Chaperone Atox1 Research Articles

Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b.

Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Copper chaperone ATOX1 is required for MAPK signaling and growth in BRAF mutation-positive melanoma

Copper (Cu) is a tightly regulated micronutrient that functions as a structural or catalytic cofactor for specific proteins essential for a diverse array of biological processes. While the study of the extremely rare genetic diseases, Menkes and Wilson, has highlighted the requirement for proper Cu acquisition and elimination in biological systems for cellular growth and proliferation, the importance of dedicated Cu transport systems, like the Cu chaperones ATOX1 and CCS, in the pathophysiology of cancer is not well defined. We found that ATOX1 was significantly overexpressed in human blood, breast, and skin cancer samples, while CCS was significantly altered in human brain, liver, ovarian, and prostate cancer when compared to normal tissue. Further analysis of genetic expression data in Cancer Cell Line Encyclopedia (CCLE) revealed that ATOX1 is highly expressed in melanoma cell lines over other cancer cell lines. We previously found that Cu is required for BRAFV600E-driven MAPK signaling and melanomagenesis. Here we show that genetic loss of ATOX1 decreased BRAFV600E-dependent growth and signaling in human melanoma cell lines. Pharmacological inhibition of ATOX1 with a small molecule, DCAC50, decreased the phosphorylation of ERK1/2 and reduced the growth of BRAF mutation-positive melanoma cell lines in a dose-dependent manner. Taken together, these results suggest that targeting the Cu chaperone ATOX1 as a novel therapeutic angle in BRAFV600E-driven melanomas.

Copper relay path through the N-terminus of Wilson disease protein, ATP7B

In human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson disease protein (ATP7B) in the Golgi for loading of Cu-dependent enzymes. ATP7B is a membrane-spanning protein which, in contrast to non-mammalian homologs, has six cytoplasmic metal-binding domains (MBDs). To address the reason for multiple MBDs, we introduced strategic mutations in which one, two or three MBDs had been blocked for Cu binding via cysteine-to-serine mutations (but all six MBDs are present in all) in a yeast system that probes Cu flow through Atox1 and ATP7B. The results, combined with earlier work, support a mechanistic model in which MBD1-3 forms a regulatory unit of ATP7B Cu transport. Cu delivery via Atox1 to this unit, followed by loading of Cu in MBD3, promotes release of inhibitory interactions. Whereas the Cu site in MBD4 can be mutated without a large effect, an intact Cu site in either MBD5 or MBD6 is required for Cu transport. All MBDs, expressed as single-domain proteins, can replace Atox1 and deliver Cu to full-length ATP7B. However, only MBD6 can deliver Cu to truncated ATP7B where all six MBDs are removed, suggesting a docking role for this structural unit.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.

