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Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin and chitosan deposition. These changes are reflected in altered macrophage activation and changes in the expression of specific virulence-associated phenotypes. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. In conclusion, our data suggest a dual role for the fungal cell wall, in particular the inner chitin / chitosan layer, in protection against toxic levels of copper and providing a source of metal ion availability during copper starvation. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein is involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.Author summaryMicroorganisms must be equipped to readily acquire essential micro-nutrients like copper from nutritionally poor environments while simultaneously shielding themselves from conditions of metal excess. We explored mechanisms of microbial copper homeostasis in the human opportunistic fungal pathogen Cryptococcus neoformans (Cn) by defining physiological roles of the newly described copper-binding and release protein Cn Cbi1/Bim1. Highly induced during copper limitation, Cbi1 has been shown to interact with the high-affinity copper transporter Ctr1. We defined Cbi1-regulated changes in the fungal cell wall, including controlling levels of the structural carbohydrates chitin and chitosan. These polysaccharides are embedded deeply in the cell wall and are known to avidly bind copper. We also defined the host immunological alterations in response to these cell wall changes. Our data suggest a model in which the fungal cell wall, especially the chito-oligomer layer, serves as a copper-binding structure to shield the cell from states of excess copper, while also serving as a copper storage site during conditions of extracellular copper depletion. Given its ability to bind and release copper, the Cbi1 protein likely shuttles copper from the cell wall to copper transporters for regulated copper acquisition.

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis. In the fungal pathogen Cryptococcus neoformans, Bim1 is a copper-binding lytic polysaccharide monooxygenase-like protein that participates in copper uptake in concert with the Ctr1 importer to drive virulence mechanisms during fungal meningitis.

The ability of cancer cells to cope with stressful conditions is critical for their survival, proliferation, and metastasis. The heat shock transcription factor 1 (HSF1) protects cells from stresses such as chemicals, radiation, and temperature. These properties of HSF1 are exploited by a broad spectrum of cancers, which exhibit high levels of nuclear, active HSF1. Functions for HSF1 in malignancy extend well beyond its central role in protein quality control. While HSF1 has been validated as a powerful target in cancers by genetic knockdown studies, HSF1 inhibitors reported to date have lacked sufficient specificity and potency for clinical evaluation. We review the roles of HSF1 in cancer, its potential as a prognostic indicator for cancer treatment, evaluate current HSF1 inhibitors and provide guidelines for the identification of selective HSF1 inhibitors as chemical probes and for clinical development.

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-ranslational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings also highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.

The ability of the human fungal pathogen Cryptococcus neoformans to adapt to variable copper (Cu) environments within the host is key for successful dissemination and colonization. During pulmonary infection, host alveolar macrophages compartmentalize Cu into the phagosome and C. neoformans Cu-detoxifying metallothioneins, MT1 and MT2, are required for survival of the pathogen. In contrast, during brain colonization the C. neoformans Cu+ importers Ctr1 and Ctr4 are required for virulence. Central for the regulation and expression of both the Cu detoxifying MT1/2 and the Cu acquisition Ctr1/4 proteins is the Cu-metalloregulatory transcription factor Cuf1, an established C. neoformans virulence factor. Due to the importance of the distinct C. neoformans Cu homeostasis mechanisms during host colonization and virulence, and to the central role of Cuf1 in regulating Cu homeostasis, we performed a combination of RNA-Seq and ChIP-Seq experiments to identify differentially transcribed genes between conditions of high and low Cu. We demonstrate that the transcriptional regulation exerted by Cuf1 is intrinsically complex and that Cuf1 also functions as a transcriptional repressor. The Cu- and Cuf1-dependent regulon in C. neoformans reveals new adaptive mechanisms for Cu homeostasis in this pathogenic fungus and identifies potential new pathogen-specific targets for therapeutic intervention in fungal infections.

Fungal cells colonize and proliferate in distinct niches, from soil and plants to diverse tissues in human hosts. Consequently, fungi are challenged with the goal of obtaining nutrients while simultaneously elaborating robust regulatory mechanisms to cope with a range of availability of nutrients, from scarcity to excess. Copper is essential for life but also potentially toxic. In this review we describe the sophisticated homeostatic mechanisms by which fungi acquire, utilize, and control this biochemically versatile trace element. Fungal pathogens, which can occupy distinct host tissues that have their own intrinsic requirements for copper homeostasis, have evolved mechanisms to acquire copper to successfully colonize the host, disseminate to other tissues, and combat host copper bombardment mechanisms that would otherwise mitigate virulence.

