MEMO1 binds iron and modulates iron homeostasis in cancer cells.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.

Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.

Connecting copper and cancer: from transition metal signalling to metalloplasia.

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles.

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.

Analysis of Wilson disease mutations revealed that interactions between different ATP7B mutants modify their properties.

Wilson disease (WD) is an autosomal-recessive disorder caused by mutations in the copper (Cu)-transporter ATP7B. Thus far, studies of WD mutations have been limited to analysis of ATP7B mutants in the homozygous states. However, the majority of WD patients are compound-heterozygous, and how different mutations on two alleles impact ATP7B properties is unclear. We characterized five mutations identified in Indian WD patients, first by expressing each alone and then by co-expressing two mutants with dissimilar properties. Mutations located in the regulatory domains of ATP7B-A595T, S1362A, and S1426I-do not affect ATP7B targeting to the trans-Golgi network (TGN) but reduce its Cu-transport activity. The S1362A mutation also inhibits Cu-dependent trafficking from the TGN. The G1061E and G1101R mutations, which are located within the ATP-binding domain, cause ATP7B retention in the endoplasmic reticulum, inhibit Cu-transport, and lower ATP7B protein abundance. Co-expression of the A595T and G1061E mutations, which mimics the compound-heterozygous state of some WD patients, revealed an interaction between these mutants that altered their intracellular localization and trafficking under both low and high Cu conditions. These findings highlight the need to study WD variants in both the homozygous and compound-heterozygous states to better understand the genotype-phenotype correlations and incomplete penetrance observed in WD.

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.

4'-Phosphopantetheine corrects CoA, iron, and dopamine metabolic defects in mammalian models of PKAN.

Pantothenate kinase-associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease-vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4'-phosphopantetheine, normalized levels of the CoA-, iron-, and dopamine-related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4'-phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron-sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4'-phosphopantetheine as a candidate therapeutic for PKAN.

Hemozoin produced by mammals confers heme tolerance.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.

Specialized cells, known as red blood cells, are responsible for transporting oxygen to various organs in the body. Each red blood cell contains over a billion molecules of heme which make up the iron containing portion of the hemoglobin protein that binds and transports oxygen. When red blood cells reach the end of their life, they are degraded, and the heme and iron inside them is recycled to produce new red blood cells. Heme, however, is highly toxic to cells, and can cause severe tissue damage if not properly removed. Scavenger cells called macrophages perform this recycling role in the spleen, liver and bone marrow. Collectively, macrophages can process around five million red blood cells every second or about 100 trillion heme molecules. But, it is unclear how they are able to handle such enormous volumes. Macrophages isolated from human and mice have been shown to transport heme from damaged red blood cells using a protein called HRG1. To investigate the role HRG1 plays in heme-iron recycling, Pek et al. used a gene editing tool known an CRISPR/Cas9 to remove the gene for HRG1 from the macrophages of mice. If HRG1 is a major part of this process, removing the gene should result in a build-up of toxic heme and eventual death of the mouse. But, rather than dying of heme-iron overload as expected, these mutant mice managed to survive. Pek et al. found that despite being unable to recycle heme, these mice were still able to make new red blood cells as long as they had a diet that was rich in iron. However, the darkening color of the spleen, bone marrow, and liver in these HRG1 deficient mice indicated that these mice were still accumulating high levels of heme. Further experiments revealed that these mice protected themselves from toxicity by converting the excess heme into crystals called hemozoin. This method of detoxification is commonly seen in blood-feeding parasites, and this is the first time it has been observed in a mammal. These crystals invite new questions about how mammals recycle heme and what happens when this process goes wrong. The next step is to ask whether humans also start to make hemozoin if the gene for HRG1 is faulty. If so, this could open a new avenue of exploration into treatments for red blood cell diseases like anemia and iron overload.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

ATP7A and ATP7B copper transporters have distinct functions in the regulation of neuronal dopamine-ß-hydroxylase.

