Copper Uptake Research Articles

Hydrogen Peroxide Mitigates Cu Stress in Wheat

Abiotic stress imposed by heavy metals (HMs) adversely influences plant growth. In crop plants, such stresses penalize grain yield and ultimately could have enduring connotations for sustainable food security. Although copper (Cu) is an essential micronutrient for crop life, excessive availability of copper impairs plant growth and/or reproductive performance. Anecdotal evidence suggests that hydrogen peroxide (H2O2) is produced in plants under either biotic or abiotic stresses to mitigate oxygen-derived cell toxicity, although the influence of H2O2 remains to be definitively quantified. Here, our aim was to investigate the effects of hydrogen peroxide (H2O2) on the growth, grain yield, and yield components, as well as copper uptake of stressed wheat grown in sandy soil. We found that applications rates of 150 or 300 mg Cu kg−1 soil significantly reduced net photosynthesis, leaf area, chlorophyll, and grain yield. Foliar application of H2O2 to plants grown under 150 and 300 mg Cu kg−1 soil had improved growth, physiological, and yield traits. For instance, foliar application of H2O2 Cu-stressed plants grown under 300 mg Cu kg−1 soil reduced detrimental effects of Cu toxicity by −12% in terms of grains per spike and −7% for 1000-grain weight in comparison to the control treatment. Foliar application of H2O2 on wheat grown under copper stress reduced accumulation of other heavy metals such as cadmium. We suggest that the potential for foliar application of H2O2 in mitigating heavy metal stress in crop plants has large global potential; however, further work is required to elucidate the environmental conditions and application rates required to attain optimal benefit.

Arabidopsis COPT1 copper transporter uses a single histidine to regulate transport activity and protein stability

Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.

Uptake and Distribution of Cadmium and Copper by Solanum lycopersicum L. and Changes in the Metabolite Production

Uptake and Distribution of Cadmium and Copper by Solanum lycopersicum L. and Changes in the Metabolite Production

Removal of copper and iron from ethanolic solutions by an anion exchange resin and its implication to rare-earth magnet recycling

In the recycling of end-of-life rare-earth magnets, the recovery of non-rare earth constituents is often neglected. In the present study, strong cation and anion exchange resins were tested batchwise for the recovery of the non-rare-earth constituents of permanent magnets (copper, cobalt, manganese, nickel and iron) from synthetic aqueous and ethanolic solutions. The cation exchange resin recovered most of metal ions from aqueous and ethanolic feeds, whereas the anion exchange resin could selectively recover copper and iron from ethanolic feeds. The highest uptake of iron and copper was found for 80 vol% and 95 vol% multi-element ethanolic feeds, respectively. A similar trend in selectivity of the anion resin was observed in breakthrough curve studies. Batch experiments, UV–Vis, FT-IR and XPS studies were performed to elucidate the ion exchange mechanism. The studies indicate that the formation of chloro complexes of copper and their exchange by the (hydrogen) sulfate counter ions of the resin have an important role in the selective uptake of copper from the 95 vol% ethanolic feed. Iron(II) was largely oxidized to iron(III) in ethanolic solutions and was expected to be recovered by the resin in the form of iron(II) and iron(III) complexes. The moisture content of the resin did not have a significant role on the selectivity for copper and iron.

Abstract 1687: ATPase copper transporting beta (ATP7B) contributes to acquired docetaxel resistance in human prostate cancer

Abstract Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but the resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel resistant mPCa cell lines exhibit less uptake of cellular copper and express markedly higher levels of ATP7B protein without significant changes in the protein expression levels of other copper transporters, such as ATPase copper transporting alpha (ATP7A) and high affinity copper uptake protein 1 (CTR1). Knock-down of ATP7B by silencing RNAs sensitized docetaxel resistant-mPCa cells to the growth inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, Disulfiram (DSF), an FDA approved drug for treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase associated protein 2 (Skp2) and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results suggest that ATP7B is a clinically relevant and key target for improving the efficacy of docetaxel for treatment of mPCa. Citation Format: Liankun Song, Vyvyan Nguyen, Jun Xie, Matthew Tippin, Le TP Truong, Christopher A. Blair, Beverly Wang, Edward Uchio, Xiaolin Zi. ATPase copper transporting beta (ATP7B) contributes to acquired docetaxel resistance in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1687.

