Copper Deprivation Research Articles

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

High-iron consumption decreases copper accumulation and colon length, and alters serum lipids

In this study, we aimed to demonstrate that a significant increase in dietary iron intake disrupts the regulation of copper availability, ultimately leading to systemic copper deficiency. To investigate this, we conducted experiments using five-week-old male weanling Sprague–Dawley rats fed diets based on AIN-93G with some modifications. These diets featured varying iron content, offering choices of adequate iron (~ 120 μg/g, near the upper limit [UL]) or high iron (~ 7544 μg/g), along with low (~ 0.3 μg/g), adequate (~ 6 μg/g), or high (~ 153 μg/g) levels of dietary copper over a 5-week period. Rats consuming the high-iron diets displayed anemia, reduced copper levels in their organs and feces, and shortened colon lengths. Increased dietary iron intake resulted in an overall reduction in copper distribution within the body, likely leading to severe copper deficiency-related disorders in the experimental rats. However, the physiological disturbances caused by a high-iron diet were prevented when additional copper was included in the rodent diet. Furthermore, high iron intake led to copper deprivation, and high iron consumption resulted in elevated serum cholesterol levels. However, increasing dietary copper consumption led to a decrease in overall serum cholesterol levels. Additionally, serum alkaline phosphate and aspartate aminotransferase levels were increased by high-iron feeding, regardless of dietary copper concentration, while alanine aminotransferase levels decreased.

Impact of biochar amendments on copper mobility, phytotoxicity, photosynthesis and mineral fluxes on (Zea mays L.) in contaminated soils

Soil copper (Cu2+) contamination is a source of phytotoxicity and biochar incorporation is a possible mitigation strategy for improving plant performance and avoiding Cu2+ contamination. This study evaluated the effect of biochar (0, 1 and 2% BC) and/or copper (0 and 100 ppm) on growth, photosynthesis, Cu2+ accumulation, and mineral content of maize grown under controlled greenhouse conditions. Biochar application increased water holding capacity (WHC), while the addition of Cu2+ improved electric conductivity (EC) in soil samples. The sole copper treatment enhanced Cu-accumulation, mineral uptake (K+, Na+, Ca++ and Mg++), dark respiration and light dependent electron flux in PSII. However, it also led to a reduction of growth and net CO2 assimilation rate and caused an enhanced ETR/Pg, indicating a threat of ROS production. The sole addition of biochar to the soil had a positive impact on the total macronutrient content but a negative impact on growth, leaf CO2/H2O gasexchange. However, the combined application of BC and Cu2+ led to a reduced tissue Cu2+ levels, higher shoot growth CO2/H2O gasexchange (Pn, WUE, COE) and finally a reduced ETR/Pg ratio. Plants treated with biochar and higher Cu2+ levels decreased about 30% ETR/Pg which is associated with the optimum growth and photosynthetic efficiency of Photosystem II. Hence, it could be clearly shown that BC and Cu2+ achieved together, and contrary to their individual mode of action, an improvement of Pn, WUE, COE and Rd. We assume that BC application in the studied soil highly restricted the availability of added Cu promoting Cu-stable forms thus reducing the environmental risk and phytotoxicity at soils high in copper (2%BC, 100 ppm Cu2+). The study proposes that BC as a suitable amendment for restoring degraded soils with toxic trace elements but not for soils with limited supply of this micronutrient or copper deprivation

Copper deprivation enhances the chemosensitivity of pancreatic cancer to rapamycin by mTORC1/2 inhibition

Cuproplasia, or copper-dependent cell proliferation, has been observed in varieties of solid tumors along with aberrant copper homeostasis. Several studies reported good response of patients to copper chelator assisted neoadjuvant chemotherapy, however, the internal target molecules are still undetermined. Unravel copper-associated tumor signaling would be valuable to forge new links to translate biology of copper into clinical cancer therapies. We evaluated the significance of high-affinity copper transporter-1 (CTR1) by bioinformatic analysis, and in 19 pairs of clinical specimens. Then, with the help of gene interference and chelating agent, enriched signaling pathways were identified by KEGG analysis and immunoblotting. Accompanying biological capability of pancreatic carcinoma-associated proliferation, cell cycle, apoptosis, and angiogenesis were investigated. Furthermore, a combination of mTOR inhibitor and CTR1 suppressor has been assessed in xenografted tumor mouse models. Hyperactive CTR1 was investigated in pancreatic cancer tissues and proven to as the key point of cancer copper homeostasis. Intracellular copper deprivation induced by CTR1 gene knock-down or systematic copper chelation by tetrathiomolybdate suppressed proliferation and angiogenesis of pancreatic cancer cell. PI3K/AKT/mTOR signaling pathway was suppressed by inhibiting the activation of p70(S6)K and p-AKT, and finally inhibited mTORC1 and mTORC2 after copper deprivation. Additionally, CTR1 gene silencing successfully improved the anti-cancer effect of mTOR inhibitor rapamycin. Our study reveals that CTR1 contributes to pancreatic tumorigenesis and progression, by up-regulating the phosphorylation of AKT/mTOR signaling molecules. Recovering copper balance by copper deprivation addresses as promising strategy for improved cancer chemotherapy.

The pathogenesis of DLD-mediated cuproptosis induced spinal cord injury and its regulation on immune microenvironment.

Spinal cord injury (SCI) is a severe central nervous system injury that leads to significant sensory and motor impairment. Copper, an essential trace element in the human body, plays a vital role in various biological functions and is strictly regulated by copper chaperones and transporters. Cuproptosis, a novel type of metal ion-induced cell death, is distinct from iron deprivation. Copper deprivation is closely associated with mitochondrial metabolism and mediated by protein fatty acid acylation. In this study, we investigated the effects of cuproptosis-related genes (CRGs) on disease progression and the immune microenvironment in acute spinal cord injury (ASCI) patients. We obtained the gene expression profiles of peripheral blood leukocytes from ASCI patients using the Gene Expression Omnibus (GEO) database. We performed differential gene analysis, constructed protein-protein interaction networks, conducted weighted gene co-expression network analysis (WGCNA), and built a risk model. Our analysis revealed that dihydrolipoamide dehydrogenase (DLD), a regulator of copper toxicity, was significantly associated with ASCI, and DLD expression was significantly upregulated after ASCI. Furthermore, gene ontology (GO) enrichment analysis and gene set variation analysis (GSVA) showed abnormal activation of metabolism-related processes. Immune infiltration analysis indicated a significant decrease in T cell numbers in ASCI patients, while M2 macrophage numbers were significantly increased and positively correlated with DLD expression. In summary, our study demonstrated that DLD affects the ASCI immune microenvironment by promoting copper toxicity, leading to increased peripheral M2 macrophage polarization and systemic immunosuppression. Thus, DLD has potential as a promising biomarker for ASCI, providing a foundation for future clinical interventions.

Moderate Copper Deprivation during Gestation and Lactation Affects Dentate Gyrus and Hippocampal Maturation in Immature Male Rats

The hippocampal formation (HF) is involved in higher brain functions including learning and declarative memory. The possibility that dietary copper has a role in the morphological development, and therefore the function of the HF, has received little attention. A rat model of tiered copper deficiency, initiated during gestation, was employed to determine the susceptibility of the HF, regions of which develop postnatally, to copper deficiency. At postnatal 23, pups whose dams had received either 1.8 or 1.4 mg Cu/kg diet during both gestation and lactation, compared with offspring of a group that had received 4.3 mg Cu/kg diet during both periods had, significantly more cell nuclei in the infrapyrimidal arm of the dentate gyrus. Offspring of rats fed 1.4 mg Cu/kg diet, but not those fed 1.8 mg/kg, compared with those fed 4.3 mg/kg, exhibited smaller, shorter, and narrower cell nuclei in the infrapyrimidal and suprapyrimidal arms of the dentate gyrus and smaller cell nuclei in region CA3c of the hippocampus. A fourth group (gestation, 1.8 mg Cu/kg diet; lactation, 0.9 mg Cu/kg diet) exhibited alterations less marked than those exhibited by the group fed 1.4 mg Cu/kg diet. All alterations in the groups fed low copper diets were consistent with slowed cell nuclear maturation. The findings indicate that copper is required for maturation of the dentate gyrus and hippocampus. Also, copper supplied at or below 1.8 mg/kg is insufficient for morphological maturation of the dentate gyrus and hippocampus. Because the HF is important for higher brain functions, further research is needed to determine whether the copper deficiency-induced alterations in dentate gyrus and hippocampus development are transient or permanent.

Dietary Copper Deficiency Leads to Changes in Gene Expression Indicating an Increased Demand for NADH in the Prefrontal Cortex of the Rat's Brain.

Copper is an essential element to brain cells as it is a cofactor and a structural component of various enzymes involved in energy metabolism pathways. Accumulating evidence points to the pivotal role of copper deficiency in neurodegeneration resulting from impaired copper homeostasis. Despite the indisputable role of copper in mitochondrial respiration, its homeostasis regulation in the brain tissue remains unclear. The assessment of changes in the expression of genes encoding key pathways of energy metabolism can greatly benefit further studies exploring copper’s role in neurodegeneration. Using a rat model, we investigate whether the replacement of the inorganic form of copper with metallic nanoparticles containing copper or complete deprivation of copper from the diet have an impact on the expression of genes involved in energy metabolism in the prefrontal cortex of the rats’ brain. Herein, we indicate that removing inorganic copper from the normal standard diet or the replacement with copper nanoparticles can lead to programmed energy metabolism changes. It can be recognized that some of these changes indicate an increased demand for NADH in the prefrontal cortex of the rat’s brain, probably as a result of adaptation effect.

Copper Metabolism in Naegleria gruberi and Its Deadly Relative Naegleria fowleri.

Although copper is an essential nutrient crucial for many biological processes, an excessive concentration can be toxic and lead to cell death. The metabolism of this two-faced metal must be strictly regulated at the cell level. In this study, we investigated copper homeostasis in two related unicellular organisms: nonpathogenic Naegleria gruberi and the “brain-eating amoeba” Naegleria fowleri. We identified and confirmed the function of their specific copper transporters securing the main pathway of copper acquisition. Adjusting to different environments with varying copper levels during the life cycle of these organisms requires various metabolic adaptations. Using comparative proteomic analyses, measuring oxygen consumption, and enzymatic determination of NADH dehydrogenase, we showed that both amoebas respond to copper deprivation by upregulating the components of the branched electron transport chain: the alternative oxidase and alternative NADH dehydrogenase. Interestingly, analysis of iron acquisition indicated that this system is copper-dependent in N. gruberi but not in its pathogenic relative. Importantly, we identified a potential key protein of copper metabolism of N. gruberi, the homolog of human DJ-1 protein, which is known to be linked to Parkinson’s disease. Altogether, our study reveals the mechanisms underlying copper metabolism in the model amoeba N. gruberi and the fatal pathogen N. fowleri and highlights the differences between the two amoebas.

Studies of High Molybdenum-Induced Copper Deprivation in P. przewalskii on the Qinghai Lake Pasture in China

The Przewalski’s Gazelles (P. przewalskii) are affected by disorders that are characterized by deprived appetites, pica, emaciation and dyskinesia. The purpose of this study was to investigate the possibility of high molybdenum (Mo) in forage leading to copper (Cu) deprivation. The mineral contents in forage, soil, and samples of hair and blood from affected ranges were compared to healthy pasture. Blood parameters were also determined. Our results showed that the mean content of Mo in forage and soil in studied pasture was 5.17 and 4.17 μg/g, respectively. The Cu to Mo ratio in forage from affected and healthy areas was 1.26 and 5.89 μg/g, respectively. The Cu concentrations in hair and blood from gazelles in affected pasture were extremely lower (p < 0.01) than those in unaffected animals. The Mo contents in hair were higher (p < 0.01) than those in unaffected gazelles. The levels of Hb, RBC, PCV, MCV, and MCH in unaffected gazelles were significantly lower (p < 0.01) than those in unaffected gazelles. The levels of TP, ALB and GLB in blood were significantly lower (p < 0.01) than those in unaffected gazelles, while the levels of AST, LDH, CPK and ALP in serum were significantly higher (p < 0.01) than those in unaffected gazelles. The activities in serum T-AOC, SOD, GSH-Px, and CAT in affected gazelles were extremely lower (p < 0.01) than those in unaffected gazelles, while MDA was significantly higher (p < 0.01) than that in unaffected gazelles. Supplementation in copper sulphate (CuSO4) has prevented and cured this disorder. In summary, high molybdenum in feed and soil may lead to secondary Cu deficiency in gazelles, which can be alleviated by supplement of copper sulfate.

Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation.

Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.

Responses of Chinese Merino Sheep (Junken Type) on Copper-Deprived Natural Pasture.

To research responses of Chinese Merino Sheep (Junken type) to copper-deprived natural pasture, we analyzed mineral contents in soil, forage, and sheep tissues. Physiological and biochemical parameters were also determined. Results showed that copper concentrations in soil and forage from affected pastures were significantly lower than those in healthy ranges (P < 0.01). Copper contents in animal tissues (blood, liver, and wool) from affected Chinese Merino Sheep were also significantly lower than those in healthy sheep (P < 0.01). Hemoglobin levels, packed cell volume, mean corpuscular volume, and mean corpuscular hemoglobin from affected Chinese Merino Sheep were significantly lower than those in healthy animals (P < 0.01). Serum ceruloplasmin was significantly lower in affected Chinese Merino Sheep than that in healthy animals, while activities of lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in serum were significantly higher in affected compared with healthy animals (P < 0.01). The levels of superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and catalase in serum were significantly lower, and the malondialdehyde content was significantly higher in affected compared with healthy sheep (P < 0.01). The levels of interleukin-2, interleukin 6, interleukin-1β, immunoglobulin A, immunoglobulin M, and immunoglobulin G in copper-deprived sheep were significantly lower than those in healthy animals (P < 0.01). Copper deprivation in forage not only influenced the mineral content in blood but also severely disrupted blood parameters (physiology, biochemistry, immunity, and antioxidant) in Chinese Merino Sheep.

Low copper availability limits Helicobacter infection in mice.

Copper is an essential element for all living organisms; however, it becomes toxic at high concentrations due to its ability to participate in many redox reactions. This vital micronutrient balance plays an important role in the battle between host and pathogen, due to its use by the host to intoxicate pathogens. In this study, we explore the effects of copper deprivation on Helicobacter infection in mice using the copper chelator tetrathiomolybdate. Our results reveal that Helicobacter infection significantly reduces copper concentration in mice stomachs without affecting its circulating levels. Moreover, in copper-deprived mice, bacteria hardly colonize the epithelium and mice show less gastric damage in comparison with the infected ones. However, when the copper chelator is administered after infection, the condition of the mouse stomachs declines. This could be explained by the lower copper availability in tetrathiomolybdate-treated mice, which would reduce macrophages' action against the pathogen. In this scenario, we observe that the protective factor trefoil factor 1 is induced upon copper-deprived conditions, probably contributing to the inefficacy of infection, whereas, when the chelator is administered after infection, the gene is already silenced by bacteria and cannot be restored. In conclusion, our data suggest that Helicobacter takes advantage of gastric copper reducing its availability for the host and that copper levels have an impact on the outcome of infection.

Copper and iron metabolism in Ostreococcus tauri - the role of phytotransferrin, plastocyanin and a chloroplast copper-transporting ATPase.

Iron and copper are essential elements for practically all living organisms. Their metabolism is frequently interconnected, and while copper is relatively abundant in the ocean, iron is often a limiting factor for the growth of many marine microorganisms. In the present study, we aimed to elucidate the metabolisms of copper and iron and the connection of both in the marine picoalga Ostreococcus tauri. We show that O. tauri adjusts its copper economy in response to copper deficiency by downregulation of the expression of plastocyanin in favor of cytochrome c oxidase without significant changes in growth and physiology. Copper deprivation leads to increased expression of copper transporting ATPase and proteins involved in tetrapyrrole synthesis, most likely to ensure higher turnover of chlorophyll and/or heme. Elucidation of the effect of copper on the incorporation of iron into O. tauri proteins led us to identify the major iron uptake mediating protein, Ot-Fea1, whose expression and binding of iron is copper dependent. Based on our investigation of the incorporation of iron into Ot-Fea1 and ferritin, we hypothesize that O. tauri possesses another Fea1-independent iron uptake system.

The copper-sensing transcription factor Mac1, the histone deacetylase Hst1, and nicotinic acid regulate de novo NAD+ biosynthesis in budding yeast

NADH (NAD+) is an essential metabolite involved in various cellular biochemical processes. The regulation of NAD+ metabolism is incompletely understood. Here, using budding yeast (Saccharomyces cerevisiae), we established an NAD+ intermediate-specific genetic system to identify factors that regulate the de novo branch of NAD+ biosynthesis. We found that a mutant strain (mac1Δ) lacking Mac1, a copper-sensing transcription factor that activates copper transport genes during copper deprivation, exhibits increases in quinolinic acid (QA) production and NAD+ levels. Similar phenotypes were also observed in the hst1Δ strain, deficient in the NAD+-dependent histone deacetylase Hst1, which inhibits de novo NAD+ synthesis by repressing BNA gene expression when NAD+ is abundant. Interestingly, the mac1Δ and hst1Δ mutants shared a similar NAD+ metabolism-related gene expression profile, and deleting either MAC1 or HST1 de-repressed the BNA genes. ChIP experiments with the BNA2 promoter indicated that Mac1 works with Hst1-containing repressor complexes to silence BNA expression. The connection of Mac1 and BNA expression suggested that copper stress affects de novo NAD+ synthesis, and we show that copper stress induces both BNA expression and QA production. Moreover, nicotinic acid inhibited de novo NAD+ synthesis through Hst1-mediated BNA repression, hindered the reuptake of extracellular QA, and thereby reduced de novo NAD+ synthesis. In summary, we have identified and characterized novel NAD+ homeostasis factors. These findings will expand our understanding of the molecular basis and regulation of NAD+ metabolism.

Blockage of SLC31A1-dependent copper absorption increases pancreatic cancer cell autophagy to resist cell death.

ObjectivesClinical observations have demonstrated that copper levels elevate in several cancer types, and copper deprivation is shown to inhibit tumour angiogenesis and growth in both animal models and preclinical trials. However, the content of copper in pancreatic duct adenocarcinoma (PDAC) and whether it is a potential therapy target is still unknown.Materials and MethodsThe levels of copper in PDAC specimens were detected by ICP‐MS assays. Copper depletion in Panc‐1 or MiaPaCa‐2 cells was conducted via copper transporter 1 (SLC31A1) interference and copper chelator tetrathiomolybdate (TM) treatment. The effects of copper deprivation on cancer cells were evaluated by cell proliferation, migration, invasion, colony formation and cell apoptosis. The mechanism of copper deprivation‐caused cancer cell quiescence was resolved through mitochondrial dysfunction tests and autophagy studies. The tumour‐suppression experiments under the condition of copper block and/or autophagy inhibition were performed both in vitro and in xenografted mice.Results SLC31A1‐dependent copper levels are correlated with the malignant degree of pancreatic cancer. Blocking copper absorption could inhibit pancreatic cancer progression but did not increase cell death. We found that copper deprivation increased mitochondrial ROS level and decreased ATP level, which rendered cancer cells in a dormant state. Strikingly, copper deprivation caused an increase in autophagy to resist death of pancreatic cancer cells. Simultaneous treatment with TM and autophagy inhibitor CQ increased cell death of cancer cells in vitro and retarded cancer growth in vivo.ConclusionsThese findings reveal that copper deprivation‐caused cell dormancy and the increase in autophagy is a reason for the poor clinical outcome obtained from copper depletion therapies for cancers. Therefore, the combination of autophagy inhibition and copper depletion is potentially a novel strategy for the treatment of pancreatic cancer and other copper‐dependent malignant tumours.

Low Ctr1p, due to lack of Sco1p results in lowered cisplatin uptake and mediates insensitivity of rho0 yeast to cisplatin

Copper and cisplatin share copper transporter 1 (Ctr1) for cellular import. Copper depletion increases sensitivity of wild type yeast to cisplatin, whereas mitochondrial DNA-deficient rho0 cells are resistant to cisplatin. In the current study, we sought to determine whether copper deprivation modulates sensitivity of rho0 yeast to cisplatin. Yeast cultures grown in low copper medium and exposed to bathocuproine disulfonic acid resulted in significant reduction of intracellular copper. We report here that low copper medium rendered wild type hypersensitive to cisplatin, but failed to sensitize rho0 yeast to cisplatin. Wild type yeast grown in low copper medium exhibited ~2.0 fold enhanced cytotoxicity in survival and colony-forming ability compared to copper adequate wild type cells. The effect of copper restriction on cisplatin sensitivity was associated with upregulation of copper transporter 1 mRNA as well as protein, facilitating enhanced uptake and accumulation of cisplatin. Rho0 yeast also showed increased copper transporter 1 mRNA upon copper restriction, but failed to increase corresponding protein. Loss of synthesis of cytochrome coxidase 1 protein (Sco1) in rho0 cells deregulated copper transporter 1, impaired Pt uptake and lowered cytotoxicity, despite lowered glutathione levels. Sco1Δ mutants exhibited low copper transporter 1, reduced Pt accumulation suggesting that Sco1 mediated regulation of copper transporter 1 is responsible for altered sensitivity to cisplatin. Rho0 cells demonstrated loss of Sco1, resulting in copper deficiency by lowering copper transporter 1 abundance, via mechanism involving increased turnover due to ubiquitination. These findings reveal that a Sco1-dependent mitochondrial signal regulates cellular cisplatin import and cytotoxicity.

CHCA-1 is a copper-regulated CTR1 homolog required for normal development, copper accumulation, and copper-sensing behavior in Caenorhabditis elegans

Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.

AB079. P051. Blockage of CTR1-dependent copper absorption increases autophagy to resist apoptosis of pancreatic ductal carcinoma cells

: Clinical observations have demonstrated that copper levels elevate in a number of cancer types, and copper deprivation is considered a prospective anti-cancer therapeutic strategy. However, the cellular mechanism underlying copper depletion in cancer therapy is still not fully understood. Here, we demonstrate that CTR1-dependent copper level is negatively correlated with the survival time of pancreatic ductal carcinoma patients and copper increase is important for pancreatic ductal adenocarcinoma progression. However, copper depletion via CTR1 knockdown or copper chelation did not induce pancreatic cancer cell apoptosis. We found that copper deprivation causes increased ROS and decreased ATP, which rendered cancer cells in a dormant state. Strikingly, copper deprivation caused an increase in autophagy to resist apoptosis of pancreatic cancer cells. Simultaneous treatment with copper chelator tetrathiomolybdate (TM) and autophagy inhibitor chloroquine diphosphate salt (CQ) increased apoptotic cancer cells in vitro and retarded cancer growth in xenotrans planted mice. These findings reveal that copper deprivation-caused cell dormancy and an increase in autophagy is a major reason for the poor clinical outcome obtained from copper depletion therapies for cancers. Therefore, the combination of autophagy inhibition and copper depletion is potentially a novel strategy for the treatment of pancreatic cancer and other copper-dependent malignant tumours.

Regression of pressure overload‐induced cardiac hypertrophy by TETA‐mediated myocardial copper supplementation in rats

Sustained pressure overload leads to cardiac hypertrophy and copper efflux from the heart. Dietary copper supplementation replenishes copper in the heart and reverses cardiac hypertrophy. On the contrary, a copper selective chelator, trientine (TETA), reverses left ventricular hypertrophy associated with diabetes. The present study was undertaken to test the hypothesis that TETA functions as a copper chaperone delivering copper to the heart leading to regression of cardiac hypertrophy. Adult male Sprague‐Dawley rats were subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Eight weeks after the surgery cardiac hypertrophy was developed and myocardial copper was reduced. TETA was then administrated by gavage at two different doses (21.9 mg/kg·day and 87.6 mg/kg·day) for six weeks. The results showed that at lower dose, TETA replenished copper contents in the heart, accompanied by a decrease in blood plasma and an increase in urine of copper concentrations. At higher dose, TETA did not replenish copper contents in the heart, but markedly increased copper concentrations in the urine, along with a decrease in serum copper concentrations. Corresponding to myocardial copper repletion, lower dose TETA reversed cardiac hypertrophy, as judged by a reduction in the left ventricle wall thickness (LVAWd and LVPWd) and a decrease in the heart size (heart weight/tibia length), and diminished cardiac fibrosis, as reflected by a decrease in collagen I content. TETA at higher dose not only did not regressed cardiac hypertrophy, but caused cardiac hypertrophy in sham‐operated rats. This study demonstrates that TETA, at lower dose, functioned as a copper chaperone delivering copper to the heart of copper deprivation and, at higher dose, simply remained its chelator function removing copper from the body by urinary excretion.Support or Funding InformationSupported by National Science Foundation of China (81230004 and 81300109).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New insights into AOX2 transcriptional regulation in Chlamydomonas reinhardtii

A feature of the mitochondrial electron transport chain in plants, some protists and many fungi is the presence of two terminal oxidases, the energy-conserving cytochrome oxidase and another termed alternative oxidase (AOX). AOX branches from the main respiratory chain, directly coupling the oxidation of ubiquinol with reduction of oxygen to water. The AOX genes can be divided into two discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In the model green alga Chlamydomonas reinhardtii, two genes have been identified that encode for AOX, AOX1 and AOX2. To test AOX2 transcriptional regulation in this alga, we used a real-time PCR analysis and an artificial microRNA approach. The C. reinhardtii AOX2 gene is up-regulated by oxygen or copper deprivation. Moreover, in dark-adapted unstressed cells, AOX2 is induced. Together, our results imply that the AOX2 gene is a stress-inducible and is regulated by the copper response regulator 1 (CRR1), probably together with yet-unknown regulatory factor(s).