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Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

ABSTRACT: Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

SUBMITTER: Prochazkova P

PROVIDER: S-EPMC4184891 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

Procházková Petra P Škanta František F Roubalová Radka R Šilerová Marcela M Dvořák Jiří J Bilej Martin M

PloS one 20141003 10

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely fold ...[more]

PMID: 25279857

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Project description:Uniformly pigmented Eisenia andrei (Ea) and striped E. fetida (Ef) lumbricid earthworms are hermaphrodites capable of self-fertilization, cross-fertilization, and asymmetrical hybridization. The latter was detected by genotyping of F1 and F2 progeny of the controlled Ea+Ef pairs by species-specific sequences of maternal mitochondrial COI genes and maternal/paternal nuclear S28 rRNA genes. Among F1offspring there were self-fertilized Ea (aAA), Ef (fFF), and cross-fertilized fertile Ea-derived hybrids (aAF); the latter mated with Ea and gave new generation of Ea and hybrids, while mated with Ef gave Ea, Ef, Ea-derived hybrids and sterile Ef-derived hybrids (fFA). Coelomic fluid of Ea exhibits unique fluorescence spectra called here the M-fluorescence considered as a molecular biomarker of this species. Since similar fluorescence was detected also in some Ef (hypothetical hybrids?), the aim of present investigations was to identify the M-positive earthworms among families genotyped previously. It was assumed that factor/s responsible for metabolic pathways leading to production of undefined yet M-fluorophore might be encoded/controlled by alleles of hypothetical nuclear gene of Eisenia sp. segregating independently from species-specific S28 rRNA nuclear genes, where 'MM' or 'Mm' alleles determine M-positivity while 'mm' alleles determine M-negative phenotypes. Spectra of M-fluorescence were detected in all 10 Ea (aAAMM) and 19 Ea-derived hybrids (aAFMm), three of four Ef-derived hybrids (fFAMm) and one 'atypical' Ef (fFFMm) among 13 Ef earthworms. Among progeny of 'atypical' M-positive Ef (fFFMm) reappeared 'typical' M-negative Ef (fFFmm), confirming such hypothesis. Alternatively, the M-fluorescence might be dependent on unknown gene products of vertically-transmitted Ea-specific symbiotic bacteria sexually transferred to the Ef partner. Hypotheses of intrinsic and external origin of M-fluorescence might complement each other. The presence/absence of M-fluorophore does not correspond with body pigmentation patterns; Ef-characteristic banding appeared in posterior parts of hybrids body. In conclusion, Ea/Ef hybridization may serve for further studies on bi-directional gene flow.

Project description:Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

Dataset Information

Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

Publications

Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

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