Grf10 regulates the response to copper, iron, and phosphate in Candida albicans.

The pathogenic yeast, Candida albicans, and other microbes must be able to handle drastic changes in nutrient availability within the human host. Copper, iron, and phosphate are essential micronutrients for microbes that are sequestered by the human host as nutritional immunity; yet high copper levels are employed by macrophages to induce toxic oxidative stress. Grf10 is a transcription factor important for regulating genes involved in morphogenesis (filamentation, chlamydospore formation) and metabolism (adenylate biosynthesis, 1-carbon metabolism). The grf10Δ mutant exhibited resistance to excess copper in a gene dosage-dependent manner but grew the same as the wild type in response to other metals (calcium, cobalt, iron, manganese, and zinc). Point mutations in the conserved residues D302 and E305, within a protein interaction region, conferred resistance to high copper and induced hyphal formation similar to strains with the null allele. The grf10Δ mutant misregulated genes involved with copper, iron, and phosphate uptake in YPD medium and mounted a normal transcriptional response to high copper. The mutant accumulated lower levels of magnesium and phosphorus, suggesting that copper resistance is linked to phosphate metabolism. Our results highlight new roles for Grf10 in copper and phosphate homeostasis in C. albicans and underscore the fundamental role of Grf10 in connecting these with cell survival.

Effect of addition of organic manures on basmati yield, nutrient content and soil fertility status in north-western India

Integrated use of chemical fertilizers with organic manure is becoming a quite promising practice to maintain higher crop productivity and to manage soil health, which is otherwise deteriorated due to intensive cultivation and imbalanced fertilizer use. Thus, the present experiment was conducted for combined application of inorganic fertilizers and organic manures for higher yield and nutrient uptake in basmati rice as well as to restore soil health. The treatments applied in the present study involve T1: control, T2: Farmyard manure (15 t ha−1), T3: Poultry manure (6 t ha−1), T4: Press mud (15 t ha−1), T5: Rice straw compost (6 t ha−1), T6: Farmyard manure (15 t ha−1) + 50% N (recommended dose of nitrogen), T7: Poultry manure (6 t ha−1) + 50% N, T8: Press mud (15 t ha−1) + 50% N, T9: Rice straw compost (6 t ha−1) + 50% N, T10: 75% N, T11: Farmyard manure (15 t ha1) + 75% N, T12: Poultry manure (6 t ha−1) + 75% N, T13: Press mud (15 t ha−1) + 75% N, T14: Rice straw compost (6 t ha−1) + 75% N, T15: 100% N. The integrated use of organic manures and inorganic fertilizers significantly increased the grain and straw yield, macronutrients as well as micronutrients uptake in basmati due to the release of nutrients from decomposition of organic manures. Among different treatments, the addition of PM+75% N showed maximum grain yield, straw yield and sustainability yield index (44.53 q ha−1, 89.67 q ha−1 and 0.91 respectively) as well as the highest uptake of nitrogen (58.29 and 65.39 kg ha−1), phosphorus (25.04 and 23.24 kg ha−1) and potassium (15.26 and 118.95 kg ha−1) in grain and straw, respectively. Similar results were observed for zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) uptake under treatment involving PM+75% N with values 183.9, 26.18, 339.3 and 355.8 g ha−1 in grain and 205.3, 25.62, 2627.3 and 278 g ha−1 in straw, respectively. Additionally, correlation studies showed that the grain and straw yield of basmati exhibited a significantly positive correlation with soil P, Cu and Fe. The study suggested that the partial substitution of inorganic fertilizers with organic manures did not lower crop yield and nutrient uptake. Thus, integrated application of organic and inorganic fertilizers can be used for the sustainability of basmati-wheat system and to retain the soil fertility which is otherwise deteriorated with sole use of inorganic fertilizers.

