Copper Chaperone Research Articles

Osa‐miR398b boosts H2O2 production and rice blast disease‐resistance via multiple superoxide dismutases

Summary miRNAs contribute to plant resistance against pathogens. Previously, we found that the function of miR398b in immunity in rice differs from that in Arabidopsis. However, the underlying mechanisms are unclear.In this study, we characterized the mutants of miR398b target genes and demonstrated that multiple superoxide dismutase genes contribute to miR398b‐regulated rice immunity against the blast fungus Magnaporthe oryzae.Out of the four target genes of miR398b, mutations in Cu/Zn‐Superoxidase Dismutase1 (CSD1), CSD2 and Os11g09780 (Superoxide DismutaseX,SODX) led to enhanced resistance to M. oryzae and increased hydrogen peroxide (H2O2) accumulation. By contrast, mutations in Copper Chaperone for Superoxide Dismutase (CCSD) resulted in enhanced susceptibility. Biochemical studies revealed that csd1, csd2 and sodx displayed altered expression of CSDs and other superoxide dismutase (SOD) family members, leading to increased total SOD enzyme activity that positively contributed to higher H2O2 production. By contrast, the ccsd mutant showed CSD protein deletion, resulting in decreased CSD and total SOD enzyme activity.Our results demonstrate the roles of different SODs in miR398b‐regulated resistance to rice blast disease, and uncover an integrative regulatory network in which miR398b boosts total SOD activity to upregulate H2O2 concentration and thereby improve disease resistance.

Measuring the Unfolding and Ligand-Binding of CusF, a Copper Chaperone

Measuring the Unfolding and Ligand-Binding of CusF, a Copper Chaperone

The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation

Copper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O2). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1·CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1·CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."

Cell type-specific differences in redox regulation and proliferation after low UVA doses.

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30–50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation. This effect was cell type and dose specific. In both cell lines the levels of reactive oxygen species and nitric oxide fluctuated with dynamics which were influenced differently by UVA; in Me45 cells decreased proliferation accompanied the changes in the dynamics of H2O2 while in HCT116 cells those of superoxide. Genes coding for proteins engaged in redox systems were expressed differently in each cell line; transcripts for thioredoxin, peroxiredoxin and glutathione peroxidase showed higher expression in HCT116 cells whereas those for glutathione transferases and copper chaperone were more abundant in Me45 cells. We conclude that these two cell types utilize different pathways for regulating their redox status. Many mechanisms engaged in maintaining cellular redox balance have been described. Here we show that the different cellular responses to a stimulus such as a specific dose of UVA may be consequences of the use of different redox control pathways. Assays of superoxide and hydrogen peroxide level changes after exposure to UVA may clarify mechanisms of cellular redox regulation and help in understanding responses to stressing factors.

Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum.

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

NosL is a dedicated copper chaperone for assembly of the CuZ center of nitrous oxide reductase.

Nitrous oxide reductase (N2OR) is the terminal enzyme of the denitrification pathway of soil bacteria that reduces the greenhouse gas nitrous oxide (N2O) to dinitrogen. In addition to a binuclear CuA site that functions in electron transfer, the active site of N2OR features a unique tetranuclear copper cluster bridged by inorganic sulfide, termed CuZ. In copper-limited environments, N2OR fails to function, resulting in truncation of denitrification and rising levels of N2O released by cells to the atmosphere, presenting a major environmental challenge. Here we report studies of nosL from Paracoccus denitrificans, which is part of the nos gene cluster, and encodes a putative copper binding protein. A Paracoccus denitrificans ΔnosL mutant strain had no denitrification phenotype under copper-sufficient conditions but failed to reduce N2O under copper-limited conditions. N2OR isolated from ΔnosL cells was found to be deficient in copper and to exhibit attenuated activity. UV-visible absorbance spectroscopy revealed that bands due to the CuA center were unaffected, while those corresponding to the CuZ center were significantly reduced in intensity. In vitro studies of a soluble form of NosL without its predicted membrane anchor showed that it binds one Cu(i) ion per protein with attomolar affinity, but does not bind Cu(ii). Together, the data demonstrate that NosL is a copper-binding protein specifically required for assembly of the CuZ center of N2OR, and thus represents the first characterised assembly factor for the CuZ active site of this key environmental enzyme, which is globally responsible for the destruction of a potent greenhouse gas.