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu+ across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu+ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu+ transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1−/− mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.

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Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect- or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes.

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition.

Cryptococcus neoformans is an opportunistic fungal pathogen whose pathogenic lifestyle is linked to its ability to cope with fluctuating levels of copper (Cu), an essential metal involved in multiple virulence mechanisms, within distinct host niches. During lethal cryptococcal meningitis in the brain, C. neoformans senses a Cu-deficient environment and is highly dependent on its ability to scavenge trace levels of Cu from its host and adapt to Cu scarcity to successfully colonize this niche. In this study, we demonstrate for this critical adaptation, the Cu-sensing transcription factor Cuf1 differentially regulates the expression of the SOD1 and SOD2 superoxide dismutases in novel ways. Genetic and transcriptional analysis reveals Cuf1 specifies 5’-truncations of the SOD1 and SOD2 mRNAs through specific binding to Cu responsive elements within their respective promoter regions. This results in Cuf1-dependent repression of the highly abundant SOD1 and simultaneously induces expression of two isoforms of SOD2, the canonical mitochondrial targeted isoform and a novel alternative cytosolic isoform, from a single alternative transcript produced specifically under Cu limitation. The generation of cytosolic Sod2 during Cu limitation is required to maintain cellular antioxidant defense against superoxide stress both in vitro and in vivo. Further, decoupling Cuf1 regulation of Sod2 localization compromises the ability of C. neoformans to colonize organs in murine models of cryptococcosis. Our results provide a link between transcription factor–mediated alteration of protein localization and cell proliferation under stress, which could impact tissue colonization by a fungal pathogen.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu+ is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2−/− mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2−/− mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu(+) transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu(+) import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.

Iron is an indispensable micronutrient for all eukaryotic organisms due to its participation as a redox cofactor in many metabolic pathways. Iron imbalance leads to the most frequent human nutritional deficiency in the world. Adaptation to iron limitation requires a global reorganization of the cellular metabolism directed to prioritize iron utilization for essential processes. In response to iron scarcity, the conserved Saccharomyces cerevisiae mRNA-binding protein Cth2, which belongs to the tristetraprolin family of tandem zinc finger proteins, coordinates a global remodeling of the cellular metabolism by promoting the degradation of multiple mRNAs encoding highly iron-consuming proteins. In this work, we identify a critical mechanism for the degradation of Cth2 protein during the adaptation to iron deficiency. Phosphorylation of a patch of Cth2 serine residues within its amino-terminal region facilitates recognition by the SCF ubiquitin ligase complex, accelerating Cth2 turnover by the proteasome. When Cth2 degradation is impaired by either mutagenesis of the Cth2 serine residues or deletion of GRR1, the levels of Cth2 rise and abrogate growth in iron-depleted conditions. Finally, we uncover that the casein kinase Hrr25 phosphorylates and promotes Cth2 destabilization. These results reveal a sophisticated posttranslational regulatory pathway necessary for the adaptation to iron depletion. IMPORTANCE Iron is a vital element for many metabolic pathways, including the synthesis of DNA and proteins, and the generation of energy via oxidative phosphorylation. Therefore, living organisms have developed tightly controlled mechanisms to properly distribute iron, since imbalances lead to nutritional deficiencies, multiple diseases, and vulnerability against pathogens. Saccharomyces cerevisiae Cth2 is a conserved mRNA-binding protein that coordinates a global reprogramming of iron metabolism in response to iron deficiency in order to optimize its utilization. Here we report that the phosphorylation of Cth2 at specific serine residues is essential to regulate the stability of the protein and adaptation to iron depletion. We identify the kinase and ubiquitination machinery implicated in this process to establish a posttranscriptional regulatory model. These results and recent findings for both mammals and plants reinforce the privileged position of E3 ubiquitin ligases and phosphorylation events in the regulation of eukaryotic iron homeostasis., This work was supported by a predoctoral contract from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) to A.M.R., by a postdoctoral fellowship from MINECO to M.M.-P., by MINECO BIO2014-56298-P, BIO2017-87828-C2-1-P, and Fondo Europeo de Desarrollo Regional (FEDER) grants to S.P., by NIH grant GM089778 to J.A.W., by NIH grant GM041840 to D.J.T., by MINECO BFU2017-85152-P and FEDER grant to E.D.N., by MINECO BFU2015-64437-P and FEDER grant to F.P., and by funding from the Catalan Government (2017 SGR 799) and the Fundación Botín by Banco Santander through its Santander Universities Global Division to F.P. F.P. is a recipient of an ICREA Acadèmia (Generalitat de Catalunya).