Novel development of zinc oxide–coated carbon nanoparticles from pineapple leaves using sol gel method for optimal adsorption of Cu2+ and reuse in latent fingerprint application

This study underlines a latest approach of preparing nitrogen carbon nanoparticles fused on zinc oxide nanoparticle nanocomposite (N-CNPs/ZnONP nanocomposite) for the uptake of copper ions (Cu2+) from wastewater using a sol gel method. The metal loaded adsorbent was then applied in the latent fingerprint application. N-CNPs/ZnONP nanocomposite proved to be a good sorbent for the optimal adsorption of Cu2+ at pH 8 and 1.0 g/L dosage. Langmuir isotherm best fitted the process with the maximum adsorption capacity of 285.71 mg/g that was superior to most values reported in other studies for the removal of Cu2+. At 25 °C, the adsorption was spontaneous and endothermic. Furthermore, Cu2+-N-CNPs/ZnONP nanocomposite revealed to be sensitive and selective for latent fingerprint (LFP) identification on a variety of porous surfaces. As a result, it is an excellent identifying chemical for latent fingerprint recognition in forensic science.Graphical

AMPK phosphorylates and stabilises copper transporter 1 to synergise metformin and copper chelator for breast cancer therapy.

Predominant roles of copper and its transporter, copper transporter 1 (CTR1), in tumorigenesis have been explored recently; however, the upstream regulation of CTR1 and combinational intervention of copper chelators in malignancies remain largely unclear. CRISPR/Cas9-based kinome screening was used to identify the CTR1 upstream kinases. Immunofluorescence assays were utilised to detect CTR1 localisation. In vitro kinase assays and mass spectrometry were performed to detect CTR1 phosphorylation. Ubiquitination assays were performed to validate CTR1 stability. Colony formation, EdU labelling, Annexin V-FITC/PI-based apoptosis assays were carried out to detect the drug effect on cell growth and apoptosis. Xenografted mouse models were employed to investigate drug effects in vivo. We identify that CTR1 undergoes AMPK-mediated phosphorylation, which enhances CTR1 stabilisation and membrane translocation by affecting Nedd4l interaction, resulting in increased oncogenic roles in breast cancer. Importantly, activation of AMPK with its agonist metformin markedly enhances CTR1 levels, and leads to the combinational usage of AMPK agonists and copper chelators for breast cancer treatment. Our findings not only reveal the crosstalk between energy response and copper uptake via AMPK-mediated CTR1 phosphorylation and stability but also highlight the strategy to combat breast cancer by a combination of AMPK agonists and copper chelators. The connection between energy response and copper homoeostasis is linked by AMPK phosphorylating and stabilising CTR1, which provides a promising strategy to combat breast cancer by combining AMPK agonists and copper chelators.

Micronutrients uptake and soil nutrients status affected by different nutrient management practices under fodder pearl millet cultivation

The study was undertaken during kharif season of 2019-20 at Agronomy research farm of ICAR-NDRI, Karnal (Haryana). The experiment was laid out in Randomized Complete Block Design with eight treatments viz., Absolute control; 100% RDF; 100% RDF + Cow urine foliar spray (10% at 30 and 45 DAS); 100% RDF + PGPR; 100% RDF + PGPR + Cow urine foliar spray; 75% RDF + Cow urine foliar spray; 75% RDF + PGPR and 75% RDF + PGPR + Cow urine foliar spray with three replications of each treatment. Study indicated that maximum iron (347.6 ppm), manganese (44.6 ppm), copper (12.9 ppm) and zinc (38.3 ppm) content as well as uptake of iron (3938.6 g ha-1), manganese (506.8 g ha-1), copper (146.1 g ha-1) and zinc (433.8 g ha-1) by fodder pearl millet recorded with 100% RDF + PGPR + Cow urine foliar spray. Soil macro and micronutrients status was found higher with application of 100% RDF + PGPR + Cow urine foliar spray, which may further strengthen and sustain fodder quality and soil fertility

Synthesis of Metakaolin Based Alkali Activated Materials as an Adsorbent at Different Na2SiO3/NaOH Ratios and Exposing Temperatures for Cu2+ Removal.

