]. In mammals, 4 copper chaperones are required to transfer copper from incoming CTR1 to CCO: COX17, SCO1, SCO2, and COX11 [41][40].

    In humans, COX17 (cytochrome c oxidase copper chaperone COX17) gene (gene ID: 10063) is localized on chromosome 3 (3q13.33) but also its pseudogene is localized on chromosome 13 (13q14-21). In mice Cox17 (gene ID: 12856) is localized on chromosome 16 [42]. Expression of Cox17 was found in all tissues but as it was shown in rodents, Cox17 mRNA level is highest in heart, kidneys, brain, and also in some endocrine cell lines. Low expression is observed in the liver, small intestine, and some fibroblast cell lines [43]. The lack of activity of Cox17 in mice leads to early prenatal lethality [39]Cox17-/- mice died between embryonic days E8.5 and E10 [41] because proper activation of CCO during embryonic development is essential [41][43]. Heterozygous mice with a mutation in this gene, which spans from the middle of exon 1 until the end of exon 2, appeared healthy, were fertile, and of normal size. Closer analysis showed, however, that in their brains, hearts, kidneys and skeletal muscles expression of the Cox17 gene was around 50% lower compared to the wild-type mice. Interestingly, the expression of Cox17 in other tissues was not changed in mutants. Also in the brains of those mice, the CCO activity was lower by 20% but there were no changes in CCO activity in kidneys or skeletal muscle [41][43].

    The second group comprises metallochaperones, cytosolic copper transporters that bind Cu ions and deliver them to the cellular organelles to prevent possible participation of Cu ions in the Fenton reaction. These metallochaperones include among others an antioxidant 1 protein (ATOX1), which binds Cu+ ions and transports them from the Golgi apparatus to ATP7A and ATP7B proteins. ATOX1-ATP7A and ATOX1-ATP7B interactions are essential for intracellular copper translocation and trafficking because ATOX1 also regulates the catalytical activities of both ATPases. The copper chaperone for superoxide dismutase 1 (CCS) is a ubiquitously expressed cytosolic metallochaperone, delivering Cu ions to Cu,Zn-SOD1 molecules in the cytoplasmic reticulum. By means of this function, CCS is responsible for the conversion of apo-SOD1 to the active holoenzyme. Cytochrome c oxidase copper chaperone (COX17) is a metallochaperone that binds Cu ions and transports them to mitochondria, where they are bound to subunits of cytochrome-c oxidase, an enzyme of complex IV of the mitochondrial respiratory chain. COX17 is a small, hydrophilic protein located in the cytoplasm and mitochondrial intramembrane space. COX17 is expressed in all tissues.

    3.2.4. The copper-transporting ATPases, ATP7A and ATP7B, are critical components of the copper metabolism 

    The third group comprises P-type Cu-transporting ATP-ases (ATP7A and ATP7B), localized in the trans-Golgi network (TGN). In the Golgi apparatus, ATP7A and ATP7B proteins transfer copper, using the energy of ATP hydrolysis, into the lumen of the secretory pathway where this metal is incorporated into the active sites of the Cu-dependent enzymes. Both ATP7A and ATP7B are also involved in the ATP-dependent transport of Cu ions across the plasma or intracellular membranes.

