Silicon induces metallochaperone-driven cadmium binding to the cell wall and restores redox status through elevated glutathione in Cd-stressed sugar beet.

Cadmium (Cd) is toxic; however, whether silicon (Si) alleviates Cd toxicity was never studied in sugar beet. The study was conducted on 2-week-old sugar beet cultivated in the presence or absence of Cd (10 µM CdSO4 ) and Si (1 mM Na2 SiO3 ) in hydroponic conditions. The morphological impairment and cellular damages observed in sugar beet upon Cd toxicity were entirely reversed due to Si. Si substantially restored the energy-providing ability, absorbed energy flux, and electron transport toward PSII, which might be correlated with the upregulation of BvIRT1 and ferric chelate reductase activity leading to the restoration of Fe status in Cd-stressed sugar beet. Although Si caused a reduction of shoot Cd, the root Cd substantially increased under Cd stress, a significant part of which was retained in the cell wall rather than in the root vacuole. While the concentration of phytochelatin and the expression of BvPCS3 (PHYTOCHELATIN SYNTHASE 3) showed no changes upon Si exposure, Si induced the expression of BvHIPP32 (HEAVY METAL-ASSOCIATED ISOPRENYLATED PLANT PROTEIN 32) in the Cd-exposed root. The BvHIPP32 and AtHIPP32 metallochaperone proteins are localized in the cell wall and they share similar sequence alignment, physiochemical properties, secondary structure, cellular localization, motif locations, domain association, and metal-binding site (cd00371) linked to the metallochaperone-like protein. It suggests that Si reduces the Cd level in shoot by retaining the excess Cd in the cell wall of roots due to the induction of BvHIPP32 gene. Also, Si stimulates glutathione-related antioxidants along with the BvGST23 expression, inferring an ascorbate-glutathione ROS detoxification pathway in Cd-exposed plants.

Iron Chaperone Poly rC Binding Protein 1 Protects Mouse Liver From Lipid Peroxidation and Steatosis.

BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.

Effective Inhibition of Cellular ROS Production by MXCXXC-Type Peptides: Potential Therapeutic Applications in Copper-Homeostasis Disorders.

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

Isolation and characterisation of cDNA encoding a wheat heavy metal-associated isoprenylated protein involved in stress responses.

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal-associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy-terminal end. To investigate the roles of wheat heavy metal-associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal-associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real-time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up-regulated by ABA exposure or wounding. Additionally, TaHIPP1 over-expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1-GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus-induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.

Redox sulfur chemistry of the copper chaperone Atox1 is regulated by the enzyme glutaredoxin 1, the reduction potential of the glutathione couple GSSG/2GSH and the availability of Cu(I).

Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)' = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)' = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport.

Chaperones CCS, ATOX and COXIV responses to copper supplementation in healthy adults.

Assessment of proteins in blood and other tissues has failed to identify markers of early copper effects on health. Studies in animal models show that chaperone of SOD (CCS) respond to changes of copper status. Evidence about other copper chaperones (COXIV, ATOX) is not clear. The aim of this study was to assess by means of an in vitro challenge the mRNA relative abundance of ccs, sod1, coxIV, mtIIa and atox in peripheral mononuclear cells (PMNCs) obtained from healthy individuals, acutely and chronically supplemented with small-to-moderate amounts of copper. Healthy participants received 8 mg Cu/d (supplemented group, SG) or placebo, (placebo group, PG) for 2 months. Biochemical indicators were assessed at basal (T0) and after 2 (T2) and 60 days (T60). At these times PMNCs were obtained, challenged with 1, 5 or 20 µM Cu-histidine for 20 h and the mRNA relative abundance of the selected genes assessed by real time PCR. The results showed that at T0, intracellular copper was not different between experimental and control groups. This increased at T2 and T60 when the copper in the media increased (two-way ANOVA, P < 0.001). In PG, CCS mRNA transcripts showed no significant changes (two-way ANOVA) at T2 and T60. In SG, CCS changed by treatment, time and interaction (two-way ANOVA, all P < 0.001). SOD, ATOX and COXIV expressions changed in both PG and SG showing various patterns of response, requiring further study. MTII responded as expected. We conclude that using healthy individuals as a human model, CCS but not SOD, ATOX or COXIV responded consistently to controlled changes of copper availability in an in vitro copper challenge.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards.

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants ß(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ∼10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.

AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.

