Potential cellular targets of platinum in the freshwater microalgae Chlamydomonas reinhardtii and Nitzschia palea revealed by transcriptomics.

Platinum group element levels have increased in natural aquatic environments in the last few decades, in particular as a consequence of the use of automobile catalytic converters on a global scale. Concentrations of Pt over tens of µg L-1 have been observed in rivers and effluents. This raises questions regarding its possible impacts on aquatic ecosystems, as Pt natural background concentrations are extremely low to undetectable. Primary producers, such as microalgae, are of great ecological importance, as they are at the base of the food web. The purpose of this work was to better understand the impact of Pt on a cellular level for freshwater unicellular algae. Two species with different characteristics, a green alga C. reinhardtii and a diatom N. palea, were studied. The bioaccumulation of Pt as well as its effect on growth were quantified. Moreover, the induction or repression factors of 16 specific genes were determined and allowed for the determination of possible intracellular effects and pathways of Pt. Both species seemed to be experiencing copper deficiency as suggested by inductions of genes linked to copper transporters. This is an indication that Pt might be internalized through the Cu(I) metabolic pathway. Moreover, Pt could possibly be excreted using an efflux pump. Other highlights include a concentration-dependent negative impact of Pt on mitochondrial metabolism for C. reinhardtii which is not observed for N. palea. These findings allowed for a better understanding of some of the possible impacts of Pt on freshwater primary producers, and also lay the foundations for the investigation of pathways for Pt entry at the base of the aquatic food web.

Role of copper and its complexes in cardiovascular diseases. / é“œåŠå ¶å¤åˆç‰©åœ¨å¿ƒè¡€ç®¡ç–¾ç— ä¸­çš„ä½œç”¨.

Copper is a trace element essential for the maintenance of normal physiological functions in cardiovascular system, and its transport and metabolisms are regulated by various copper proteins such as copper-based enzymes, copper chaperones and copper transporters. The disturbance of copper level or abnormal expression of copper proteins are closely associated with the development of cardiovascular diseases such as atherosclerosis, hypertension, ischemic heart disease, myocardial hypertrophy and heart failure. Thus, intervention of copper ion signaling pathways is expected to be an effective measure for treating cardiovascular diseases. Some copper complexes, such as trientine, copper-aspirinate complex and copper (II) diethyldithiocarbamate, have been found to play a role in the prevention and treatment of cardiovascular diseases and possess potential prospects. Exploring the role of copper in maintaining normal cardiovascular status and the potential application of copper complexes in the treatment of cardiovascular diseases may lay a foundation for finding new targets for prevention and treatment of various cardiovascular diseases, and provide new ideas for clinical treatment of cardiovascular diseases.

Interference between copper transport systems and platinum drugs.

Cisplatin, or cis-diamminedichloridoplatinum(II) cis-[PtCl2(NH3)2], is a platinum-based anticancer drug largely used for the treatment of various types of cancers, including testicular, ovarian and colorectal carcinomas, sarcomas, and lymphomas. Together with other platinum-based drugs, cisplatin triggers malignant cell death by binding to nuclear DNA, which appears to be the ultimate target. In addition to passive diffusion across the cell membrane, other transport systems, including endocytosis and some active or facilitated transport mechanisms, are currently proposed to play a pivotal role in the uptake of platinum-based drugs. In this review, an updated view of the current literature regarding the intracellular transport and processing of cisplatin will be presented, with special emphasis on the plasma membrane copper permease CTR1, the Cu-transporting ATPases, ATP7A and ATP7B, located in the trans-Golgi network, and the soluble copper chaperone ATOX1. Their role in eliciting cisplatin efficacy and their exploitation as pharmacological targets will be addressed.

Copper in the tumor microenvironment and tumor metastasis.

Copper (Cu), an essential micronutrient, plays an essential role in several physiological processes, including cell proliferation and angiogenesis; however, its dysregulation induces oxidative stress and inflammatory responses. Significant Cu accumulation is observed in several tumor tissues. The bioavailability of intracellular Cu is tightly controlled by Cu transporters, including Cu transporter 1 (CTR1) and Cu-transporting P-type ATPase α and ß (ATP7A and ATP7B), and Cu chaperones, including Cu chaperone for superoxide dismutase 1 (CCS) and antioxidant-1 (Atox-1). In several tumor tissues, these abnormalities that induce intra-cellular Cu accumulation are involved in tumor progression. In addition, functional disturbance in Cu-containing secretory enzymes, such as superoxide dismutase 3 (SOD3), and lysyl oxidase enzymes (LOX and LOXL1-4) with abnormal Cu dynamics plays a key role in tumor metastasis. For example, the loss of SOD3 in tumor tissues induces oxidative stress, which promotes neovascularization and epithelial-to-mesenchymal transition (EMT). LOX promotes collagen crosslinking, which functions in the metastatic niche formation. Accordingly, restricted Cu regulation may be a novel strategy for the inhibition of tumor metastasis. However, it is unclear how these Cu disturbances occur in tumor tissues and the exact molecular mechanisms underlying Cu secretory enzymes. In this review article, I discuss the role of Cu transporters, Cu chaperones, and Cu-containing secretory enzymes in tumor progression to better understand the role of Cu homeostasis in tumor tissues.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Copper transporters and copper chaperones: roles in cardiovascular physiology and disease.