The heat shock transcription factors (HSFs) were discovered over 30 years ago as direct transcriptional activators of genes regulated by thermal stress, encoding heat shock proteins. The accepted paradigm posited that HSFs exclusively activate the expression of protein chaperones in response to conditions that cause protein misfolding by recognizing a simple promoter binding site referred to as a heat shock element. However, we now realize that the mammalian family of HSFs comprises proteins that independently or in concert drive combinatorial gene regulation events that activate or repress transcription in different contexts. Advances in our understanding of HSF structure, post-translational modifications and the breadth of HSF-regulated target genes have revealed exciting new mechanisms that modulate HSFs and shed new light on their roles in physiology and pathology. For example, the ability of HSF1 to protect cells from proteotoxicity and cell death is impaired in neurodegenerative diseases but can be exploited by cancer cells to support their growth, survival and metastasis. These new insights into HSF structure, function and regulation should facilitate the development tof new disease therapeutics to manipulate this transcription factor family.

The response by Brewer & Althaus to our recent review on biomarkers for Cu(1) bears testimony that the subject is topical and of public, scientific and commercial interest.

Cu is an essential nutrient for man, but can be toxic if intakes are too high. In sensitive populations, marginal over- or under-exposure can have detrimental effects. Malnourished children, the elderly, and pregnant or lactating females may be susceptible for Cu deficiency. Cu status and exposure in the population can currently not be easily measured, as neither plasma Cu nor plasma cuproenzymes reflect Cu status precisely. Some blood markers (such as ceruloplasmin) indicate severe Cu depletion, but do not inversely respond to Cu excess, and are not suitable to indicate marginal states. A biomarker of Cu is needed that is sensitive to small changes in Cu status, and that responds to Cu excess as well as deficiency. Such a marker will aid in monitoring Cu status in large populations, and will help to avoid chronic health effects (for example, liver damage in chronic toxicity, osteoporosis, loss of collagen stability, or increased susceptibility to infections in deficiency). The advent of high-throughput technologies has enabled us to screen for potential biomarkers in the whole proteome of a cell, not excluding markers that have no direct link to Cu. Further, this screening allows us to search for a whole group of proteins that, in combination, reflect Cu status. The present review emphasises the need to find sensitive biomarkers for Cu, examines potential markers of Cu status already available, and discusses methods to identify a novel suite of biomarkers.

SummaryRecalcitrant microbial infections demand new therapeutic options. Here we present an approach that exploits two prongs of the host immune cell antimicrobial response: the oxidative burst and the compartmentalization of copper (Cu) within phagolysosomes. The prochelator QBP is a nontoxic protected form of 8-hydroxyquinoline (8HQ) in which a pinanediol boronic ester blocks metal ion coordination by 8HQ. QBP is deprotected via reactive oxygen species produced by activated macrophages, creating 8HQ and eliciting Cu-dependent killing of the fungal pathogen Cryptococcus neoformans in vitro and in mouse pulmonary infection. 8HQ ionophoric activity increases intracellular Cu, overwhelming the Cu-resistance mechanisms of C. neoformans to elicit fungal killing. The Cu-dependent antimicrobial activity of 8HQ against a spectrum of microbial pathogens suggests that this strategy may have broad utility. The conditional activation of Cu ionophores by innate immune cells intensifies the hostile antimicrobial environment and represents a promising approach to combat infectious disease.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

The copper transporter 1, Ctr1, is part of a major pathway for cellular copper (Cu) uptake in the intestinal epithelium, in hepatic and cardiac tissue, and likely in many other mammalian cells and tissues. Here, we summarize what is currently known about how extracellular Cu travels across the plasma membrane to enter the cytoplasm for intracellular distribution and for use by proteins and enzymes, the physiological roles of Ctr1, and its regulation. As a critical Cu importer, Ctr1 occupies a strategic position to exert a strong modifying influence on diseases and pathophysiological states caused by imbalances in Cu homeostasis. A more thorough understanding of the mechanisms that regulate Ctr1 abundance, trafficking, and function will provide new insights and opportunities for disease therapies.

