ATP7A Trafficking Research Articles

A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival

Copper (Cu) is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of Cu are known to be utilized by host species against various pathogenic invasions. Leishmania, in both free-living and intracellular forms, exhibits appreciable tolerance toward Cu stress. While determining the mechanism of Cu-stress evasion employed by Leishmania, we identified and characterized a hitherto unknown Cu-ATPase in Leishmania major and established its role in parasite survival in host macrophages. This novel L. major Cu-ATPase, LmATP7, exhibits homology with its orthologs at multiple motifs. In promastigotes, LmATP7 primarily localized at the plasma membrane. We also show that LmATP7 exhibits Cu-dependent expression patterns and complements Cu transport in a Cu-ATPase-deficient yeast strain. Promastigotes overexpressing LmATP7 exhibited higher survival upon Cu stress, indicating efficacious Cu export compared with Wt and heterozygous LmATP7 knockout parasites. We further explored macrophage–Leishmania interactions with respect to Cu stress. We found that Leishmania infection triggers upregulation of major mammalian Cu exporter, ATP7A, in macrophages, and trafficking of ATP7A from the trans-Golgi network to endolysosomes in macrophages harboring amastigotes. Simultaneously, in Leishmania, we observed a multifold increase in LmATP7 transcripts as the promastigote becomes established in macrophages and morphs to the amastigote form. Finally, overexpressing LmATP7 in parasites increases amastigote survivability within macrophages, whereas knocking it down reduces survivability drastically. Mice injected in their footpads with an LmATP7-overexpressing strain showed significantly larger lesions and higher amastigote loads as compared with controls and knockouts. These data establish the role of LmATP7 in parasite infectivity and intramacrophagic survivability.

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

Homozygous Loss-of-Function Mutations in AP1B1, Encoding Beta-1 Subunit of Adaptor-Related Protein Complex 1, Cause MEDNIK-like Syndrome

MEDNIK syndrome (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma) is an autosomal-recessive disorder caused by bi-allelic mutations in AP1S1, encoding the small σ subunit of the AP-1 complex. Central to the pathogenesis of MEDNIK syndrome is abnormal AP-1-mediated trafficking of copper transporters; this abnormal trafficking results in a hybrid phenotype combining the copper-deficiency-related characteristics of Menkes disease and the copper-toxicity-related characteristics of Wilson disease. We describe three individuals from two unrelated families in whom a MEDNIK-like phenotype segregates with two homozygous null variants in AP1B1, encoding the large β subunit of the AP-1 complex. Similar to individuals with MEDNIK syndrome, the affected individuals we report display abnormal copper metabolism, evidenced by low plasma copper and ceruloplasmin, but lack evidence of copper toxicity in the liver. Functional characterization of fibroblasts derived from affected individuals closely resembles the abnormal ATP7A trafficking described in MEDNIK syndrome both at baseline and in response to copper treatment. Taken together, our results expand the list of inborn errors of copper metabolism.

Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological Cu I

Full elucidation of the functions and homeostatic pathways of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as CuI. Buffered at attomolar concentrations, intracellular CuI is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, we reveal a chelator design strategy in which phosphine sulfides aid in CuI coordination while simultaneously stabilizing aliphatic phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissociation constant and 1017-fold selectivity for CuI over ZnII, FeII, and MnII. As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring CuI in a broad range of biological systems.

Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological CuI.

Full elucidation of the functions and homeostatic pathways of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as CuI . Buffered at attomolar concentrations, intracellular CuI is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, we reveal a chelator design strategy in which phosphine sulfides aid in CuI coordination while simultaneously stabilizing aliphatic phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissociation constant and 1017 -fold selectivity for CuI over ZnII , FeII , and MnII . As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring CuI in a broad range of biological systems.

Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) Complexes on Cellular Copper Uptake, Copper-Mediated ATP7A Trafficking, and P-Glycoprotein Mediated Efflux.