Amylotrophic Lateral Sclerosis-Like Motor Impairment in Prion Diseases

Neurodegenerative diseases are collective diseases that affect different parts of the brain with common or distinct disease phenotype. In almost all of the Prion diseases, motor impairments that are characterized by motor derangement, apathy, ataxia, and myoclonus are documented and again are shared by motor neuron diseases (MND). Proteins such as; B-Cell lymphoma 2 (BCL2), Copper chaperone for superoxide dismutase (CCS), Amyloid beta precursor protein (APP), Amyloid Precursor-Like Protein1/2 (APLP1/2), Catalase (CAT), and Stress induced phosphoprotein 1 (STIP1), are common interactomes of Prion and superoxide dismutase 1 (SOD1). Although there is no strong evidence to show the interaction of SOD1 and Prion, the implicated common interacting proteins indicate the potential bilateral interaction of those proteins in health and disease. For example, down-regulation of Heat shock protein A (HSPA5), a Prion interactome, increases accumulation of misfolded SOD1 leading to MND. Loss of Cu uptake function disturbs normal function of CCS. Over-expressed proteasome subunit alpha 3 (PSMA3) could fatigue its normal function of removing misfolded proteins. Studies showed the increase in CAT and lipid oxidation both in Prion-knocked out animal and in catalase deficiency cases. Up regulation, down regulation or direct interaction with their interactomes are predicted molecular mechanisms by which Prion and SOD exert their effect. The loss of protective function or the gain of a novel toxic property by the principal proteins is shared in Prion and MND. Thus, it might be possible to conclude that the interplay of proteins displayed in both diseases could be a key phenomenon in motor dysfunction development.

Targeting of copper-trafficking chaperones causes gene-specific systemic pathology inDrosophila melanogaster: prospective expansion of mutational landscapes that regulate tumor resistance to cisplatin

ABSTRACTCopper, a transition metal, is an essential component for normal growth and development. It acts as a critical co-factor of many enzymes that play key roles in diverse cellular processes. The present study attempts to investigate the regulatory functions decisively controlling copper trafficking during development and aging of the Drosophila model system. Hence, through engagement of the GAL4/UAS genetic platform and RNAi technology, we herein examined the in vivo significance of Atox1 and CCS genes, products of which pivotally govern cellular copper trafficking in fly tissue pathophysiology. Specifically, we analyzed the systemic effects of their targeted downregulation on the eye, wing, neuronal cell populations and whole-body tissues of the fly. Our results reveal that, in contrast to the eye, suppression of their expression in the wing leads to a notable increase in the percentage of malformed organs observed. Furthermore, we show that Atox1 or CCS gene silencing in either neuronal or whole-body tissues can critically affect the viability and climbing capacity of transgenic flies, while their double-genetic targeting suggests a rather synergistic mode of action of the cognate protein products. Interestingly, pharmacological intervention with the anti-cancer drug cisplatin indicates the major contribution of CCS copper chaperone to cisplatin's cellular trafficking, and presumably to tumor resistance often acquired during chemotherapy. Altogether, it seems that Atox1 and CCS proteins serve as tissue/organ-specific principal regulators of physiological Drosophila development and aging, while their tissue-dependent downregulation can provide important insights for Atox1 and CCS potential exploitation as predictive gene biomarkers of cancer-cell chemotherapy responses.