    ATP7A and ATP7B belong to the large family of P-type ATPases and form a separate subgroup within the P-type ATPase family (P1B-ATPases) which has distinct structural and mechanistic characteristics [3]. ATP7A and ATP7B share significant (50 – 60%) primary sequence homology, have similar architecture and are composed of several functional domains. An N-terminal cytosolic portion of these proteins contains six cysteine-rich copper-binding domains (MBD1-MBD6) with characteristic MetxCysxxCys motifs that bind reduced form of copper (Cu+) [3][4]. Eight transmembrane domains (TM1-TM8) stabilize the protein in the membrane and form the intramembrane channel, with a highly conserved CysProCys sequence in the TM6 for metal ions transport. ATP7A and ATP7B proteins play a crucial role in the maintenance of cellular Cu homeostasis. Both ATPases use energy from the ATP hydrolysis for Cu+ ions transport [3][44][45]. During the catalytic cycle, ATP7ases are autophosphorylated at a conserved aspartic acid in the ATP-binding domain to form the cytoplasmic loop between TM6 and TM7 and subsequently to bind Cu to transmembrane domains [3]. After the release of Cu, ATPases are dephosphorylated by A-domain which is located between TM4 and TM5 [4][44]. C-terminus of the ATP7A contains dileucine (LeuLeu) and the ATP7B trileucine (LeuLeuLeu) motif, that are responsible for the process of exocytosis [4][3][44].   By means of different mechanisms, both ATP7A and ATP7B prevent toxic copper accumulation by expelling Cu ions from the cells. When the cells are exposed to increased copper concentrations, ATP7A moves to the cytoplasm and plasma membrane (in polarized cells to the basolateral membrane). Under the same conditions, ATP7B is transported to the plasma membrane in vesicles (in polarized cells to the apical membrane). It has been proposed that this Cu-induced trafficking of both ATPases is fundamental for maintaining cellular copper homeostasis. Another important role of ATP7A involves delivering of Cu+ ions to the secretory pathway where they are incorporated into the Cu-dependent enzymes such as lysyl oxidase, tyrosinase, dopamine-β-hydroxylase, peptidylglycine-α-amidating monooxygenase, and extracellular dismutase (SOD3). ATP7A and ATP7B are encoded by different genes localized in separate chromosomes. In humans and laboratory rodents, ATP7A protein is encoded by the X-linked ATP7A (Atp7a in rodents) gene (gene ID: 538 in human and 11977 in mouse) and its expression has been reported in nearly all cells in the body. For this reason, ATP7A ATP7A/Atp7a is considered to be a housekeeping gene. Nevertheless, its expression differs among the cells and tissues and is age-dependent. The highest expression levels have been found in the brain, kidney, small intestine, and heart. In humans, lack of ATP7A activity caused by a mutation in the ATP7A ATP7A gene leads to severe metabolic syndrome—Menkes disease. Mice with mutations in the Atp7a gene are called mottled mutants. Many mottled mutants have arisen spontaneously in different laboratories  or have been induced by chemical or radiation mutagenesis. Currently, about 44 different mutations in the mottled locus have been described and 15 of them have been characterized at the molecular level [48]. Mottled mutants exhibit defects in copper metabolism and hemizygous mottled males exhibit a severe and often lethal phenotype. As in humans, the severity of the phenotype in Atp7a mutant mice is dependent on the mottled allele and varies between mottled mutations [48][49].  In general, affected males belong to one of three classes of phenotypic severity: (1) Mutant males that die in utero; (2) mutant males that die in the 3rd week of postnatal life; and (3) mutant males that die within a few postnatal months. Mutants from the second class (brindled (Atp7a mo–br) macular(Atp7a mo–ml) and mosaic (Atp7a mo–ms))  have been extensively studied and are widely accepted as good animal models of the severe form of Menkes disease [50][49][51]. Lack of activity of the ATP7A protein in mottled mutants lead to a defect in copper transport and absorption. Analyses of the copper content in organs of these mutants indicate that copper is accumulated in the small intestine and kidneys, while the brain, liver and heart suffer from copper deficiency. Macular, mosaic and bridled mutant males exhibit many clinical features characteristic for defective copper metabolism, including defects in pigmentation and hair structure, decrease in body weight, poor viability  During the second week of life they developed neurological symptoms as tremor, seizures, ataxia and progressive paresis of the hind limbs and they die at third week postpartum[52][53][54][55][56][57]. This pathological symptoms are connected to the specific function of ATP7A in several cell types, which consists of expelling Cu from cells to the extracellular environment. Consequently, dysfunctional ATP7A in absorptive enterocytes and epithelial cells of the renal tubules does not only lead to the accumulation of Cu in these cells, but also limits this metal to other cells in the body [58][51][54]. Furthermore, because ATP7A plays an imported roles in the delivery of Cu to Cu-containing enzymes, a large number of essential enzymes in mottled mutants are dysfunctional or exhibit decreasing in activity [56][59][57][60][61]. The phenotypic diversity of the mottled alleles is a valuable source of knowledge not only about the molecular basis of Menkes disease, but also about the role of copper in metabolism and development.

    ATP7B is encoded by the autosomal ATP7B gene. In humans the ATP7B (ATPase copper transporting beta) gene (gene ID: 540) is located on chromosome 13, in mice Atp7b (gene ID: 11979) on chromosome 8. In mammals, ATP7B protein is primarily synthesized in the liver in the hepatocytes, but its expression has been also reported in other tissues such as the placenta, mammary gland, eye, lung, and brain. ATP7B protein in hepatocytes is responsible for copper binding to apo-ceruloplasmin, resulting in the formation of the redox-active holo-ceruloplasmin, responsible for the transport of copper into the bloodstream. ATP7B also participates in the excretion of excess copper to the bile. Besides the liver, ATP7B is also responsible for the return transport of copper from the placenta to the maternal compartment, to prevent excess copper accumulation in the fetus. During lactation in mammary gland cells, ATP7B expression exhibits a granular, diffuse cytoplasmic pattern, and participates in copper export to the milk. Mutation in the ATP7B gene results in disturbances in copper binding to ceruloplasmin (CP) by the ATP7B protein and leads to copper accumulation in the liver up to the toxic level in patients with Wilson disease. Mice with mutation in Atp7b gene (tx – toxic milk mice) and Atp7b-/- knockout mice are the animal model of Wilson disease [62][63][64]. In Atp7b knockout and mutant mice, lack of  ATP7B activity leads to progressive copper accumulation in the liver up to the toxic level resulting in the development of a wide spectrum of hepatic pathological symptoms [65][66][67]. Moreover in the tx mutants, copper concentration is increased in th e spleen, kidney and in the brain, whereas infant mice are copper deficient and display increased mortality [62][63][66]. Tx mutant mice during the progress of disease  also developed some neurological symptoms as motor and cognitive disturbances [68]. Decreasing in ceruloplasmin level and its ferroxidase activity in Tx mutant mice lead to disturbances in iron metabolism. Adult tx mutants develop mild anaemia caused by functional iron deficiency. Mutant mice showed decreased plasma iron level with concomitant iron accumulation in hepatocytes and liver macrophages [69].

    In cells, ATP7A and ATP7B are found predominantly in membranes of the TGN and endocytic vesicles, and to a lesser extent at the plasma membrane. ATP7A and ATP7B circulate between these compartments in response to changes in intracellular copper levels. The Cu-dependent regulation of intracellular localization of ATPases determines the fate of this metal, which is either used for the biosynthesis of Cu-dependent enzymes in the secretory pathway or is sequestered in or released from vesicles into the extracellular milieu.

