The DpsA-homologue of the archaeon Halobacterium salinarum is a ferritin

An iron-rich protein, DpsA(Hsal), was isolated from the archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC7942. It consists of 20-kDa subunits forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 100 Fe ions per mole of holoprotein. CD spectra and secondary structure calculations are consistent with an alpha-helical contribution of 60%. The UV/VIS spectrum provides no evidence for the presence of heme groups. This protein exhibits features of a non-heme type bacterial ferritin (Ftn) although it shares only little sequence homology with Ftn. Molecular modelling disclosed a high structural similarity to E. coli Dps.     

Characterization of a non-haem ferritin of the Archaeon Halobacterium salinarum,homologous to Dps (starvation-induced DNA-binding protein)

An iron-rich protein was isolated from the Archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC 7942. It consists of 20 kDa subunits, forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 103 Fe ions/mol of holoprotein. CD spectra are consistent with an alpha-helical contribution of 58%. The UV/visible spectrum provides no evidence for the presence of haem groups. This protein exhibits features of a non-haem-type bacterial ferritin although it shares only little sequence homology with non-haem bacterial ferritin.     

Expression and mutagenesis of the dpsA gene of Synechococcus sp. PCC7942, encoding a DNA-binding protein involved in oxidative stress protection

The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long-term nutrient limitation. Some members of the Dps protein family have been shown to form abundant, large (∼150 kD) hexameric complexes that bind chromosomal DNA with little sequence specificity. Previous work from this lab has demonstrated that the Dps proteins are divergent members of the bacterioferritin/bacterioferritin superfamily, and that the Synechococcus sp. PCC7942 Dps homolog, named DpsA, is a DNA-binding hemoprotein having heme-dependent catalytic activity. We speculated that this protein may yield a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggested that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Current work has examined the expression of dpsA both under nutrient stress and during the growth phase; whereas dpsA mRNA is detectable in the exponential phase, transition to stationary phase yields a 20-fold increase in steady-state mRNA levels. Mapping the promoter region identifies a TAGAAT −10 sequence likely recognized by a cyanobacterial RpoS homolog. Lastly, site-directed mutants lacking dpsA function exhibit a severe phenotype impaired under all conditions yielding photooxidative stress; these include high light and treatment with paraquat. This supports our contention that the DpsA protein serves an important protective function in an obligate photoautotroph.     

Identification of an oxidative stress-sensitive protein from Campylobacter jejuni, homologous to rubredoxin oxidoreductase/rubrerythrin

An oxidative stress-sensitive protein was found in the microaerophile Campylobacter jejuni. A novel 27-kDa protein was found to decrease concomitantly with a decrease in viability from either exogenous H(2)O(2) stress or endogenous oxidative stresses in aerobic conditions. Sequence analyses revealed that the 27-kDa protein was identical to Cj0012c in C. jejuni NCTC11168 and its deduced 215 amino acid sequence has similarity to two non-heme iron proteins found in other bacteria, rubredoxin oxidoreductase (Rbo) and rubrerythrin (Rbr). Thus, we designated the protein as Rrc (Rbo/Rbr-like protein of C. jejuni). In H(2)O(2)-treated cells, Western blot analysis showed some bands smaller than Rrc, and RT-PCR showed similar expression of Rrc mRNA to the control without treatment, suggesting that the sensitive response of Rrc to oxidative stress is due to degradation of the protein.     

Ferritin and ferritin isoforms II: protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes

Ferritin is a major iron storage protein involved in the regulation of iron availability. Each ferritin molecule comprises 24 subunits. Various combinations of H-subunits and L-subunits make up the 24-subunit protein structure and these ferritin isoforms differ in their H-subunit to L-subunit ratio, as well as in their metabolic properties. Ferritin is an acute-phase protein and its expression is up-regulated in conditions such as uncontrolled cellular proliferation, in any condition marked by excessive production of toxic oxygen radicals, and by infectious and inflammatory processes. Under such conditions ferritin up-regulation is predominantly stimulated by increased reactive oxygen radical production and by cytokines. The major function of ferritin in these conditions is to reduce the bio-availability of iron in order to stem uncontrolled cellular proliferation and excessive production of reactive oxygen radicals. Ferritin is not, however, indiscriminately up-regulated in these conditions as a marked shift towards a predominance in H-subunit rich ferritins occurs. Preliminary indications are that, while the L-subunit primarily fulfils the conventional iron storage role, the H-subunit functions primarily as rapid regulator of iron availability, and perhaps indirectly as regulator of other cellular processes. It is suggested that the optimum differential expression of the two subunits differ for different cells and under different conditions and that the expression of appropriate isoferritins offers protection against uncontrolled cellular proliferation, oxidative stress and against side effects of infectious and inflammatory conditions.     