UNLABELLED: Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD. With the exception of p.S1363F, all mutations tested (p.G85V, p.R778L, p.H1069Q, p.C1104F, p.V1262F, p.G1343V, and p.S1363F) resulted in reduced ATP7B protein expression, whereas messenger RNA abundance was unaffected. Retention of mutant ATP7B in the endoplasmic reticulum, increased protein expression, and normalization of localization after culturing cells at 30 degrees C, and homology modeling suggested that these proteins were misfolded. Four distinct mutations exhibited residual copper export capacity, whereas other mutations resulted in complete disruption of copper export by ATP7B. Treatment with pharmacological chaperones 4-phenylbutyrate (4-PBA) and curcumin, a clinically approved compound, partially restored protein expression of most ATP7B mutants. CONCLUSION: These findings might enable novel treatment strategies in WD by directly enhancing the protein expression of mutant ATP7B with residual copper export activity. 1795.).

The N-terminal metal-binding site 2 of the Wilson's Disease Protein plays a key role in the transfer of copper from Atox1.

The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver. Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease. The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP. We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process. The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe. Such selectivity is not observed when free copper is added to N-WNDP. Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper. The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites. Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process. X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1. Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1. The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed.

Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper.

Impaired uptake of cisplatin (DDP) consistently accompanies the acquisition of resistance to the platinum drugs. The pathways by which DDP enters or exits from cells remain poorly defined. Using three pairs of human ovarian carcinoma cell lines, each consisting of a sensitive parental line and a stably DDP-resistant subline derived by in vitro selection, resistance to DDP was found to be accompanied by cross-resistance to Cu. Accumulation of DDP in the resistant sublines ranged from 38 to 67% of that in the parental line at 1 h, and DNA adduct formation varied from 10 to 38% of that in the sensitive cells. The DDP-resistant cells had 22-56% lower basal levels of copper, and the copper levels were only 27-46% of those observed in the sensitive parental lines after a 24-h exposure to medium supplemented with copper. The initial influx rate for DDP in the three resistant cell lines ranged from 23 to 55% of that in the sensitive cells of each pair; the initial influx rate for copper in the resistant cells varied from 56 to 75% of control. Studies performed using one pair of cell lines demonstrated that for both copper and DDP the initial efflux rate was lower, whereas the terminal efflux rate was higher in the resistant cells. On Western blot analysis all three resistant lines exhibited increased expression of one or the other of the two copper export pumps (ATP7A or ATP7B) with no change in the HAH1 chaperone. We conclude that the acquisition of DDP resistance in ovarian carcinoma is accompanied by alterations in the cellular pharmacology of DDP that are paralleled by similar changes in the uptake and efflux of copper. These results are consistent with the concept that DDP enters and exits from the cell via transporters that normally mediate copper homeostasis.

Structure, expression, and chromosomal localization of the mouse Atox1 gene.

Copper trafficking in eukaryotes involves small proteins termed metallochaperones, which mediate copper delivery to specific intracellular sites. Previous studies in yeast and human cell lines have suggested that Atox1 plays a critical role in copper delivery to the secretory pathway. In the present study, a mouse Atox1 (mAtox1) cDNA was cloned and shown to encode an open reading frame with 85% amino acid identity to human Atox1. RNA blot analysis revealed that mAtox1 was expressed as a single transcript in multiple tissues, and immunoblotting indicated that the relative abundance of mAtox1 mRNA directly correlated with mAtox1 protein. Analysis of the mAtox1 gene locus revealed a genomic structure with four exons encompassing a total of 14.5 kb. RFLP and haplotype analyses indicated that the mAtox1 locus was tightly linked to the Trhr and D15Bir7 loci on mouse chromosome 15. Taken together, these data reveal marked evolutionary conservation of Atox1 structure and provide a genomic organization and localization that will aid in the genetic deciphering of the molecular role of this protein in copper homeostasis.

Expression profile of the copper homeostasis gene, rAtox1, in the rat brain.

In humans the regulation of cellular copper homeostasis is essential for proper organ development and function. A novel cytosolic protein, named Atox 1, was recently identified in yeast that functions in shuttling intracellular mononuclear copper [Cu(I)] to copper-requiring proteins. Atox 1 and its human homolog, hAtox1, are members of an emerging family of proteins termed copper chaperones that are involved in the maintenance of copper homeostasis. Northern blot analysis demonstrates that Atox 1 is widely expressed at varying levels in a variety of rat tissues including brain. Using in situ hybridization histochemistry, we characterized the expression profile for the rat homolog of Atox1 (rAtox1) in the normal adult rat brain. There is widespread expression within the brain that appears to be primarily neuronal. The highest levels of Atox1 message consists of distinct neuronal subtypes that are also characterized by their high levels of metals like copper, iron, and zinc, which include the pyramidal neurons of the cerebral cortex and hippocampus in addition to the neurons of the locus coeruleus. The high levels of a metal chaperone like Atox1 in subsets of neurons that also sequester metals suggests that Atox1 may be important in maintaining the functionality of metal requiring enzymes. A detailed analysis of the restricted expression profile for a novel copper chaperone, rAtox1, is described in the adult rat CNS. Further analysis shows that Atoxl expression is associated with neuronal populations that sequester copper.