Bis(thiosemicarbazonato)copper(II) complexes are of interest as potential therapeutics for cancer and neurodegenerative diseases as well as imaging agents for positron emission tomography (PET). The cellular uptake of six bis(thiosemcarbazonato)copper(II)complexes derived from glyoxal, with different functional groups Cu(gtsx) where x = different functional groups, was investigated in SKOV-3, HEK293, and HEK293 P-gp cell lines. Treatment of the cells with the copper complexes increased intracellular copper and increased levels of p-ERK due to activation of the Ras-Raf-MEK-ERK pathway. Treatment of SKOV-3 cells with low concentrations (μM) of two of the copper complexes led to trafficking of the endogenous copper transporter ATP7A from the Golgi network to the cell membrane. Experiments in HEK293 and HEK293-P-gp cells suggest that Cu(gtsm) and Cu(gtse) are substrates for the P-gp efflux protein but the complex with a pyrrolidine functional group, Cu(gtspyr), is not. A PET experiment in mice showed that [64Cu]Cu(gtspyr) has reasonable brain uptake but high liver uptake.

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Omeprazole, a Gastric Proton Pump Inhibitor, Inhibits Melanogenesis by Blocking ATP7A Trafficking

Omeprazole is a proton pump inhibitor used in the treatment of peptic ulcer disease and gastrosophageal reflux disease and acts by irreversibly blocking ATP4A, a P-type H+/K+ ATPase in gastric parietal cells. We found that omeprazole and its closely related congeners inhibited melanogenesis at micromolar concentrations in B16 mouse melanoma cells, normal human epidermal melanocytes, and in a reconstructed human skin model. Omeprazole topically applied to the skin of UV-irradiated human subjects significantly reduced pigment levels after 3 weeks compared with untreated controls. Omeprazole had no significant inhibitory effect on the activities of purified human tyrosinase or on the mRNA levels of tyrosinase, dopachrome tautomerase, Pmel17, or MITF mRNA levels. Although melanocytes do not express ATP4A, they do express ATP7A, a copper transporting P-type ATPase in the trans-Golgi network that is required for copper acquisition by tyrosinase. ATP7A relocalization from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations in melanocytes was inhibited by omeprazole. Omeprazole treatment increased the proportion of EndoH sensitive tyrosinase, indicating that tyrosinase maturation was impaired. In addition, omeprazole reduced tyrosinase protein abundance in the presence of cycloheximide, suggestive of increased degradation. Our findings are consistent with the hypothesis that omeprazole reduces melanogenesis by inhibiting ATP7A and by enhancing degradation of tyrosinase.

Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking.

ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7A(P1386S) causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7A(P1386S) partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy.

ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A.

Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A missense mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7A(T994I) and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of valosin-containing protein corrected ATP7A(T994I) mislocalization. For ATP7A(P1386S) , flow cytometry documented that nonpermeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the eighth transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7A(P1386S) with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop.

Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)-mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

The ADP-ribosylation factor 1 (Arf1) is involved in regulating copper uptake

Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.

Altered microglial copper homeostasis in a mouse model of Alzheimer’s disease

Alzheimer's disease (AD) is characterized by progressive neurodegeneration associated with the aggregation and deposition of β-amyloid (Aβ(40) and Aβ(42) ) peptide in senile plaques. Recent studies suggest that copper may play an important role in AD pathology. Copper concentrations are elevated in amyloid plaques and copper binds with high affinity to the Aβ peptide and promotes Aβ oligomerization and neurotoxicity. Despite this connection between copper and AD, it is unknown whether the expression of proteins involved in regulating copper homeostasis is altered in this disorder. In this study, we demonstrate that the copper transporting P-type ATPase, ATP7A, is highly expressed in activated microglial cells that are specifically clustered around amyloid plaques in the TgCRND8 mouse model of AD. Using a cultured microglial cell line, ATP7A expression was found to be increased by the pro-inflammatory cytokine interferon-gamma, but not by TNF-α or IL-1β. Interferon-gamma also elicited marked changes in copper homeostasis, including copper-dependent trafficking of ATP7A from the Golgi to cytoplasmic vesicles, increased copper uptake and elevated expression of the CTR1 copper importer. These findings suggest that pro-inflammatory conditions associated with AD cause marked changes in microglial copper trafficking, which may underlie the changes in copper homeostasis in AD. It is concluded that copper sequestration by microglia may provide a neuroprotective mechanism in AD.

Missense Mutations in the Copper Transporter Gene ATP7A Cause X-Linked Distal Hereditary Motor Neuropathy

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporter's known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.

Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.

Copper transport into the secretory pathway is regulated by oxygen in macrophages

Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A.