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Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker’s yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10–100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr 90 . Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts. Existing heavy metal bioremediation systems are mainly based on plants, which require long growing time in specific conditions. Here, the authors mimic the characteristics of plant hyperaccumulators to engineer more tractable baker’s yeast and achieve 10–100-fold higher accumulation of chromium, arsenic, or cadmium.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3 −/− mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function.

Copper ions are needed for several hallmarks of cancer. However, the involved pathways, mechanisms, and copper-binding proteins are mostly unknown. We recently found that cytoplasmic Antioxidant 1 copper chaperone (Atox1), which is up-regulated in breast cancer, is localized at the lamellipodia edges of aggressive breast cancer cells. To reveal molecular insights into a putative role in cell migration, we here investigated breast cancer cell (MDA-MB-231) migration by video microscopy as a function of Atox1. Tracking of hundreds of individual cells (per condition) over a 9-h time series revealed that cell migration velocity and directionality are significantly reduced upon Atox1 silencing in the cells. Because silencing of the copper transporter ATP7A also reduced cell migration, these proteins appear to be on the same pathway, suggesting that their well-known copper transport activity is involved. In-cell proximity ligation assays demonstrated that Atox1, ATP7A, and the proenzyme of lysyl oxidase (LOX; copper-loaded via ATP7A) are all in close proximity and that LOX activity is reduced upon Atox1 silencing in the cells. Since LOX is an established player in cancer cell migration, our results imply that Atox1 mediates breast cancer cell migration via coordinated copper transport in the ATP7A-LOX axis. Because individual cell migration is an early step in breast cancer metastasis, Atox1 levels in tumor cells may be a predictive measure of metastasis potential and serve as a biomarker for copper depletion therapy.

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials. Copper depletion has been reported to improve survival in patients with triple negative breast cancer (TNBC) but the underlying mechanisms are not completely understood. Here, the authors show that copper chelation reduces mitochondrial oxidative phosphorylation leading to decreased TNBC metastasis.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

The copper transport (COPT/Ctr) family plays an important role in maintaining metal homeostasis in organisms, and many species rely on Ctrs to achieve transmembrane transport via copper (Cu) uptake. At present, the Ctr family is widely studied in plants. However, there are few reports on the use of Ctrs in edible mushrooms. In this study, the Pleurotus ostreatus CCMSSC00389 strain was used as the research object, and the addition of exogenous copper ions (Cu2+) increased the temperature tolerance of mycelia, maintained the integrity of cell membranes, and increased mycelial density. In addition, four PoCtr genes were further identified and subjected to bioinformatics analysis. Further research revealed that there were differences in the expression patterns of the PoCtr genes under different temperature stresses. In addition, the biological function of PoCtr4 was further explored by constructing transformed strains. The results showed that OE-PoCtr4 enhanced the tolerance of mycelia to heat stress and H2O2. After applying heat stress (40 °C), OE-PoCtr4 promoted the recovery of mycelia. Under mild stress (32 °C), OE-PoCtr4 promoted mycelial growth, maintained cell membrane integrity, and reduced the degree of cell membrane damage caused by heat stress. It is speculated that OE-PoCtr4 may maintain the integrity of the cell membrane and enhance the heat resistance of mycelia by regulating the homeostasis of Cu2+.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

The search for relevant molecular targets is one of the main tasks of modern tumor chemotherapy. To successfully achieve this, it is necessary to have the most complete understanding of the functioning of a transcriptional apparatus of the cell, particularly related to proliferation. The p53 protein plays an important role in regulating processes such as apoptosis, repair, and cell division, and the loss of its functionality often accompanies various types of tumors and contributes to the development of chemoresistance. Additionally, the proliferative activity of tumor cells is closely related to the metabolism of transition metals. For example, the metallochaperone Atox1 – a copper transporter protein – acts as a transcription activator for cyclin D1, promoting progression through the G1/S phase of the cell cycle. On the other hand, p53 suppresses cyclin D1 at the transcriptional level, thereby these proteins have divergent effects on cell cycle progression. However, the contribution of the interaction between these proteins to cell survival is poorly understood. This work demonstrates that not only exists a positive feedback loop between Atox1 and cyclin D1 but also that the activity of this loop depends on the status of the TP53 gene. Upon inactivation of TP53 in A549 and HepG2 cell lines, the expression of ATOX1 and CCND1 genes is enhanced, and their suppression in these cells leads to pronounced apoptosis. This fundamental observation may be useful in selecting more precise interventions for combined therapy of p53-negative tumors.

Copper (Cu) dyshomeostasis plays a pivotal role in several neuropathologies, such as Parkinson’s disease (PD). Metal accumulation in the central nervous system (CNS) could result in loss-of-function of proteins involved in Cu metabolism and redox cycling, generating reactive oxygen species (ROS). Moreover, neurodegenerative disorders imply the presence of an excess of misfolded proteins known to lead to neuronal damage. In PD, Cu accumulates in the brain, binds α-synuclein, and initiates its aggregation. We assessed the correlation between neuronal differentiation, Cu homeostasis regulation, and α-synuclein phosphorylation. At this purpose, we used differentiated SHSY5Y neuroblastoma cells to reproduce some of the characteristics of the dopaminergic neurons. Here, we reported that differentiated cells expressed a significantly higher amount of a copper transporter protein 1 (CTR1), increasing the copper uptake. Cells also showed a significantly more phosphorylated form of α-synuclein, further increased by copper treatment, without modifications in α-synuclein levels. This effect depended on the upregulation of the polo-like kinase 2 (PLK2), whereas the levels of the relative protein phosphatase 2A (PP2A) remained unvaried. No changes in the oxidative state of the cells were identified. The Cu dependent alteration of α-synuclein phosphorylation pattern might potentially offer new opportunities for clinical intervention.