The copper (Cu) transporters ATPase copper-transporting alpha (ATP7A) and ATPase copper-transporting beta (ATP7B) are essential for the normal function of the mammalian central nervous system. Inactivation of ATP7A or ATP7B causes the severe neurological disorders, Menkes disease and Wilson disease, respectively. In both diseases, Cu imbalance is associated with abnormal levels of the catecholamine-type neurotransmitters dopamine and norepinephrine. Dopamine is converted to norepinephrine by dopamine-ß-hydroxylase (DBH), which acquires its essential Cu cofactor from ATP7A. However, the role of ATP7B in catecholamine homeostasis is unclear. Here, using immunostaining of mouse brain sections and cultured cells, we show that DBH-containing neurons express both ATP7A and ATP7B. The two transporters are located in distinct cellular compartments and oppositely regulate the export of soluble DBH from cultured neuronal cells under resting conditions. Down-regulation of ATP7A, overexpression of ATP7B, and pharmacological Cu depletion increased DBH retention in cells. In contrast, ATP7B inactivation elevated extracellular DBH. Proteolytic processing and the specific activity of exported DBH were not affected by changes in ATP7B levels. These results establish distinct regulatory roles for ATP7A and ATP7B in neuronal cells and explain, in part, the lack of functional compensation between these two transporters in human disorders of Cu imbalance.

Altered zinc balance in the Atp7b-/- mouse reveals a mechanism of copper toxicity in Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder caused by mutation in the ATP7B gene that affects copper transport in the body. ATP7B mutation damages copper transporter function, ultimately resulting in excessive copper accumulation and subsequent toxicity in both the liver and brain. Mechanisms of copper toxicity, however, are not well defined. The Atp7b-/- mouse model is well-characterized and presents a hepatic phenotype consistent with WD. In this study, we found that the untreated Atp7b-/- mice accumulate approximately 2-fold excess hepatic zinc compared to the wild type. We used targeted transcriptomics and proteomics to analyze the molecular events associated with zinc and copper accumulation in the Atp7b-/- mouse liver. Altered gene expression of Zip5 and ZnT1 zinc transporters indicated a transcriptional homeostatic response, while increased copper/zinc ratios associated with high levels of metallothioneins 1 and 2, indicated altered Zn availability in cells. These data suggest that copper toxicity in Wilson disease includes effects on zinc-dependent proteins. Transcriptional network analysis of RNA-seq data reveals an interconnected network of transcriptional activators with over-representation of zinc-dependent and zinc-responsive transcription factors. In the context of previous research, these observations support the hypothesis that mechanisms of copper toxicity include disruption of intracellular zinc distribution in liver cells. The translational significance of this work lies in oral zinc supplementation in treatment for WD, which is thought to mediate protective effects through the induction of metallothionein synthesis in the intestine. This work indicates broader impacts of altered zinc-copper balance in WD, including global transcriptional responses and altered zinc balance in the liver.

Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes.

Copper (Cu) has emerged as an important modifier of body lipid metabolism. However, how Cu contributes to the physiology of fat cells remains largely unknown. We found that adipocytes require Cu to establish a balance between main metabolic fuels. Differentiating adipocytes increase their Cu uptake along with the ATP7A-dependent transport of Cu into the secretory pathway to activate a highly up-regulated amino-oxidase copper-containing 3 (AOC3)/semicarbazide-sensitive amine oxidase (SSAO); in vivo, the activity of SSAO depends on the organism's Cu status. Activated SSAO oppositely regulates uptake of glucose and long-chain fatty acids and remodels the cellular proteome to coordinate changes in fuel availability and related downstream processes, such as glycolysis, de novo lipogenesis, and sphingomyelin/ceramide synthesis. The loss of SSAO-dependent regulation due to Cu deficiency, limited Cu transport to the secretory pathway, or SSAO inactivation shifts metabolism towards lipid-dependent pathways and results in adipocyte hypertrophy and fat accumulation. The results establish a role for Cu homeostasis in adipocyte metabolism and identify SSAO as a regulator of energy utilization processes in adipocytes.

Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model.