Copper (Cu) is an essential micronutrient but excess Cu is potentially toxic. Its important propensity to cycle between two oxidation states accounts for its frequent presence as a cofactor in many physiological processes through Cu-containing enzymes, including mitochondrial energy production (via cytochrome c-oxidase), protection against oxidative stress (via superoxide dismutase), and extracellular matrix stability (via lysyl oxidase). Since free Cu is potentially toxic, the bioavailability of intracellular Cu is tightly controlled by Cu transporters and Cu chaperones. Recent evidence reveals that these Cu transport systems play an essential role in the physiological responses of cardiovascular cells, including cell growth, migration, angiogenesis and wound repair. In response to growth factors, cytokines, and hypoxia, their expression, subcellular localization, and function are tightly regulated. Cu transport systems and their regulators have also been linked to various cardiovascular pathophysiologies such as hypertension, inflammation, atherosclerosis, diabetes, cardiac hypertrophy, and cardiomyopathy. A greater appreciation of the central importance of Cu transporters and Cu chaperones in cell signaling and gene expression in cardiovascular biology offers the possibility of identifying new therapeutic targets for cardiovascular disease.

Cell-surface copper transporters and superoxide dismutase 1 are essential for outgrowth during fungal spore germination.

During fungal spore germination, a resting spore returns to a conventional mode of cell division and resumes vegetative growth, but the requirements for spore germination are incompletely understood. Here, we show that copper is essential for spore germination in Schizosaccharomyces pombe Germinating spores develop a single germ tube that emerges from the outer spore wall in a process called outgrowth. Under low-copper conditions, the copper transporters Ctr4 and Ctr5 are maximally expressed at the onset of outgrowth. In the case of Ctr6, its expression is broader, taking place before and during outgrowth. Spores lacking Ctr4, Ctr5, and the copper sensor Cuf1 exhibit complete germination arrest at outgrowth. In contrast, ctr6 deletion only partially interferes with formation of outgrowing spores. At outgrowth, Ctr4-GFP and Ctr5-Cherry first co-localize at the spore contour, followed by re-location to a middle peripheral spore region. Subsequently, they move away from the spore body to occupy the periphery of the nascent cell. After breaking of spore dormancy, Ctr6 localizes to the vacuole membranes that are enriched in the spore body relative to the germ tube. Using a copper-binding tracker, results showed that labile copper is preferentially localized to the spore body. Further analysis showed that Ctr4 and Ctr6 are required for copper-dependent activation of the superoxide dismutase 1 (SOD1) during spore germination. This activation is critical because the loss of SOD1 activity blocked spore germination at outgrowth. Taken together, these results indicate that cell-surface copper transporters and SOD1 are required for completion of the spore germination program.

Cisplatin resistance and opportunities for precision medicine.

Cisplatin is one of the most commonly used chemotherapy drugs, treating a wide range of cancer types. Unfortunately, many cancers initially respond to platinum treatment but when the tumor returns, drug resistance frequently occurs. Resistance to cisplatin is attributed to three molecular mechanisms: increased DNA repair, altered cellular accumulation, and increased drug inactivation. The use of precision medicine to make informed decisions on a patient's cisplatin resistance status and predicting the tumor response would allow the clinician to tailor the chemotherapy program based on the biology of the disease. In this review, key biomarkers of each molecular mechanism will be discussed along with the current clinical research. Additionally, known polymorphisms for each biomarker will be discussed in relation to their influence on cisplatin resistance.

The mechanism of copper transporters in ovarian cancer cells and the prospect of cuproptosis.

Copper transporters can not only carry copper (Cu) to maintain the homeostasis of Cu in cells but also transport platinum-based chemotherapy drugs. The effect of copper transporters on chemosensitivity has been demonstrated in a variety of malignancies. In addition, recent studies have reported that copper transporters can act as vectors to induce cuproptosis. Therefore, copper transporters can act on cells through different mechanisms to achieve different purposes. This review mainly describes the current research progress of the intracellular transport mechanism of copper transporters and cuproptosis, and prospects for the application of them in the treatment of ovarian cancer (OC).

Copper homeostasis and the ubiquitin proteasome system.