Excess copper promotes photoinhibition and modulates the expression of antioxidant-related genes in Zostera muelleri

Copper (Cu) is an essential micronutrient for plants and as such is vital to many metabolic processes. Nevertheless, when present at elevated concentrations, Cu can exert toxic effects on plants by disrupting protein functions and promoting oxidative stress. Due to their proximity to the urbanised estuaries, seagrasses are vulnerable to chemical contamination via industrial runoff, waste discharges and leachates. Zostera muelleri is a common seagrass species that forms habitats in the intertidal areas along the temperate coast of Australia. Previous studies have shown the detrimental effects of Cu exposure on photosynthetic efficiency of Z. muelleri. The present study focuses on the impacts of sublethal Cu exposure on the physiological and molecular responses. By means of a single addition, plants were exposed to 250 and 500 μg Cu L−1 (corresponding to 3.9 and 7.8 μM, respectively) as well as uncontaminated artificial seawater (control) for 7 days. Chlorophyll fluorescence parameters, measured as the effective quantum yield (ϕPSII), the maximum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) were assessed daily, while Cu accumulation in leaf tissue, total reactive oxygen species (ROS) and the expression of genes involved in antioxidant activities and trace metal binding were determined after 1, 3 and 7 days of exposure. Z. muelleri accumulated Cu in the leaf tissue in a concentration-dependent manner and the bioaccumulation was saturated by day 3. Cu exposure resulted in an acute suppression of ϕPSII and Fv/Fm. These two parameters also showed a concentration- and time-dependent decline. NPQ increased sharply during the first few days before subsequently decreasing towards the end of the experiment. Cu accumulation induced oxidative stress in Z. muelleri as an elevated level of ROS was detected on day 7. Lower Cu concentration promoted an up-regulation of genes encoding Cu/Zn-superoxide dismutase (Cu/Zn-sod), ascorbate peroxidase (apx), catalase (cat) and glutathione peroxidase (gpx), whereas no significant change was detected with higher Cu concentration. Exposure to Cu at any concentration failed to induce regulation in the expression level of genes encoding metallothionein type 2 (mt2), metallothionein type 3 (mt3) and cytochrome c oxidase copper chaperone (cox17). It is concluded that chlorophyll fluorescence parameters provide timely probe of the status of photosynthetic machinery under Cu stress. In addition, when exposed to a moderate level of Cu, Z. muelleri mitigates any induced oxidative stress by up-regulating transcripts coding for antioxidant enzymes.

Regulation through MicroRNAs in Response to Low-Energy N+ Ion Irradiation in Oryza sativa.

MicroRNAs (miRNAs) are a non-coding regulatory RNAs that play significant roles in plant growth and development, especially in the stress response. Low-energy ion radiation, a type of environmental stress, can cause multiple biological effects. To understand the roles of miRNAs in response to low-energy N+ ion radiation in Oryza sativa, high-throughput sequencing of small RNAs was carried out to detect the expression of miRNAs in the shoots of the rice after 2 × 1017 N+/cm2 irradiation. The differentially expressed 28 known miRNAs were identified, 17 of these identified miRNAs were validated by real-time quantitative fluorescent PCR (q-PCR), including 9 up-regulated miRNAs (miR1320-3p, miR1320-5p, miR156b-3p, miR156c-5p, miR156c-3p/g-3p, miR1561-5p, miR398b and miR6250) and 8 down-regulated miRNAs (miR156a/e/i, miR156k, miR160f-5p, miR166j-5p, miR1846e and miR399d). In addition, 45 novel radiationresponsive miRNAs were predicted, and 8 of them were verified by q-PCR. The target genes of radiation-responsive miRNAs were predicted and gene function enrichment analysis was performed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The expression of 9 targets of 4 known miRNA families (miR156, miR399, miR1320 and miR398) and 2 targets of 2 novel miRNAs were quantified by q-PCR, and a strong negative regulation relation between miRNAs and their targets were observed. Those targets including SQUAMOSA promoterbinding-like protein (SPL) genes, copper/zinc superoxide dismutase (Cu/Zn-SOD), copper chaperone for SOD (CCS1) and electron transporter/ heat-shock protein binding protein (HSP), which are involved in growth and defense against various stresses, especially associated with reactive oxygen species (ROS) scavenging. This work provides important information for understanding the ROS generation and elimination mechanisms closely related to miRNAs in rice seedlings after low-energy N+ radiation exposure.