Menkes disease is a lethal neurodegenerative disorder of copper metabolism caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Based on our prior clinical and animal studies, we seek to develop a therapeutic approach suitable for application in affected human subjects, using the mottled-brindled (mo-br) mouse model that closely mimics the Menkes disease biochemical and clinical phenotypes. Here, we evaluate the efficacy of low-, intermediate-, and high-dose recombinant adeno-associated virus serotype 9 (rAAV9)-ATP7A delivered to the cerebrospinal fluid (CSF), in combination with subcutaneous administration of clinical-grade copper histidinate (sc CuHis, IND #34,166). Mutant mice that received high-dose (1.6 × 1010 vg) cerebrospinal fluid-directed rAAV9-rsATP7A plus sc copper histidinate showed 53.3% long-term (≥300-day) survival compared to 0% without treatment or with either treatment alone. The high-dose rAAV9-rsATP7A plus sc copper histidinate-treated mutant mice showed increased brain copper levels, normalized brain neurochemical levels, improvement of brain mitochondrial abnormalities, and normal growth and neurobehavioral outcomes. This synergistic treatment effect represents the most successful rescue to date of the mo-br mouse model. Based on these findings, and the absence of a large animal model, we propose cerebrospinal fluid-directed rAAV9-rsATP7A gene therapy plus subcutaneous copper histidinate as a potential therapeutic approach to cure or ameliorate Menkes disease.

The Function of ATPase Copper Transporter ATP7B in Intestine.

BACKGROUND & AIMS: Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver-the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues. METHODS: We collected intestinal tissues from ATP7B-knockout (Atp7b-/-) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy. RESULTS: ATP7B maintains a Cu gradient along the duodenal crypt-villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b-/- mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons. CONCLUSIONS: ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.

Copper, zinc and calcium: imaging and quantification in anterior pituitary secretory granules.

The anterior pituitary is specialized for the synthesis, storage and release of peptide hormones. The activation of inactive peptide hormone precursors requires a specific set of proteases and other post-translational processing enzymes. High levels of peptidylglycine α-amidating monooxygenase (PAM), an essential peptide processing enzyme, occur in the anterior pituitary. PAM, which converts glycine-extended peptides into amidated products, requires copper and zinc to support its two catalytic activities and calcium for structure. We used X-ray fluorescence microscopy on rat pituitary sections and inductively coupled plasma mass spectrometry on subcellular fractions prepared from rat anterior pituitary to localize and quantify copper, zinc and calcium. X-ray fluorescence microscopy indicated that the calcium concentration in pituitary tissue was about 2.5 mM, 10-times more than zinc and 50-times more than copper. Although no higher than cytosolic levels, secretory granule levels of copper exceeded PAM levels by a factor of 10. Atp7a, which transports copper into the lumen of the secretory pathway, was enriched in endosomes and Golgi, not in secretory granules. If Atp7a transfers copper directly to PAM, this pH-dependent process is likely to occur in Golgi and endosomes.

Systemic Delivery and Biodistribution of Cisplatin in Vivo.

Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

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.

Oral zinc reduces amyloid burden in Tg2576 mice.

The aggregation of amyloid-ß in Alzheimer's disease can be affected by free transition metals such as copper and zinc in the brain. Addition of copper and zinc with amyloid acts to increase aggregation and copper additionally promotes the formation of reactive oxygen species. We propose that reduction of brain copper by blocking uptake of copper from the diet is a viable strategy to regulate the formation of insoluble amyloid-ß in the brain of Tg2576 mice. Mice were treated with regimens of zinc acetate, which acts with metallothionein to block copper uptake in the gut, at various times along their lifespan to model prevention and treatment paradigms. We found that the mice tolerated zinc acetate well over the six month course of study. While we did not observe significant changes in cognition and behavior, there was a reduction in insoluble amyloid-ß in the brain. This observation coincided with a reduction in brain copper and interestingly no change in brain zinc. Our findings show that blocking copper uptake from the diet can redistribute copper from the brain and reduce amyloid-ß aggregation.