Highlights of This Issue

Treatment of adult glioma has not changed for over a decade, and it remains unclear how to best translate insights from genomic studies to bedside. Zheng and colleagues tested the utility of panel sequencing for assisting in glioma diagnosis, prognosis prediction, and informing targeted therapy using a prospective cohort of 237 gliomas. The study showed that the platform can identify IDH mutations that align with disease histopathology and outcome, and had high specificity and modest sensitivity in predicting 1p/19q codeletion. It also enabled stratification of hypermutators. Three out of ten patients whose treatments were tailored based on this panel achieved exceptional responses.Treatment of aggressive breast cancer remains challenging. Finding novel anti-cancer targets is critical for progress. In this study, Karginova and colleagues evaluate the efficacy and biological activity of the small molecule DCAC50, designed to inhibit intracellular copper transport through chaperones ATOX1 and CCS. DCAC50 triggers apoptosis in breast cancer cells and suppresses tumor angiogenesis in xenograft models. Co-treatment with paclitaxel enhances cytotoxicity in breast cancer cells. Mechanistically, DCAC50 disrupts copper homeostasis and generates oxidative stress. The ability of DCAC50 to induce apoptosis in cancer cells and suppress angiogenesis supports intracellular copper transport as a promising target for breast cancer therapy.This study by Shatsky and colleagues compares and contrasts solid tumor tissue NGS and ctDNA NGS technology to assess for potentially actionable genomic alterations in advanced breast malignancies. Both technologies were found to be useful and potentially complementary in the discovery of actionable alterations. In addition, ctDNA was found to have prognostic significance, as patients with greater than or equal to 5% ctDNA had a poorer overall survival. Both types of NGS have potential value that may add to targeted treatment options and further our understanding of advanced breast cancer.Peptide receptor radiotherapies with β-emitters are currently used to treat neuroendocrine tumors in patients. However, not all patients respond to treatment and most patients experience hematological toxicity. α-emitters, however, have a shorter pathlength and higher linear energy transfer allowing them to be more potent agents. This study by Stallons and colleagues examined 212Pb-DOTAMTATE in a preclinical neuroendocrine model and was more effective at controlling tumor growth in a single dose efficacy than previously published data with a beta-emitting somatostatin analog with little hematological toxicity. These studies support the therapeutic potential and possible advantage of 212Pb-DOTAMTATE and have led to the initiation of a phase I clinical trial.

Recent advances in copper chaperone and cancer

Copper is a redox-active transition metal essential for most living organisms and serves as a catalytic cofactor for enzymes that function in antioxidant defence, iron homeostasis, cellular respiration and a variety of biochemical processes. However, intracellular free copper must be strictly limited because of its potential toxic side effects. Copper dyshomeostasis is involved in the pathogenesis and progression of diseases including cancer and neurodegenerative diseases. The uncontrolled accumulation of copper could lead to increased oxidative stress and inappropriate binding to macromolecules. Most cells evolve complex systems of copper regulation and trafficking to satisfy the cellular copper requirements and simultaneously minimize the potential toxicity. This review presents the importance of copper transporter Ctr1, copper chaperone CCS, Atox1 and COX17 for the maintenance of copper ion balance in cells. Moreover, we discuss recent highlights in the development of copper chelators for the treatment of cancer. Finally, we introduce the development of small molecules that alter copper homeostasis by inhibiting copper chaperones.

Monitoring Interactions Inside Cells by Advanced Spectroscopies: Overview of Copper Transporters and Cisplatin.

Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum- based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation. The use of powerful spectroscopic, spectrometric and computational methods has allowed a deep structural insight into the mode of interaction of platinum drugs with the metal-binding domains of the transporter proteins. This review article focuses on the mode in which platinum drugs can compete with copper ion for binding to transport proteins and consequent structural and biological effects. Three types of transporters are discussed in detail: copper transporter 1 (Ctr1), the major responsible for Cu+ uptake; antioxidant-1 copper chaperone (Atox1), responsible for copper transfer within the cytoplasm; and copper ATPases (ATP7A/B), responsible for copper export into specific subcellular compartments and outside the cell. The body of knowledge summarized in this review can help in shaping current chemotherapy to optimize the efficacy of platinum drugs (particularly in relation to resistance) and to mitigate adverse effects on copper metabolism.

Interaction of the Copper Chaperone Atox1 with Alpha-Synuclein

Interaction of the Copper Chaperone Atox1 with Alpha-Synuclein

Copper Chaperone Atox1 Interacts with Cell Cycle Proteins

The anaphase-promoting complex (APC) is involved in several processes in the cell cycle, most prominently it facilitates the separation of the sister chromatids during mitosis, before cell division. Because of the key role in the cell cycle, APC is suggested as a putative target for anticancer agents. We here show that the copper chaperone Atox1, known for shuttling copper in the cytoplasm from Ctr1 to ATP7A/B in the secretory pathway, interacts with several APC subunits. Atox1 interactions with APC subunits were discovered by mass spectrometry of co-immunoprecipitated samples and further confirmed using proximity ligation assays in HEK293T cells. Upon comparing wild-type cells with those in which the Atox1 gene had been knocked out, we found that in the absence of Atox1 protein, cells have prolonged G2/M phases and a slower proliferation rate. Thus, in addition to copper transport for loading of copper-dependent enzymes, Atox1 may modulate the cell cycle by interacting with APC subunits.