Targeting the Mitochondrial Metallochaperone Cox17 Reduces DNA Methylation and Promotes AML Differentiation through a Copper Dependent Mechanism

The majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and imported into the mitochondria. A subset of cysteine-rich proteins destined for the mitochondrial intermembrane space are oxidized and folded by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML by targeting the FAD linked sulfhydryl oxidase ALR, an integral part of the MIA machinery.Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, ALR knockdown reduced engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6 killed AML cells (IC50 of 5-10 mM) and preferentially reduced the clonogenic growth of primary AML cells over normal hematopoietic cells. MitoBloCK-6 treatment (80 mg/kg i.p. 5 of 7 days x 2 weeks) of mice engrafted with primary AML cells strongly reduced the leukemic burden without changing mouse body weight, serum chemistries, or organ histology. In contrast, MitoBloCK-6 did not change engraftment of normal cord blood in similar experiments. As evidenced by secondary transplants, MitoBloCK-6 also targeted leukemic stem cells.As expression levels of ALR substrates are increased in AML stem cells we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by changes in gene expression, increased differentiation associated CD surface marker expression and increased non-specific esterase.Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Genetic or chemical inhibition of ALR selectively reduced levels of Cox17 protein. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation and induced AML differentiation.Cox17 is a copper metallochaperone that promotes respiratory chain complex IV assembly by loading copper into the respiratory complex. COX17 knockdown slightly reduced the complex IV enzymatic activity, but did not change basal oxygen consumption or ROS production. However, COX17 knockdown increased intracellular levels of free copper 16-fold as measured by atomic mass spectrometry. Copper is a known inhibitor of adenosylhomocysteinase, a key enzyme involved in the preservation of S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio in cells. SAM is a global methyl donor and is critical for DNA methylation. Knockdown of COX17 or ALR inhibition with MitoBloCK-6 decreased levels of SAM and reduced DNA methylation in AML cells. Likewise, the enzymatic activity of adenosylhomocysteinase was reduced in OCI-AML2 cells after MitoBloCK-6 treatment. Importantly, co-treatment with the copper chelator, penicillamine, rescued reductions in SAM, DNA methylation, and cell viability after COX17 knockdown or MitoBloCK-6 treatment.Thus, we have discovered a novel copper-dependent mechanism by which mitochondrial pathways regulate epigenetics and stemness in AML. Moreover, inhibitors of ALR or COX17 may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. DisclosuresSchimmer:Otsuka Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Cytotoxic phenanthroline derivatives alter metallostasis and redox homeostasis in neuroblastoma cells.

Copper homeostasis is generally investigated focusing on a single component of the metallostasis network. Here we address several of the factors controlling the metallostasis for neuroblastoma cells (SH-SY5Y) upon treatment with 2,9-dimethyl-1,10-phenanthroline-5,6-dione (phendione) and 2,9-dimethyl-1,10-phenanthroline (cuproindione). These compounds bind and transport copper inside cells, exert their cytotoxic activity through the induction of oxidative stress, causing apoptosis and alteration of the cellular redox and copper homeostasis network. The intracellular pathway ensured by copper transporters (Ctr1, ATP7A), chaperones (CCS, ATOX, COX 17, Sco1, Sco2), small molecules (GSH) and transcription factors (p53) is scrutinised.

178 - Structural studies on SOD1 zinc site coordination with the new mutant T135S,K136E-SOD1

Cu,Zn-Superoxide Dismutase (SOD1) has two metal ions on its active site: Cu which plays catalytic role and Zn that has a structural function. Cu ion is placed at SOD1 site through action of CCS (Copper chaperone for SOD) but Zn entrance in SOD1 is still unknown. In this work, we used the native wild type SOD1 and we constructed a mutant enzyme using site directed mutation approach to study Zn complexation and its influence on enzyme activity. This mutant SOD1 resulted in a double amino acid mutation around Zn ion T135S and K136E, resulting in a metal non-stabilization coordination, and a lower superoxide dismutase activity. The CD spectra with metal titration showed a huge difference between the two entries of Zn in the native enzyme, highly different from the binding event of Cu ions in the two metal sites of SOD1, since Cu presents a simultaneous entrance in the protein. Nevertheless, the mutant T135S,K136E-SOD1 showed a metal complexation instability. This found is a plausible explanation for the observed not fully folded SOD1 in absence of zinc and its lability.

