Distorted copper homeostasis with decreased sensitivity to cisplatin upon chaperone Atox1 deletion in Drosophila

Copper is an integral part of a number of proteins and thus an essential trace metal. However, free copper ions can be highly toxic and every organism has to carefully control its bioavailability. Eukaryotes contain three copper chaperones; Atx1p/Atox1 which delivers copper to ATP7 transporters located in the trans-Golgi network, Cox17 which provides copper to the mitochondrial cytochrome c oxidase, and CCS which is a copper chaperone for superoxide dismutase 1. Here we describe the knockout phenotype of the Drosophila homolog of mammalian Atox1 (ATX1 in yeast). Atox1−/− flies develop normally, though at reduced numbers, and the eclosing flies are fertile. However, the mutants are unable to develop on low-copper food. Furthermore, the intestinal copper importer Ctr1B, which is regulated by copper demand, fails to be induced upon copper starvation in Atox1−/− larvae. At the same time, intestinal metallothionein is upregulated. This phenotype, which resembles the one of the ATP7 mutant, is best explained by intestinal copper accumulation, combined with insufficient delivery to the rest of the body. In addition, compared to controls, Drosophila Atox1 mutants are relatively insensitive to the anticancer drug cisplatin, a compound which is also imported via Ctr1 copper transporters and was recently found to bind mammalian Atox

Human copper transporter Ctr1 is functional in Drosophila , revealing a high degree of conservation between mammals and insects

Living cells have to carefully control the intracellular concentration of trace metals, especially of copper, which is at the same time essential but owing to its redox activity can also facilitate generation of reactive oxygen species. Mammals have two related copper transporters, Ctr1 and Ctr2, with Ctr1 playing the major role. The fruit fly Drosophila has three family members, termed Ctr1A, Ctr1B, and Ctr1C. Ctr1A is expressed throughout development, and a null mutation causes lethality at an early stage. Ctr1B ensures efficient copper uptake in the intestinal tract, whereas Ctr1C is mainly expressed in male gonads. Ectopic expression of Ctr1 transporters in Drosophila causes toxic effects due to excessive copper uptake. Here, we compare the effects of human Ctr1 (hCtr1) with those of the Drosophila homologs Ctr1A and Ctr1B in two overexpression assays. Whereas the overexpression of Drosophila Ctr1A and Ctr1B results in strong phenotypes, expression of hCtr1 causes only a very mild phenotype, indicating a low copper-import efficiency in the Drosophila system. However, this can be boosted by coexpressing the human copper chaperone CCS. Surprisingly, hCtr1 complements a lethal Ctr1A mutation at least as well as Ctr1A and Ctr1B transgenes. These findings reveal a high level of conservation between the mammalian and insect Ctr1-type copper importers, and they also demonstrate that the Drosophila Ctr1 proteins are functionally interchangeabl

Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione

Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein

Mercury and cadmium trigger expression of the copper importer Ctr1B, which enables Drosophila to thrive on heavy metal-loaded food

Organisms from insects to mammals respond to heavy metal load (copper, zinc, cadmium, and mercury) by activating the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to short DNA sequence motifs, termed metal response elements, and boosts transcription of a number of genes, notably those for metallothioneins. In Drosophila, MTF-1 somewhat counter-intuitively also activates transcription of a copper importer gene (Ctr1B) in response to copper starvation. Here, we report that mutant flies lacking Ctr1B are extremely sensitive to cadmium and mercury treatment, but can be rescued by excess copper in the food. We thus propose that copper, by competing for binding sites on cellular proteins, alleviates the toxic effects of mercury and cadmium. Such a scenario also explains a seemingly fortuitous metal response, namely, that cadmium and mercury strongly induce the expression of a Ctr1B reporter gene. Thus, the transcription enhancer/promoter region of the Ctr1B copper importer gene is subject to three modes of regulation. All of them depend on MTF-1 and all make biological sense, namely, (i) induction by copper starvation, (ii) repression by copper abundance, and (iii), as shown here, induction by cadmium or mercury at normal copper suppl

Characterization of MtnE, the fifth metallothionein member in Drosophila

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missin

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural ‘linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regu­latory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manne

Toxicity of Alzheimer's disease-associated Aβ peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability

Amyloid plaques consisting of aggregated Aβ peptide are a hallmark of Alzheimer's disease. Among the different forms of Aβ, the one of 42aa length (Aβ42) is most aggregation-prone and also the most neurotoxic. We find that eye-specific expression of human Aβ42 in Drosophila results in a degeneration of eye structures that progresses with age. Dietary supplements of zinc or copper ions exacerbate eye damage. Positive effects are seen with zinc/copper chelators, or with elevated expression of MTF-1, a transcription factor with a key role in metal homeostasis and detoxification, or with human or fly transgenes encoding metallothioneins, metal scavenger proteins. These results show that a tight control of zinc and copper availability can minimize cellular damage associated with Aβ42 expressio

Transcriptional repression by methylation: cooperativity between a CpG cluster in the promoter and remote CpG-rich regions

AbstractCytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA-VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation-free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PlGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PlGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PlGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlG

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro. It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis-trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domaincontaining splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcriptio