All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion were determined. Furthermore, the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant was analyzed. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in this study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification (Yepiskoposyan, 2006).
An unexpected result of the present study is the induction of ferritin heavy and light chain homologue genes by heavy metals. Ferritin is well known to detoxify, store and transport iron. It was also shown to bind other metal ions and has been speculated to function as a general metal detoxicant in mammals. The results suggest an induction of Drosophila ferritins by metals other than iron, notably zinc. The latter induction depends on MTF-1, based on the following findings: (1) the presence of multiple MREs, (2) the loss of zinc induction in the MTF-1 mutant, and (3) elevated ferritin transcripts upon MTF-1 overexpression. In contrast, iron inducibility must be regulated independently since it is not altered by the absence of MTF-1. In line with different signaling pathways responding to iron load versus excess of zinc, copper or cadmium, it is noted that the classical MTF-1 dependent MtnA gene is not inducible by iron. Yet these pathways apparently overlap in the ferritin genes (Yepiskoposyan, 2006).
The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. IREs have been identified and characterized in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. These results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis (Gray, 1996; Missirlis, 2007).
As in vertebrates, the IRE/IRP system functions in Drosophila (Rothenberger, 1990; Missirlis, 2003). A functional IRE is present in the 5ķUTR of the Fer1HCH mRNA, but only in certain splice variants that are preferentially encoded under iron-limiting conditions (Lind, 1998; Georgieva, 1999). In contrast, no IRE is present in Fer2LCH mRNA (Georgieva, 2002). IRP homologs are expressed in the fly (Muckenthaler, 1998) and one homolog (IRP-1A) has been shown to bind to IREs from both Drosophila and mammals (Lind, 2006; Missirlis, 2007).
Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans, have been identifed. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network (Muckenthaler, 1998).
Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, a genetic analysis of the interaction between iron and oxygen metabolism was undertaken in Drosophila. Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. Exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, it was found that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. It is concluded that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments (Missirlis, 2003).
In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs. The Drosophila genome offers an evolutionary intermediate containing two IRP1-like proteins (IRP-1A and IRP-1B) and target genes with IREs. This study used purified recombinant IRP-1A and IRP-1B from Drosophila melanogaster and showed that only IRP-1A can bind to IREs, although both proteins possess aconitase activity. These results were also corroborated in whole-fly homogenates from transgenic flies that overexpress IRP-1A and IRP-1B in their fat bodies. Ubiquitous and muscle-specific overexpression of IRP-1A, but not of IRP-1B, resulted in pre-adult lethality, underscoring the importance of the biochemical difference between the two proteins. Domain-swap experiments showed that multiple amino acid substitutions scattered throughout the IRP1 domains are synergistically required for conferring IRE binding activity. These data suggest that as a first step during the evolution of the IRP/IRE system, the ancient cytosolic aconitase was duplicated in insects with one variant acquiring IRE-specific binding (Lind, 2006).
A GFP-containing P-element integrated into the second intron of Fer1HCH was used to study ferritin expression. Intron/exon donor and acceptor splice sites flank the GFP sequence on the P-element; consequently all Fer2LCHG188 mRNA types (IRE +/-) are predicted to contain the GFP exon, which should encode GFP in frame with the Fer1HCH protein. Therefore, post-transcriptional regulation of GFP-Fer1HCH mediated by the IRP-1A (Lind, 2006) would remain unaffected by the GFP exon. GFP is inserted two amino acids downstream of the predicted cleavage site for the Fer1HCH signal peptide (Charlesworth, 1997), which is encoded from the second exon of the gene. Thus, the full-length mature polypeptide is predicted to translocate to the endoplasmic reticulum and contain the GFP attached to the N-terminus, after the signal peptide is cleaved. To test whether the GFP-Fer1HCH fusion polypeptide was generated as predicted from the locus containing the G188 P-element, extracts from adult wild type (+/+) flies or flies heterozygous for the GFP insertion line (G188/+) were separated by SDS-page electrophoresis under reducing conditions. Western blots were probed with antisera raised against Fer1HCH or Fer2LCH peptides. Results indicated the presence of two immuno-reactive bands with the Fer1HCH antibody in Fer2LCHG188/+ lysates. The higher-molecular weight species migrated at the predicted size of 50kDa, consistent with a 27kDa GFP addition to the 23kDa Fer1HCH chain. Levels of the Fer2LCH did not change compared to wild type lysates (Missirlis, 2007).