Water contamination is a major issue due to industrial releases of hazardous heavy metals. Copper ions are among the most dangerous heavy metals owing to their carcinogenicity and harmful effects on the environment and human health. Adsorption of copper ions using alkali activated materials synthesized through the polycondensation reaction of an alkali source and aluminosilicates is the most promising technique, and has a high adsorption capability owing to a large surface area and pore volume. This research focuses on the effect of the alkaline activator ratio, which is a sodium silicate to sodium hydroxide ratio. Various exposing temperatures on metakaolin based alkali activated materials on a surface structure with excellent functional properties can be used as adsorbent materials for the removal of copper ions. A variety of mix designs were created with varying sodium silicate to sodium hydroxide ratios, with a fixed sodium hydroxide molarity, metakaolin to alkali activator ratio, hydrogen peroxide, and surfactant content of 10 M, 0.8, 1.00 wt%, and 3.0 wt%, respectively. Most wastewater adsorbents need high sintering temperatures, requiring an energy-intensive and time-consuming manufacturing process. In this way, metakaolin-based alkali activated materials are adsorbent and may be produced easily by solidifying the sample at 60 °C without using much energy. The specific surface area, water absorption, microstructure, phase analysis, functional group analysis, and adsorption capability of copper ions by metakaolin based alkali activated materials as adsorbents were evaluated. The water absorption test on the samples revealed that the sodium silicate to sodium hydroxide 0.5 ratio had the highest water absorption percentage of 36.24%, superior pore size distribution, and homogeneous porosity at 60 °C, with a surface area of 24.6076 m2/g and the highest copper ion uptake of 63.726 mg/g with 95.59% copper ion removal efficiency at adsorption condition of pH = 5, a dosage of 0.15 g, 100 mg/L of the initial copper solution, the temperature of 25 °C, and contact time of 60 min. It is concluded that self-supported metakaolin based alkali activated material adsorbents synthesized at low temperatures effectively remove copper ions in aqueous solutions, making them an excellent alternative for wastewater treatment applications.

Copper Uptake and Its Effects on Two Riparian Plant Species, the Native Urtica dioica, and the Invasive Fallopia japonica.

Copper accumulating in stream sediments can be transported to adjacent riparian habitats by flooding. Although being an essential element for plants, copper is toxic at high concentrations and restricts, among other things, plant growth. Besides copper, invasive plants, such as Fallopia japonica, which are known to be tolerant toward heavy metals, modify riparian habitats. If the tolerance of F. japonica is higher compared to native plants, this could accelerate invasion under high heavy metal stress. Therefore, we aimed to compare the effect of copper on two common riparian plants, the invasive F. japonica and the native Urtica dioica. We performed a pot experiment with a gradient from 0 to 2430 mg kg-1 of soil copper. We hypothesized that (i) negative effects on plant growth increase with increasing soil copper concentrations with F. japonica being less affected and (ii) accumulating higher amounts of copper in plant tissues compared to U. dioica. In support of our first hypothesis, growth (height, leaf number) and biomass (above- and belowground) of F. japonica were impacted at the 810 mg kg-1 treatment, while the growth of U. dioica was already impacted at 270 mg kg-1. Due to 100% mortality of plants, the 2430 mg kg-1 treatment was omitted from the analysis. In contrast, chlorophyll content slightly increased with increasing copper treatment for both species. While U. dioica accumulated more copper in total, the copper uptake by F. japonica increased more strongly after exposure compared to the control. In the 810 mg kg-1 treatment, copper concentrations in F. japonica were up to 2238% higher than in the control but only up to 634% higher in U. dioica. Our results indicate that F. japonica might be able to more efficiently detoxify internal copper concentrations controlling heavy metal effects compared to the native species. This could give F. japonica a competitive advantage particularly in polluted areas, facilitating its invasion success.

Elevated CO2 reduces copper accumulation and toxicity in the diatom Thalassiosira pseudonana.

The projected ocean acidification (OA) associated with increasing atmospheric CO2 alters seawater chemistry and hence the bio-toxicity of metal ions. However, it is still unclear how OA might affect the long-term resilience of globally important marine microalgae to anthropogenic metal stress. To explore the effect of increasing pCO2 on copper metabolism in the diatom Thalassiosira pseudonana (CCMP 1335), we employed an integrated eco-physiological, analytical chemistry, and transcriptomic approach to clarify the effect of increasing pCO2 on copper metabolism of Thalassiosira pseudonana across different temporal (short-term vs. long-term) and spatial (indoor laboratory experiments vs. outdoor mesocosms experiments) scales. We found that increasing pCO2 (1,000 and 2,000 μatm) promoted growth and photosynthesis, but decreased copper accumulation and alleviated its bio-toxicity to T. pseudonana. Transcriptomics results indicated that T. pseudonana altered the copper detoxification strategy under OA by decreasing copper uptake and enhancing copper-thiol complexation and copper efflux. Biochemical analysis further showed that the activities of the antioxidant enzymes glutathione peroxidase (GPX), catalase (CAT), and phytochelatin synthetase (PCS) were enhanced to mitigate oxidative damage of copper stress under elevated CO2. Our results provide a basis for a better understanding of the bioremediation capacity of marine primary producers, which may have profound effect on the security of seafood quality and marine ecosystem sustainability under further climate change.