Copper is involved in many physiological pathways and important biological processes as a cofactor of several copper-dependent enzymes. Given the requirement for copper and its potential toxicity, intracellular copper levels are tightly controlled. Disturbances of human copper homeostasis are characterized by disorders of copper overload (Wilson's disease) or copper deficiency (Menkes disease). The maintenance of cellular copper levels involves numerous copper transporters and copper chaperones. Recently, accumulating evidence has revealed that components of the ubiquitin proteasome system (UPS) participate in the posttranslational regulation of these proteins, suggesting that they might play a role in maintaining copper homeostasis. Cellular copper levels could also affect the activity of the UPS, indicating that copper homeostasis and the UPS are interdependent. Copper homeostasis and the UPS are essential to the integrity of normal brain function and while separate links between neurodegenerative diseases and UPS inhibition/copper dyshomeostasis have been extensively reported, there is growing evidence that these two networks might contribute synergistically to the occurrence of neurodegenerative diseases. Here, we review the role of copper and the UPS in the development of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, and discuss the genetic interactions between copper transporters/chaperones and components of the UPS.

The implications and prospect of cuproptosis-related genes and copper transporters in cancer progression.

Currently, cancer has become one of the major public health problems worldwide. Apoptosis is an important anti-cancer defense mechanism, which is used in the development of targeted drugs. Because cancer cells have endogenous resistance to apoptosis,the clinical efficacy of related drugs is not ideal. Therefore, non-apoptotic regulatory cell death may bring new therapeutic strategies for cancer treatment. Cuproptosis is a novel form of regulatory cell death which is copper-dependent, regulated and distinct from other known cell death regulatory mechanisms. FDX1,LIAS,and DLAT named cuproptosis-related genes play an essential role in regulating cuproptosis. Meanwhile, abnormal accumulation of copper can be observed in various malignant tumors. The correlation has been established between elevated copper levels in serum and tissues and the progression of several cancers. Copper transporters, CTR1 and Copper-transporting ATPases(ATP7A and ATP7B), are mainly involved in regulating the dynamic balance of copper concentration to maintain copper homeostasis. Thus,cuproptosis-related genes and copper transporters will be the focus of cancer research in future. This review elaborated the basic functions of cuproptosis-related genes and copper transporters by retrievalling PubMed. And then we analyzed their potential relationship with cancer aiming to provide theoretical support and reference in cancer progression, diagnosis and treatment for future study.

Cadmium alters whole animal ionome and promotes the re-distribution of iron in intestinal cells of Caenorhabditis elegans.

The chronic exposure of humans to the toxic metal cadmium (Cd), either occupational or from food and air, causes various diseases, including neurodegenerative conditions, dysfunction of vital organs, and cancer. While the toxicology of Cd and its effect on the homeostasis of biologically relevant elements is increasingly recognized, the spatial distribution of Cd and other elements in Cd toxicity-caused diseases is still poorly understood. Here, we use Caenorhabditis elegans as a non-mammalian multicellular model system to determine the distribution of Cd at the tissue and cellular resolution and its effect on the internal levels and the distribution of biologically relevant elements. Using inductively coupled plasma-mass spectrophotometry (ICP-MS), we show that exposure of worms to Cd not only led to its internal accumulation but also significantly altered the C. elegans ionome. Specifically, Cd treatment was associated with increased levels of toxic elements such as arsenic (As) and rubidium (Rb) and a decreased accumulation of essential elements such as zinc (Zn), copper (Cu), manganese (Mn), calcium (Ca), cobalt (Co) and, depending on the Cd-concentration used in the assay, iron (Fe). We regarded these changes as an ionomic signature of Cd toxicity in C. elegans. We also show that supplementing nematode growth medium with Zn but not Cu, rescues Cd toxicity and that mutant worms lacking Zn transporters CDF-1 or SUR-7, or both are more sensitive to Cd toxicity. Finally, using synchrotron X-Ray fluorescence Microscopy (XRF), we showed that Cd significantly alters the spatial distribution of mineral elements. The effect of Cd on the distribution of Fe was particularly striking: while Fe was evenly distributed in intestinal cells of worms grown without Cd, in the presence of Cd, Fe, and Cd co-localized in punctum-like structures in the intestinal cells. Together, this study advances our understanding of the effect of Cd on the accumulation and distribution of biologically relevant elements. Considering that C. elegans possesses the principal tissues and cell types as humans, our data may have important implications for future therapeutic developments aiming to alleviate Cd-related pathologies in humans.

Bioactive copper(II) agents and their potential involvement in the treatment of copper deficiency-related orphan diseases.

The deregulation of copper homoeostasis can promote various diseases such as Menkes disease or hypertrophic cardioencephalomyopathy. We have recently synthesized solid copper(II) complexes ([Cu(His)2Cl2] and [Cu(Ser)2]), stable in physiological media and with potential as therapeutic agents. This report describes: i) the biocompatibility of these complexes at concentrations up to 100 µM using a differentiated Caco-2 cells model; ii) their transport across the intestinal epithelium using a transepithelial resistance assay and monitoring the amount of copper complexes at the apical and basolateral sides of the cells. The results suggest that the flow occurs through paracellular routes. The intracellular copper retention was <2.7% with no significant differences in intracellular copper content between 6 h and 48 h, suggesting an early copper retention process. Furthermore, this is the first evidence that demonstrates [Cu(His)2Cl2] and [Cu(Ser)2] induce transcriptional downregulation of the four major copper transporters (CTR1, DMT1, ATP7A, ATP7B), and the upregulation of the metallothionein gene expression. A remarkable finding was the increase in cytochrome c oxidase activity observed after the treatment of differentiated Caco-2 cells with copper(II) complexes at concentrations of 50-100 µM. The understanding of the transport mechanisms of these copper(II) complexes across the intestinal epithelium and of their subsequent biological activities could contribute to the development of optimal pharmaceutical formulations for the therapy of copper deficiency-related diseases.

Copper chelation therapy inhibits renal fibrosis by modulating copper transport proteins.

The copper (Cu) transporter proteins play an important role in the maintenance of the Cu homeostasis in the body. Lysyl oxidase (LOX) proteins are involved in crosslinking of collagens and elastin molecules resulting in the establishment of extracellular matrix (ECM) and require Cu for their functional activity. Although there are few reports showing the protective effects of Cu chelators, the mechanism behind protection remains unknown. The present study investigated the role of Cu transporter proteins in renal fibrosis. We used tubular epithelial cells and three different animal models of renal injury to investigate the induction of Cu transporter proteins in renal injury with different etiology. We used disulfiram, clioquinol as two Cu chelators and ammonium tetrathiomolybdate as a standard Cu chelator. In addition, ß-aminopropionitrile (BAPN) was used as a standard LOX inhibitor. We demonstrated that renal fibrosis is associated with the induction of Cu transporter proteins such as ATP7A and Copper Transporter 1 (CTR1) but the Cu overload did not induce renal fibrosis. In addition, the Cu chelators inhibited renal fibrosis by inhibiting the Cu transporter proteins.

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.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system.

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions-however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Regulation of murine copper homeostasis by members of the COMMD protein family.

Copper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under low- or high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.

NMR spectroscopy to study the fate of metallodrugs in cells.

Metal-based drugs can modulate various biological processes and exhibit a rich variety of properties that foster their use in biomedicine and chemical biology. On the way to intracellular targets, ligand exchange and redox reactions can take place, thus making metallodrug speciation in vivo a challenging task. Advances in NMR spectroscopy have made it possible to move from solution to live-cell studies and elucidate the transport of metallodrugs and interactions with macromolecular targets in a physiological setting. In turn, the electronic properties and supramolecular chemistry of metal complexes can be exploited to characterize drug delivery nanosystems by NMR. The recent evolution of in-cell NMR methodology is presented with special emphasis on metal-related processes. Applications to paradigmatic cases of platinum and gold drugs are highlighted.

The expression of copper transporters associated with the ototoxicity induced by platinum-based chemotherapeutic agents.

BACKGROUND: Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. METHODS: We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. RESULTS: CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79  µM, compared to 611.7 µM for L-OHP and 882.9 µM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 µM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. CONCLUSIONS: The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.

Copper Tolerance Mediated by FgAceA and FgCrpA in Fusarium graminearum.

All organisms must secure essential trace elements (e.g., Cu) for survival and reproduction. However, excess trace element accumulation in cells is highly toxic. The maintenance of copper (Cu) homeostasis has been extensively studied in mammals, bacteria, and yeast but not in plant pathogens. In this study, we investigated the molecular mechanisms of copper tolerance in Fusarium graminearum, the important wheat head scab fungus. RNA-seq revealed induced expression of the P-type ATPase transporter FgCrpA and metallothionein (MT) FgCrdA after excess Cu treatment. Deletion of FgCrpA but not FgCrdA resulted in reduced tolerance to Cu toxicity. The "Cu fist" transcription factor FgAceA was involved in Cu detoxification through activation of FgCrpA. △FgAceA was more sensitive to copper toxicity than △FgCrpA and overexpression of FgCrpA restored copper tolerance in △FgAceA. FgAceA negatively regulated aurofusarin production and its biosynthetic gene expression. △FgCrpA and △FgAceA were reduced in virulence in flowering wheat heads and synthesized decreased amounts of the mycotoxin deoxynivalenol when challenged with excess Cu. Taken together, these results suggest that mediation of Cu tolerance in F. graminearum mainly relies on the Cu efflux pump and that FgAceA governs Cu detoxification through activation of FgCrpA.