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1.

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH4)2MoS4], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells.

The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.

Interactions between human copper chaperone Atox1 and cisplatin, carboplatin, nedaplatin and oxaliplatin studied by ESI mass spectrometry

Increasing evidence indicates that the copper chaperone Atox1 involves the mechanism of Pt-drugs. Here, the interactions of Atox1 with cisplatin and cisplatin analogues were studied. Results show that, like cisplatin, the other three Pt complexes can interact with Atox1 and CuI coordination obviously enhances the platination of Atox1. Cisplatin, carboplatin and nedaplatin formed some of adducts with the same composition due to having the same cis-diammine carrier ligands and differ only in the leaving groups. Nedaplatin, similar to carboplatin, was relatively inert with respect to cisplatin. Remarkably, an unexpected adduct with loss of the 1, 2-diaminocyclohexane (DACH) carrier ligand were observed in the reaction of oxaliplatin with holo-Atox1. These results indicate that both platination adducts and rates of platination are significantly influenced by the ligand and cuprous ion, suggesting the probably different influence of Atox1 on Pt-drugs in vivo.

Copper chaperone ATOX1 regulates pluripotency factor OCT4 in preimplantation mouse embryos

Despite of the importance of copper (Cu) during pregnancy, the roles of Cu-binding proteins during early embryonic development are unknown. The Cu chaperone ATOX1 was recently suggested to have additional functions related to transcription and cancer. When we analyzed single-cell RNA transcript data from early mouse embryos, Atox1 transcript levels increased dramatically at the 8-cell stage and, at 16- and 32-cell embryo stages, matched those of Oct4 which expresses a transcription factor essential for pluripotency in the inner cell mass. To explore this, we probed Atox1 expression during the first week of development of mouse embryos. ATOX1 appeared ubiquitously expressed throughout the cells until compaction; in subsequent embryo stages, ATOX1 relocalized to cytoplasmic perinuclear domains in the inner cell mass. Silencing of Oct4 did not affect Atox1 expression, but silencing of Atox1 at the 2-cell stage strongly diminished Oct4 expression in 16-cell embryos.

Binding of Copper and Cisplatin to Atox1 Is Mediated by Glutathione through the Formation of Metal-Sulfur Clusters.

Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

Thiol‐based copper handling by the copper chaperone Atox1

Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017.

Defining the human copper proteome and analysis of its expression variation in cancers.

Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is associated with increased cellular Cu concentrations, whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer versus normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-molecular studies of Cu-dependent processes in cancer.

Copper chaperone Atox1 plays role in breast cancer cell migration

Copper (Cu) is an essential transition metal ion required as cofactor in many key enzymes. After cell uptake of Cu, the metal is transported by the cytoplasmic Cu chaperone Atox1 to P1B-type ATPases in the Golgi network for incorporation into Cu-dependent enzymes in the secretory path. Cu is vital for many steps of cancer progression and Atox1 was recently suggested to have additional functionality as a nuclear transcription factor. We here investigated the expression level, cellular localization and role in cell migration of Atox1 in an aggressive breast cancer cell line upon combining immunostaining, microscopy and a wound healing assay. We made the unexpected discovery that Atox1 accumulates at lamellipodia borders of migrating cancer cells and Atox1 silencing resulted in migration defects as evidenced from reduced wound closure. Therefore, we have discovered an unknown role of the Cu chaperone Atox1 in breast cancer cell migration.

ChemInform Abstract: A Copper Story: From Protein Folding and Metal Transport to Cancer

AbstractReview: [73 refs.