To address whether the expressed GFP-Fer1HCH polypeptide contributes to the formation of the holoferritin in the transgenic insect, the same extracts were separated by SDS-page electrophoresis under non-reducing conditions, which have previously been shown to preserve higher molecular weight ferritin heteropolymers (Missirlis, 2006). In extracts from wild type flies a single ferritin species was observed that migrated close to mouse liver ferritin. As expected from the lower levels of both ferritin subunits in Fer1HCH451/+ heterozygotes, these flies expressed less total ferritin. Conversely, in extracts from heterozygous Fer2LCHG188/+ flies that expressed GFP-tagged ferritin, several higher-molecular weight bands could be detected. Thus, ferritin polymers are formed in these flies and the different molecular weight species most likely reflect varying ratios of Fer1HCH and GFP-Fer1HCH chains in polymers. Importantly, no ferritin composed solely of native Fer1HCH and Fer2LCH was detected in the heterozygous Fer2LCHG188/+ flies, suggesting that the GFP-tagged ferritins are the functional ferritins of these animals. However, homozygous Fer2LCHG188/G188 and hetero-allelic Fer2LCHG188/451 embryos failed to develop into larvae, indicating that the fly cannot survive when all Fer1HCH subunits are tagged with GFP. It was of interest to find out if GFP tagging of all Fer1HCH subunits hindered ferritin assembly, or whether it obstructed access of an iron carrier to the assembled ferritin. If failure to assemble ferritin with 12 GFP-Fer1HCH subunits was the cause of lethality, rescue or the lethality of Fer2LCHG188/451 flies by overexpression of the mutant ferroxidase construct UAS-Fer1HCH* was expected. This expectation arose from the fact that GFP-Fer1HCH should provide intact ferroxidase centers to the holomer, and mutant Fer1HCH* should allow normal assembly. However, overexpression of wild type UAS-Fer1HCH rescued the lethality of Fer2LCHG188/451 flies, but UAS-Fer1HCH* did not, suggesting that a more likely explanation for the lethality of embryos expressing only GFP-Fer1HCH subunits is that the GFP tag blocks Fer1HCH-interacting proteins from accessing the ferroxidase centers of holoferritin (Missirlis, 2007).
Attempts were made to demonstrate that the GFP-tagged ferritin in the Fer2LCHG188/+ heterozygous flies is indeed able to sequester iron and substitute for non-tagged holoferritin. To this end, adult flies were fed with radioactive iron in the form of 55FeCl3. Nitrilotriacetate was added to maintain 55Fe3+ in solution at the neutral pH of food, and whole-fly homogenates were prepared after the insects were allowed to feed for 24 hours. Autoradiographs of the non-denaturing SDS-page gels clearly indicated that iron is incorporated in ferritin from wild type or Fer1HCH451/+ flies but also in GFP-tagged ferritin. The observation that the same amount of iron is associated with half the amount of ferritin protein in the heterozygous Fer1HCH451/+ strain suggests that the iron load per ferritin holomer can increase when total ferritin decreases (Missirlis, 2007).
Also, the absence of 55Fe incorporation in the heterozygous Fer2LCHG188/+ strain at the same molecular weight where putative residual normal ferritins would run suggests that these flies survive on ferritin containing both GFP-tagged and non-tagged Fer1HCH subunits. It is speculated that GFP may partially inhibit iron loading is consistent with findings that assembled ferritin molecules with fewer GFP-tags (distinguished by their faster migration) are more heavily iron loaded than assembled ferritin molecules with many GFP-Fer1HCH subunits (Missirlis, 2007).