THE EFFECT OF HEAVY METAL IONS ON THE PEROXIDASE ACTIVITY IN ARABIDOPSIS THALIANA

The biosphere pollution with the heavy metals (HM) has increased significantly in recent decades due to human activity. Plants can accumulate and concentrate HM, which negatively affects their growth, productivity and quality of agricultural products. Some HM, such as copper, belong to the group of biogenic elements that, in low concentrations, are essential for the normal functioning of plant organisms. Other HM such as cadmium are toxic even in low concentrations. The toxicity of HM is related to oxidative damage. In the plant cell, the antioxidant system provides protection against this kind of stress. However, data on changes in antioxidant enzyme activities in the early stage of the cellular response to HM-induced stress remain scarce. Therefore, we focused our research on studying peroxidase (POD) activity changes in Arabidopsis thaliana under conditions of rapid uptake of copper and cadmium ions into leaf tissue. For the experiments, 4.5–5-week-old A. thaliana plants were used. The plants were incubated on 0.5x MS liquid medium containing copper or cadmium chloride at concentrations of 0.1, 0.5 and 5 mM. The HM salt treatment was carried out in the dark at 20 °C for 2 (short-term stress) and 12 (long-term stress) hours. After that, the leaves were frozen and the POD activity was measured. Evaluation of the effects of Cd2+ and Cu2+ ions shows that these HM cause a decrease in POD activity after 2 hours and its increase after 12 hours of treatment. Therefore, modulation of POD activity is a component of the HM stress response in A. thaliana. Analysis of the available data revealed that the enzymes POD and CAT, which eliminate hydrogen peroxide, can partially replace each other and thus provide cellular protection in different phases of the stress response.

Polymer/copper nanocomplex-induced lysosomal cell death promotes tumor lymphocyte infiltration and synergizes anti-PD-L1 immunotherapy for triple-negative breast cancer.

Our previous research discovered that combining the PDA-PEG polymer with copper ions can selectively kill cancer cells. However, the precise mechanism by which this combination functions was not fully understood. This study revealed that the PDA-PEG polymer and copper ions form complementary PDA-PEG/copper (Poly/Cu) nanocomplexes by facilitating copper ion uptake and lysosomal escape. An in vitro study found that Poly/Cu killed 4T1 cells through a lysosome cell death pathway. Furthermore, Poly/Cu inhibited both the proteasome function and autophagy pathway and induced immunogenic cell death (ICD) in 4T1 cells. The Poly/Cu induced ICD coupled with the checkpoint blockade effect of the anti-PD-L1 antibody (aPD-L1) synergistically promoted immune cell penetration into the tumor mass. Benefiting from the tumor-targeting effect and cancer cell-selective killing effect of Poly/Cu complexes, the combinatory treatment of aPD-L1 and Poly/Cu effectively suppressed the progression of triple-negative breast cancer without inducing systemic side effects.

Biological Uptake of Chromium and Copper from Aqueous Systems by Penicillium notatum Biomass

Fungi have been shown to play an important role in the bioremediation of contaminated water and soils. The biosorption potential of a fungus, Penicillium notatum for chromium and copper ions in aqueous solution of their salts was investigated in this study. The effects of various parameters on the biosorption process were evaluated. These parameters which include biosorbent dose, solution pH, contact time and initial metal ion concentration were evaluated. The study showed that the maximum removal efficiency for chromium ions was recorded at pH 5 while that for copper was attained at pH 9. There was also an increase in biosorptive amount with increase in initial metal ion concentration for both ions. The equilibrium data for copper fitted better to the Langmuir isotherm compared to the Freundlich isotherm while that of chromium had a better fit for Freundlich isotherm compared to the Langmuir model. The maximum biosorption capacity, Qo, for chromium and copper obtained from the Langmuir plots are 23.15 and 4.02 (mg/g) respectively. This shows that Penicillium notatum biomass has the ability to remove these metal ions from contaminated waters under suitable conditions.

Targeting Copper in Cancer Imaging and Therapy: A New Theragnostic Agent

Copper is required for cancer cell proliferation and tumor angiogenesis. Copper-64 radionuclide (64Cu), a form of copper chloride (64CuCl2), is rapidly emerging as a diagnostic PET/CT tracer in oncology. It may also represent an interesting alternative to gallium-68 (68Ga) as a radionuclide precursor for labelling radiopharmaceuticals used to investigate neuroendocrine tumors and prostate cancer. This emerging interest is also related to the nuclear properties of 64CuCl2 that make it an ideal theragnostic nuclide. Indeed, 64CuCl2 emits β+ and β- particles together with high-linear-energy-transfer Auger electrons, suggesting the therapeutic potential of 64CuCl2 for the radionuclide cancer therapy of copper-avid tumors. Recently, 64CuCl2 was successfully used to image prostate cancer, bladder cancer, glioblastoma multiforme (GBM), and non-small cell lung carcinoma in humans. Copper cancer uptake was related to the expression of human copper transport 1 (hCTR1) on the cancer cell surface. Biodistribution, toxicology and radiation safety studies showed its radiation and toxicology safety. Based on the findings from the preclinical research studies, 64CuCl2 PET/CT also holds potential for the diagnostic imaging of human hepatocellular carcinoma (HCC), malignant melanoma, and the detection of the intracranial metastasis of copper-avid tumors based on the low physiological background of radioactive copper uptake in the brain.

Carbon dioxide enrichment effects on concentration, partitioning and uptake of metallic micronutrient elements in soybean under varied nitrogen application rates

Information on the fate of micronutrients in plant tissue and total uptake from soil upon exposure to elevated carbon dioxide (CO2) is meager. Hence, this field study was conducted in open top field chambers to investigate the effects of elevated CO2 and applied nitrogen (N) on partitioning and uptake of zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn) in soybean crop under two CO2 and four N levels during 2016 and 2018 crop seasons. The two CO2 concentrations were ambient and elevated (∼550 µmol mol−1). Nitrogen treatments included application at 0, 50, 100, and 150% of the recommended dose. Significant effects of CO2 and N were observed on grain yield, biomass and uptake of the four micronutrients. Seed Fe concentration was significantly increased by 12% under CO2 enrichment. Tukey’s post-hoc test revealed significantly higher grain yield, biomass and uptake of micronutrients in seed and straw under CO2 elevation and at higher N application. Uptake of Fe, Zn, Cu, and Mn increased by 53, 42, 36, and 41%, respectively, in seed and by 25, 26, 31, and 18% in straw under elevated CO2. Total uptake of Fe, Zn, Cu, and Mn increased by 39, 38, 34, and 29%, respectively. Nitrogen at 100% level enhanced uptake of Fe, Zn, Cu, and Mn by 25, 20, 32, and 28%. The study bears implications in micronutrient management for sustaining soil health under changing climate.

Principles to recover copper-conducting CTR proteins for the purpose of structural and functional studies

Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.

Molecular Mechanisms of Cellular Copper Homeostasis in Mammals

Copper (Cu) is a trace element necessary for the growth and development of all living organisms, and is the third most abundant trace metal in the body after iron and zinc. Copper is essential for maintaining the life processes in all living cells, because several copper-dependent enzymes play an important role in key physiological processes like cellular respiration, oxygen radical scavenging, the transport of iron and neurotransmitter synthesis. Maintaining copper homeostasis implies maintaining the constancy of copper levels in the cells and fluids throughout the body, in order to support the enzymes and other factors that underlie normal life processes. Therefore, living organisms have developed complex mechanisms for maintaining their physiological copper level, because an excess copper level can be toxic for the cells. In the cell, copper homeostasis is controlled by a network of copper-binding proteins and transporters. Furthermore, copper uptake is mediated by the membrane transporter CTR1 and CTR2 proteins. In the cytoplasm, it is bound to a unique group of metallochaperones (ATOX1, CCS COX17) and transported to different cell compartments, where it is linked to the recipient proteins. The Cu-transporting ATPases (ATP7A and ATP7B) are responsible for transferring copper into the Golgi apparatus, where the copper is added to the active sites of enzymes, and it is also directed onto the path of excess cellular copper removal to prevent the occurrence of toxicity.