The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence

Ferritins are ubiquitous iron (Fe) storage proteins that play a fundamental role in cellular Fe homeostasis. The enteric pathogen Salmonella enterica serovar Typhimurium possesses four ferritins: bacterioferritin, ferritin A, ferritin B and Dps. The haem-containing bacterioferritin (Bfr) accounts for the majority of stored Fe, followed by ferritin A (FtnA). Inactivation of bfr elevates the intracellular free Fe concentration and enhances susceptibility to H2O2 stress. The DNA-binding Dps protein provides protection from oxidative damage without affecting the steady-state intracellular free Fe concentration. FtnB appears to be particularly important for the repair of oxidatively damaged Fe-sulphur clusters of aconitase and, in contrast to Bfr and FtnA, is required for Salmonella virulence in mice. Moreover, ftnB and dps are repressed by the Fe-responsive regulator Fur and induced under conditions of Fe limitation, whereas bfr and ftnA are maximally expressed when Fe is abundant. The absence of a conserved ferroxidase domain and the potentiation of oxidative stress by FtnB in some strains lacking Dps suggest that FtnB serves as a facile cellular reservoir of Fe2+.     

Cloning and expression of 32 kDa ferritin from Galleria mellonella

We have sequenced a cDNA clone encoding 32-kDa ferritin subunit in the Wax Moth, Galleria mellonella. The 32-kDa ferritin subunit cDNA was obtained from PCR using identical primer designed from highly conserved regions of insect ferritins. RACE PCR was used to obtain the complete protein coding sequence. The 32-kDa ferritin subunit encoded a 232 amino acid polypeptide, containing a 19 leader peptide. The iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region of the wax moth 32-kDa ferritin subunit mRNA. The 32-kDa sequence alignment had 78 and 69% identity with Manduca sexta and Calpodes ethlius (G), respectively. The G. mellonella ferritin subunits showed minimal identity with each other (19%). The glycosylation site (Asn-X-Ser/Thr) was found in the 32-kDa subunit but not in the 26-kDa subunit. Northern blot analysis showed that the mRNA expression of the 32-kDa ferritin was detected in the fat body and midgut. The fat body expression increased after 6 h and the mRNA in midgut dramatically increased about 3-fold the expression level at 12 h after iron feeding. Western blot revealed that a protein level of the 32-kDa subunit is abundant in midgut after 12 and 24 h iron feeding.     

Molecular cloning,expression and isolation of ferritins from two tick species--Ornithodoros moubata and Ixodes ricinus

Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively. The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity). The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5' untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Streptomyces coelicolor Dps-like proteins: differential dual roles in response to stress during vegetative growth and in nucleoid condensation during reproductive cell division

The Dps protein, a member of the ferritin family, contributes to DNA protection during oxidative stress and plays a central role in nucleoid condensation during stationary phase in unicellular eubacteria. Genome searches revealed the presence of three Dps-like orthologues within the genome of the Gram-positive bacterium Streptomyces coelicolor . Disruption of the S. coelicolor dpsA , dpsB and dpsC genes resulted in irregular condensation of spore nucleoids in a gene-specific manner. These irregularities are correlated with changes to the spacing between sporulation septa. This is the first example of these proteins playing a role in bacterial cell division. Translational fusions provided evidence for both developmental control of DpsA and DpsC expression and their localization to sporogenic compartments of aerial hyphae. In addition, various stress conditions induced expression of the Dps proteins in a stimulus-dependent manner in vegetative hyphae, suggesting stress-induced, protein-specific protective functions in addition to their role during reproductive cell division. Unlike in other bacteria, the S. coelicolor Dps proteins are not induced in response to oxidative stress.     

Isolation and Expression Pattern Analysis of Two Ferritin Genes in Tobacco

For understanding of the ferritin gene expression pattern and the mechanism of iron homeostasis in tobacco (Nicotiana tabaccum L.) plants, two full-length ferritin cDNAs, NtFerl and NtFer2, were isolated from tobacco seedlings and characterized. These cDNAs are 1 214 and 1 125 bp nucleotides and encode 25 1 and 259 amino acid residues, respectively. The deduced amino acid sequences showed that two tobacco ferritins share the same characteristics as the plant ferritins from Arabidopsis, soybean, and maize.Southern blotting analysis indicated that both NtFerl and NtFer2 were probably multicopy genes in the tobacco genome. Northern blotting analysis indicated that iron loading of tobacco plantlets increased the ferritin mRNA abundance and that NtFerl expression was higher and more sensitive to iron than NtFer2expression. Furthermore, NtFerl was expressed in both leaves and roots, whereas NtFer2 was expressed mainly in leaves.     

Changes in the characteristics and distribution of ferritin in iron-loaded cell cultures.

When Chang liver cells are grown in an iron-rich medium for up to 20 weeks, iron loading up to 50 times the normal cellular iron content may be obtained, although ferritin increases only to about 10 times normal. Ferritin has been isolated from such cells, and the isoferritin pattern found on elution from DEAE-Sephadex A-50 by increasing chloride concentrations has been used as a basis for studying changes in the properties of ferritin under conditions of cellular loading. A consistent shift of peak ferritin-elution position to higher chloride concentrations (lower pI) occurs when cells are loaded with ferric nitrilotriacetate for increasing lengths of time. A change in immunoreactivity also takes place on loading, the ratio of ferritin reacting with heart and spleen ferritin antibodies increasing at any particular value of pI. Cells were pulse-labelled with [59Fe]ferric nitrilotriacetate and [3H]leucine followed by non-radioactive iron in the same form. During the 72 h after the synthesis of new protein and its incorporation of iron, there is a slight acid shift in its isoelectric point. This effect is seen in both normal and loaded cells, with the whole spectrum being shifted towards lower pI in the loaded state. These findings suggest that the shift to more acidic ferritins on iron loading and the associated changes in antigenicity may be unrelated to subunit composition.