Thus, although the GFP-Fer1HCH in solo is not fully functional, it coassembles in vivo with Fer1HCH to form functional fluorescent ferritin (Missirlis, 2007).
Since iron overload is known to induce ferritin expression in vertebrates and flies alike, attempts were made to test the consequences of overexpression of either ferritin H or L genes under normal or low iron levels. Immunoblotting of lysates prepared from whole fly extracts revealed that ubiquitous overexpression of either of the two single chains individually is not sufficient to significantly alter total ferritin amounts or the relative ratio between the two chains. In contrast, when both UAS-Fer1HCH and UAS-Fer2LCH are simultaneously activated with the same Actin-Gal4 driver, robust overexpression is achieved in both sexes. Expression of the ferroxidase inactive UAS-Fer1HCH* was also demonstrated when co-expressed with UAS-Fer2LCH. Overexpression of ferritin was also revealed when the proteins were separated in nonreducing gels. Successful expression from the transgenes was confirmed by performing RT-PCR using primers that were specific to the transgenic mRNA (Missirlis, 2007).
Phenotypes associated with ferritin overexpression: Ferritin overexpression in mammalian cells causes functional iron deficiency due to iron chelation (Cozzi, 2000; Wilkinson, 2006). However, no significant alteration of total radioactive iron associated with overexpressed ferritin was detected after 24-hours of feeding. If the feeding period was extended for 5 days, a slight increase was seen in sequestration of radiolabeled iron into the ferritin of overexpressors, but overall the results show that, at least in Drosophila, ferritin protein levels are not the determining factor controlling iron storage into ferritin. Overexpression of ferroxidase-inactive UAS-Fer1HCH* together with UAS-Fer2LCH in the presence of functional endogenous Fer1HCH subunits results in ferritin heteropolymers that are still potent iron storage complexes (Missirlis, 2007).
It has been shown that overexpression of Fer3HCH, a homopolymeric mitochondrial ferritin composed entirely of Fer3HCH chains, causes female-specific resistance to paraquat (Missirlis, 2006). This study shows that overexpression of Fer1HCH or Fer2LCH alone is not sufficient to confer paraquat resistance, but coexpression of either Fer1HCH or Fer1HCH* in concert with Fer2LCH confers greater survival to a paraquat challenge, underscoring the functional cooperation of the ferritin subunits in vivo. As with flies overexpressing mitochondrial ferritin, resistance to oxidative stress is not observed in males that overexpress Fer1HCH and Fer2LCH. These results were corroborated by using different driver lines (FB-Gal4 and Elav-Gal4) and also by using hydrogen peroxide as a stressor. It is speculated that overexpression of ferritin triggers a developmental signal, possibly related to iron availability, that is specific in females and could relate to the complex nutritional regulation that allocates resources towards reproduction, energy storage or metabolic activity, all adaptive traits in females. In contrast to Fer3HCH overexpression, which does not affect development, flies overexpressing Fer1HCH and Fer2LCH are at a developmental disadvantage compared to their siblings. The progeny from the cross Actin-Gal4/Cyo x UASFer1HCH, UAS-Fer2LCH/UAS-Fer1HCH, UAS-Fer2LCH were scored by gender and by presence or absence of the Actin-Gal4 driver. Two independent sets of recombinant chromosomes were used (one on the X and one on the 3rd) for UAS-Fer1HCH and UAS-Fer2LCH; also flies were used that carried both recombinant chromosomes, allowing for testing if the phenotype was dosage-sensitive. In male flies, the competitive disadvantage of ferritin overexpression is more pronounced than in females. Importantly, the effects of ferritin overexpression are more dramatic under iron-limiting conditions induced by addition of the iron chelator Bathophenanthroline disulfate in the food, whereas the lethal effects can be rescued by dietary iron supplementation. Therefore, the results indicate that ubiquitous ferritin overexpression in the absence of iron overload can be deleterious, by a mechanism which implicates the iron-chelating properties of ferritin (Missirlis, 2007).
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