Proteomic analysis of Zhangzagu3 (Setaria italica) and its parents based on iTRAQ technique

Zhangzagu3, derived from Zhangzagu3fu and A2 (photoperiod- and thermo-sensitive genic male-sterile line) is well-known for its strong adaptability, good stability, disease, and lodging resistance. To evaluate the parental contributions to its elite traits, isobaric tags for relative and absolute quantitation (iTRAQ) and mass spectrometry (MS) were used to analyze the protein expression profile of these three lines. Enrichment analysis of differentially expressed proteins (DEPs) was performed through GO term and KEGG pathway. A total of 4015 proteins were detected, among which 504/390 DEPs between Zhangzagu3 and the female/male parent, respectively. The cluster analysis using Cluster 3.0 showed that the DEP expression profiles of Zhangzagu3 were closer to those of the male parent. GO functional classification divided the DEPs into three functional categories (cellular component, biological process and molecular function) and 71 subfamilies, among which three subfamilies had 100% difference between Zhangzagu3 and its parents. KEGG metabolic pathway exhibited significant enrichment of differentially expressed genes (DEGs) in Carbon metabolism (mainly Carbon fixation, Glycolysis and Gluconeogenesis), Photosynthesis and Oxidative phosphorylation. qRT-PCR verification confirmed the differential expression data and revealed that the expression level of 17 selected genes (such as putative receptor-like protein kinase, copper chaperone for superoxide dismutase etc.) was higher in Zhangzagu3 than that in its male parent, but significantly lower than that in A2. Thus, A2 can potentially optimize the agricultural qualities of Zhangzagu3. Taken together, we hope that this study provides valuable information for the further utilization and study on the molecular mechanisms of heterosis in hybrid millet.AbbreviationsiTRAQisobaric tags for relative and absolute quantitationDEPsdifferentially expressed proteinsDEGsdifferentially expressed genesPMSFphenylmethanesulfonyl fluorideHEPES4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acidDTTdithiothreitolEDTAethylene diamine tetraacetic acidGOgene ontologyIAMiodoacetamideMSmass spectrometryKEGGKyoto Encyclopedia of Genes and GenomesPVPPpolyvinylpolypyrrolidoneHPLChigh performance liquid chromatographyTCAtrichloroacetic acidTEABtetraethylammonium bicarbonateACNacetonitrileHCDhigher collision energy dissociationFDRfalse discovery rateSDSsodium dodecyl sulphateqRT-PCRquantitative real-time polymerase chain reaction

Significance of MccR, MccC, MccD, MccL and 8-methylmenaquinone in sulfite respiration of Wolinella succinogenes

Reduction of sulfite to sulfide is an essential step in the biogeochemical sulfur cycle. The Epsilonproteobacterium Wolinella succinogenes uses the copper-containing octahaem cytochrome c sulfite reductase MccA to respire sulfite. MccA is encoded by the first gene of the mcc gene cluster, whose transcription is apparently induced by the two-component regulatory system MccRS. It has been proposed that the iron‑sulfur protein MccC, the putative quinol dehydrogenase MccD, the copper chaperone MccL as well as menaquinone-6 (MK6) and/or 8-methylmenaquinone-6 (8-MMK6) are involved in the electron transport chain of W. succinogenes sulfite respiration. Here, non-polar W. succinogenes mutants were constructed that lacked MccC, MccD, MccL or the 8-MMK6-producing MK6 methyltransferase MqnK. Each mutant possessed a frameshift-corrected mccR gene, thus inducing mcc expression in the presence of a mixture of fumarate and sulfite as terminal electron acceptors. Under these conditions, growth by sulfite respiration of cells lacking MccA, MccC or MccD was found to be abolished. However, cells lacking MccL or 8-MMK6 still coupled formate oxidation to sulfite reduction and grew by sulfite respiration to some extent. The results indicate that MccR, MccC, MccD, MccL and 8-MMK6 are essential or significant components of W. succinogenes sulfite respiration.

Recent advances in copper chaperone and cancer

Copper is a redox-active transition metal essential for most living organisms and serves as a catalytic cofactor for enzymes that function in antioxidant defence, iron homeostasis, cellular respiration and a variety of biochemical processes. However, intracellular free copper must be strictly limited because of its potential toxic side effects. Copper dyshomeostasis is involved in the pathogenesis and progression of diseases including cancer and neurodegenerative diseases. The uncontrolled accumulation of copper could lead to increased oxidative stress and inappropriate binding to macromolecules. Most cells evolve complex systems of copper regulation and trafficking to satisfy the cellular copper requirements and simultaneously minimize the potential toxicity. This review presents the importance of copper transporter Ctr1, copper chaperone CCS, Atox1 and COX17 for the maintenance of copper ion balance in cells. Moreover, we discuss recent highlights in the development of copper chelators for the treatment of cancer. Finally, we introduce the development of small molecules that alter copper homeostasis by inhibiting copper chaperones.

Trientine selectively delivers copper to the heart and suppresses pressure overload-induced cardiac hypertrophy in rats.

Our study reveals that TETA, traditionally regarded as a copper chelator, in lower doses delivers copper selectively to the heart through a mechanism independent of copper transporter-1 or -2. Copper supplementation by a lower dose of TETA suppresses pressure overload-induced cardiac hypertrophy. Since ischemic heart disease and hypertrophic cardiomyopathy are accompanied by myocardial copper loss, this approach of using a lower dose of TETA to supplement copper to the heart would help treat the disease condition of patients with such cardiac events.

Identification of a novel zinc-binding protein, C1orf123, as an interactor with a heavy metal-associated domain.

Heavy metal-associated (HMA) domains bind metal ions at its Cys-x-x-Cys (CxxC) motif and constitute an intracellular network for trafficking of metal ions for utilization and detoxification. We thus expect that novel metalloproteins can be identified by screening proteins interacting with a HMA domain. In this study, we performed yeast two-hybrid screening of the human proteome and found an uncharacterized protein encoded as open reading frame 123 in chromosome 1 (C1orf123) that can interact specifically with the HMA domain of a copper chaperone for superoxide dismutase (CCSdI). Our X-ray structural analysis of C1orf123 further revealed that it binds a Zn2+ ion in a tetrahedral coordination with four thiolate groups from two conserved CxxC motifs. For the interaction between C1orf123 and CCSdI, the CxxC motifs in both C1orf123 and CCSdI were required, implying metal-mediated interaction through the CxxC motifs. Notably, C1orf123 did not interact with several other HMA domains containing CxxC motifs, supporting high specificity in the interaction between C1orf123 and CCSdI. Based upon these results, we further discuss functional and structural significance of the interaction between C1orf123 and CCS.

Isolation and Transcriptome Analysis of Phenol-Degrading Bacterium From Carbon-Sand Filters in a Full-Scale Drinking Water Treatment Plant.

Phenol is a typical organic contaminant in the environment. To date, the biodegradation of phenol by microorganisms remains the preferred method for its removal and remediation, but data on phenol removal by drinking water biofilters are lacking. In this study, we used high-throughput sequencing to investigate the microbial community structure in a carbon–sand biofilter. The results indicated that the predominant bacterial group was Bacilli, followed by Gammaproteobacteria, Clostridia, and Alphaproteobacteria. In addition, a strain was capable of degrading phenol at low concentrations of 500 μg/L within 100 min was isolated and identified as Rhodococcus sp. CS-1. Transcriptome analysis results showed that Rhodococcus sp. CS-1 was able to degrade phenol via both the catechol and protocatechuate branch of the β-ketoadipate pathway. Furthermore, some novel candidate biomarkers (copper oxidase, copper chaperone, and MarR/DeoR/TetR family transcriptional regulators) were successfully identified to be potentially involved in phenol biodegradation. This study indicates that carbon–sand filters have the potential for remediation of phenol. The application of native microorganisms to drinking water treatment system is an adaptive strategy in oligotrophic water environments.

Lack of superoxide dismutase in a rad51 mutant exacerbates genomic instability and oxidative stress-mediated cytotoxicity in Saccharomyces cerevisiae

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.