    4. Partnership of copper-dependent ferroxidases with ferroportin in cellular iron export

    Physiological and pathological consequences of the interactions between copper and iron metabolisms have been known for many years  [70]. The best known interplay between these microelements, showing similar physiochemical properties, is demosntrated by the fact that in case of copper deficiency, iron is retained in the duodenum, liver, and spleen. This leads to decreased iron availability for erythropoiesis and may contribute to the development of iron deficiency anemia (IDA). Such tissular iron arrest in copper-defcient individuals clearly indicates a major defect in iron release into the circulation. At the molecular level this process relies, from the copper side, on two copper-dependent ferroxidases, ceruloplasmin (CP, encoded by human CP gene, ID: 1356) and its homologue hephaestin (HEPH, encoded by human HEPH gene, ID: 9843), and, from the iron side, on solute carrier family 40 member 1 protein, widely knonw as ferroprtin (SLC40A1, previously known as FPN1), encoded by human SLC40A1 gene (gene ID: 30061), the only known vertebrate iron exporter [71]. The importance of CP in human iron homeostasis has been confirmed by the description of the congenital disease, aceruloplasminemia, in which mutations in the CP gene (gene ID: 1356) lead to a marked accumulation of iron in the affected parenchymal tissues including brain and liver [72]. CP is mainly synthesized in hepatocytes in the form of apo-CP. Incorporation of Cu into apo-CP results in the formation of the redox-active holo-enzyme and is mediated by ATP7B during transit through the TGN. Recycling of iron from senescent and/or damaged erytrocytes by hepatic and splenic macrophages is a proces recovering approximately 25 mg iron per day (in humans), which corresponds to the daily requirement of iron for erythropoiesis. Cooperation of an alternative spliced glycosylphosphatidylinositol (GPI) variant of CP (GPI-CP) anchored on a cellular membrane, originally characterized in the brain [73] with ferroportin is of particular importance for iron efflux from macrophages. Ferrous ions transported into the circulation by ferroportin are oxidized by GPI-CP and ferric ions are delivered to extracellular transferrin. Then in this complex are suppiled to the bone marrow and other sites of iron utilization in the body. Importantly, ferroxidase activity of GPI-CP is also required for the stability of cell surface ferroportin [74]. Iron release mechanisms involving copper and iron are also linked through the second transmembrane Cu-containing ferroxidase, HEPH, necessary for effective iron transport from intestinal enterocytes into the circulation. [70]. In postnatal life, exogenous iron can only enter the body across the wall of the small intestine. In humans, under physiological conditions, the amount of iron absorbed depends on the period of postnatal development declining to around 1-2 mg per day in adults. Most of iron from the diet is moved across the enterocyte brush border membrane by the iron transporter divalent metal-ion transporter 1, a process enhanced by the prior reduction of the iron by duodenal cytochrome B . HEPH co-localizes with ferroportin on the basolateral membranę of duodenal enterocytes, where it oxidizes ferrous iron to facilitate its binding to plasma transferrin. The sla (sex-linked anemia) mice have a defect in the basolateral export of iron from intestinal enterocytes into the circulation due to mutation in the Heph gene (gene ID: 15203), resulting in iron deficiency and microcytic hypochromic anemia [75]. Systemic iron deficiency has also been reported in Cu-deficient mice that display decreased HEPH ferroxidase activity in the intestine [76]. It has been postulated that decreased expression of both dudodenal Heph and Slc40a1 can be directly caused by changes in the distribution of copper ions within the enterocyte of anemic piglets and downregulation of Atp7b expression [77].

    5. Summary

    However mammalian organs have distinct physiological functions and consist of different cell types, both CTR transporters described above, metallochaperones, and Cu-transporting ATPases are expressed in all tissues and organs, and their expression and activity are in majority regulated by the copper level. Cellular copper homeostasis is controlled in several levels: copper uptake by CTR transporters (CTR1 and CTR2), intracellular molecules engaged in handling Cu and delivering it to its specific sites by metallochaperones (ATOX1, CCS, and COX17), and copper efflux by ATP7A and ATP7B protein. Interactions between all these groups of proteins allow for copper transfer from the intramembrane side of the transporter to the binding side of the acceptor proteins and also for modulating intracellular copper level. Lack of activity of copper transporters caused by mutation or genetic knockout leads to lethality and pathological symptoms in laboratory mice. However, experiments on those mutant and knockout animals give us the possibility to analyze multiple copper-dependent pathways of regulation of Cu metabolism during physiological and pathological conditions. In humans mutations in genes encoding copper transporting proteins  ATP7A and ATP7B results in severe metabolic disorders called Menkes and Wilson disease. 

    The entry is from 10.3390/ijms21239053


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