We report here the full characterization of the metal binding abilities of CnMT1 and CnMT2, two Cryptococcus neoformans proteins recently identified as metallothioneins (MTs), which have been shown to play a crucial role in the virulence and pathogenicity of this human-infecting fungus. In this work, we first performed a thorough in silico study of the CnMT1 and CnMT2 genes, cDNAs and corresponding encoded products. Subsequently, the Zn(II)-, Cd(II)- and Cu(I) binding abilities of both proteins were fully determined through the analysis of the metal-to-protein stoichiometries and the structural features (determined by ESI-MS, CD, ICP-AES and UV-vis spectroscopies) of the corresponding recombinant Zn-, Cd- and Cu-MT preparations synthesized in metal-enriched media. Finally, the analysis of the Zn/Cd and Zn/Cu replacement processes of the respective Zn-MT complexes when allowed to react with Cd(II) or Cu(I) aqueous solutions was performed. Comprehensive consideration of all gathered results allows us to consider both isoforms as genuine copper-thioneins, and led to the identification of unprecedented Cu5-core clusters in MTs. CnMT1 and CnMT2 polypeptides appear to be evolutionarily related to the small fungal MTs, probably by ancient tandem-duplication events responding to a highly selective pressure to chelate copper, and far from the properties of Zn- and Cd-thioneins. Finally, we propose a modular structure of the Cu-CnMT1 and Cu-CnMT2 complexes on the basis of Cu5 clusters, concordantly with the modular structure of the sequence of CnMT1 and CnMT2, constituted by three and five Cys-rich units, respectively.

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001

Transition metals such as iron, zinc, copper, and manganese are essential for the growth and development of organisms ranging from bacteria to mammals. Numerous studies have focused on the impact of iron availability during bacterial and fungal infections, and increasing evidence suggests that copper is also involved in microbial pathogenesis. Not only is copper an essential cofactor for specific microbial enzymes, but several recent studies also strongly suggest that copper is used to restrict pathogen growth in vivo. Here, we review evidence that animals use copper as an antimicrobial weapon and that, in turn, microbes have developed mechanisms to counteract the toxic effects of copper.

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and prion-based neurodegeneration are associated with the accumulation of misfolded proteins, resulting in neuronal dysfunction and cell death. However, current treatments for these diseases predominantly address disease symptoms, rather than the underlying protein misfolding and cell death, and are not able to halt or reverse the degenerative process. Studies in cell culture, fruitfly, worm and mouse models of protein misfolding-based neurodegenerative diseases indicate that enhancing the protein-folding capacity of cells, via elevated expression of chaperone proteins, has therapeutic potential. Here, we review advances in strategies to harness the power of the natural cellular protein-folding machinery through pharmacological activation of heat shock transcription factor 1 — the master activator of chaperone protein gene expression — to treat neurodegenerative diseases.

Summary Life on Earth has evolved within a complex mixture of organic and inorganic compounds. While organic molecules such as amino acids, carbohydrates and nucleotides form the backbone of proteins and genetic material, these fundamental components of macromolecules are enzymatically synthesized and ultimately degraded. Inorganic elements, such as copper (Cu), iron and zinc, once solubilized from the Earth's crust, are neither created nor destroyed and therefore their homeostatic regulation is under strict control. In the fascinating field of ‘metals in biology', by virtue of direct interactions with amino acid side-chains within polypeptide chains, metals play unique and critical roles in biology, promoting structures and chemistries that would not otherwise be available to proteins alone.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

The yeast Cth2 protein is a CX(8)CX(5)CX(3)H tandem zinc finger protein that binds AU-rich element (ARE)-containing transcripts to enhance their decay in response to iron (Fe) deficiency. Mammalian members of this family of proteins are known to undergo nucleocytoplasmic shuttling, but little is known about the role of shuttling in the mechanism of ARE-dependent mRNA decay. Here we demonstrate that, like its mammalian homologues, Cth2 is a nucleocytoplasmic shuttling protein whose nuclear export depends on mRNA transport to the cytosol. The nuclear import information of Cth2 is contained within its tandem zinc finger domain, but it is independent of mRNA-binding function. Moreover, we also demonstrate that nucleocytoplasmic shuttling of Cth2 requires active transcription and that disruption of shuttling leads to defects in Cth2 function in mRNA decay under Fe deficiency. Taken together, our data suggest that under conditions of Fe deficiency Cth2 travels into the nucleus to recruit target mRNAs, perhaps cotranscriptionally, that are destined for cytosolic degradation as part of the mechanism of adaptation to growth under Fe limitation. These data also suggest an important role for nucleocytoplasmic shuttling in this conserved family of proteins in the mechanism of ARE-mediated mRNA decay.

SummaryCopper (Cu) is an essential cofactor for a variety of metabolic functions, and the regulation of systemic Cu metabolism is critical to human health. Dietary Cu is absorbed through the intestine, stored in the liver, and mobilized into the circulation; however, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac-specific knockout of the Ctr1 Cu transporter (Ctr1hrt/hrt), resulting in cardiac Cu deficiency and severe cardiomyopathy. Unexpectedly, Ctr1hrt/hrt mice exhibited increased serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1hrt/hrt mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand, and illuminate a systemic regulation in which the Cu status of the heart is signaled to organs that take up and store Cu.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

In mammalian and Drosophila cells, heat stress strongly reduces general protein translation while activating cap-independent translation mechanisms to promote the expression of stress-response proteins. In contrast, in Saccharomyces cerevisiae general translation is only mildly and transiently reduced by heat stress and cap independent translation mechanisms have not been correlated with the heat stress response. Recently we have identified direct target genes of the heat shock transcription factor, HSF, including genes encoding proteins thought to be important for general translation. One gene activated by HSF during heat stress encodes the enhancer of decapping protein, Edc2, previously shown to enhance mRNA decapping under conditions when the decapping machinery is limited. In this report we show that strains lacking Edc2, as well as the paralogous protein Edc1, are compromised for growth under persistent heat stress. This growth deficiency can be rescued by expression of a mutant Edc1 protein deficient in mRNA decapping indicative of a decapping independent function during heat stress. Yeast strains lacking Edc1 and Edc2 are also sensitive to the pharmacological inhibitor of translation paromomycin and exposure to heat stress and paromomycin functions synergistically to reduces yeast viability, suggesting that in the absence of Edc1 and Edc2 translation is compromised under heat stress conditions. Strains lacking Edc1 and Edc2 have significantly reduced rates of protein translation during growth under heat stress conditions, but not under normal growth conditions. We propose that Edc1 and the stress responsive isoform Edc2 play important roles in protein translation during stress.

Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated.

Summary Iron (Fe) is an essential cofactor for a wide range of cellular processes. We have previously demonstrated in yeast that Cth2 is expressed during Fe deficiency and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe storage. We report here that the Cth2-homologous protein Cth1 is transiently expressed during Fe deprivation and participates in the response to Fe deficiency through the degradation of mRNAs primarily involved in mitochondrially localized activities including respiration and amino acid biosynthesis. In parallel, wild-type cells, but not cth1 Δ cth2 Δ cells, accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe deficiency leads to phosphorylation of Snf1, an AMP-activated protein kinase family member required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe starvation that is dependent on the coordinated activities of two mRNA-binding proteins.

Copper (Cu) is a redox-active metal ion essential for most aerobic organisms. Cu serves as a catalytic and structural cofactor for enzymes that function in energy generation, iron acquisition, oxygen transport, cellular metabolism, peptide hormone maturation, blood clotting, signal transduction and a host of other processes. The inability to control Cu balance is associated with genetic diseases of overload and deficiency and has recently been tied to neurodegenerative disorders and fungal virulence. The essential nature of Cu, the existence of human genetic disorders of Cu metabolism and the potential impact of Cu deposition in the environment have been driving forces for detailed investigations in microbial and eukaryotic model systems. Here we review recent advances in the identification and function of cellular and systemic molecules that drive Cu accumulation, distribution and sensing.

Heat Shock Transcription Factor (HSF) family members function in stress protection and in human disease including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, co-factor recruitment and target gene activation for specific HSF paralogs are unknown. We present high-resolution crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a novel carboxyl-terminal helix that directs the coiled-coil domain to wrap around DNA, exposing paralog-specific sequences of the DBD surface, for differential post-translational modifications and co-factor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation to influence the transcriptional response to cellular stress.

Fungal infections are responsible for millions of human deaths annually. Copper, an essential but toxic trace element, plays an important role at the host-pathogen axis during infection. In this review, we describe how the host uses either Cu compartmentalization within innate immune cells or Cu sequestration in other infected host niches such as in the brain to combat fungal infections. We explore Cu toxicity mechanisms and the Cu homeostasis machinery that fungal pathogens bring into play to succeed in establishing an infection. Finally, we address recent approaches that manipulate Cu-dependent processes at the host-pathogen axis for antifungal drug development.

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.