An Overview of Dps: Dual Acting Nanovehicles in Prokaryotes with DNA Binding and Ferroxidation Properties.

DNA binding proteins under starvation (Dps) are proteins belonging to the ferritin family with the capacity for DNA binding, in addition to iron storage and ferroxidation. Present only in the prokaryotes, these multifaceted proteins have been assigned with a number of roles, from pathogenesis to nucleoid condensation and protection. They have a significant role in protecting the cells from free radical assaults, indirectly by sequestration of iron and by directly binding to the DNA. Due to their symmetry, stability and biomineralization capacity, these proteins have ever increasing potential applications in biotechnology and drug delivery. This chapter tries to bring together all these aspects of Dps in the view of current understanding and older perspectives by studies of our group as well as other experts in the field.

Characterization of the Fe metalloproteome of a ubiquitous marine heterotroph, Pseudoalteromonas (BB2-AT2): multiple bacterioferritin copies enable significant Fe storage.

Fe is a critical nutrient to the marine biological pump, which is the process that exports photosynthetically fixed carbon in the upper ocean to the deep ocean. Fe limitation controls photosynthetic activity in major regions of the oceans, and the subsequent degradation of exported photosynthetic material is facilitated particularly by marine heterotrophic bacteria. Despite their importance in the carbon cycle and the scarcity of Fe in seawater, the Fe requirements, storage and cytosolic utilization of these marine heterotrophs has been less studied. Here, we characterized the Fe metallome of Pseudoalteromonas (BB2-AT2). We found that with two copies of bacterioferritin (Bfr), Pseudoalteromonas possesses substantial capacity for luxury uptake of Fe. Fe : C in the whole cell metallome was estimated (assuming C : P stoichiometry ∼51 : 1) to be between ∼83 µmol : mol Fe : C, ∼11 fold higher than prior marine bacteria surveys. Under these replete conditions, other major cytosolic Fe-associated proteins were observed including superoxide dismutase (SodA; with other metal SOD isoforms absent under Fe replete conditions) and catalase (KatG) involved in reactive oxygen stress mitigation and aconitase (AcnB), succinate dehydrogenase (FrdB) and cytochromes (QcrA and Cyt1) involved in respiration. With the aid of singular value decomposition (SVD), we were able to computationally attribute peaks within the metallome to specific metalloprotein contributors. A putative Fe complex TonB transporter associated with the closely related Alteromonas bacterium was found to be abundant within the Pacific Ocean mesopelagic environment. Despite the extreme scarcity of Fe in seawater, the marine heterotroph Pseudoalteromonas has expansive Fe storage capacity and utilization strategies, implying that within detritus and sinking particles environments, there is significant opportunity for Fe acquisition. Together these results imply an evolved dedication of marine Pseudoalteromonas to maintaining an Fe metalloproteome, likely due to its dependence on Fe-based respiratory metabolism.

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas.

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519 bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-ß-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20 kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP-rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd(2+) stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.

Use of structural phylogenetic networks for classification of the ferritin-like superfamily.

In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the "twilight zone" where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.

Single subunit type of ferritin from visceral mass of Saccostrea cucullata: cloning, expression and cisplatin-subunit analysis.

Ferritin, the iron storage protein, plays a key role in iron metabolism. Here, we have cloned an inducible ferritin cDNA with 516 bp within the open reading frame fragment from the visceral mass of Saccostrea cucullata. The subunit sequence of the ferritin was predicted to be a polypeptide of 171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-ß-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The recombinant ScFer should prove to be useful for further study of the structure and function of ferritin in S. cucullata.

A role for ferritin in the antioxidant system in coffee cell cultures.

Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 µM) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 µM, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.

Molecular cloning and characterization of a ferritin gene upregulated by cold stress in Chorispora bungeana.

The ability of iron to accept and donate electrons makes it important for plant growth, but it can also damage plants when they are under environmental stress. Ferritin, a protein encoded by the gene Fer, catalyzes the oxidation of Fe(2+) and subsequent storage of Fe(3+) within the mineral core. Ferritin may reduce the adverse effects of iron on Chorispora bungeana Fisch. & C.A. May during the course of cold stress. C. bungeana is a rare alpine subnival plant species that is highly resistant to a freezing environment. We have isolated and characterized the ferritin cDNA (CbFer) from C. bungeana. It is 975 bp in length with an open reading frame of 260 amino acids, corresponding to a protein of predicted molecular mass of 29.17 kDa and an isoelectric point of 5.44. Amino acid analysis of the polypeptides indicated that CbFer codes for a ferritin subunit plus a chloroplast-targeting transit peptide. Reverse transcription polymerase chain reaction analysis confirmed that CbFer was a tissue-specific gene since the expression could only be detected in leaves. The gene expression patterns were investigated in relation to cold stress (4 degrees C and -4 degrees C) and to various exogenous signals, including excessive iron, hydrogen peroxide (H(2)O(2)), and nitrogen monoxidum (NO). The amount of CbFer mRNA increased in response to low temperatures and gene expression at -4 degrees C was both more distinct and quicker than that at 4 degrees C. Two exogenous signals, excessive iron and H(2)O(2), upregulated the expression of the CbFer gene, but NO had no effect. The CbFer gene may play an important role in response to cold stress, while the expression of the gene during stress may be influenced by major and minor factors such as iron and H(2)O(2), respectively.

Estrategias diagnósticas utilizadas para detectar deficiencias de hierro subclínicas y asociadas a enfermedades crónicas / Diagnostic strategies for detecting subclinical iron deficiencies and associated to chronic diseases

La homeostasis y las variaciones fisiológicas horarias en el metabolismo del hierro se constituyen en verdaderos desafíos para los expertos, quienes intentan diseñar pruebas altamente sensibles y específicas que cuantifiquen los niveles circulantes y de depósito de este elemento, su repercusión en la eritropoyesis,cuantificación de las proteínas, transportadores y receptores involucrados en el proceso, a fin de descartar la presencia de estados carenciales. Las deficiencias de hierro pasan por tres fases; las dos primeras son lasmás difíciles de identificar porque son subclínicas, más aún, cuando pueden cursar simultáneamente con enfermedades crónicas inflamatorias, infecciosas y neoplásicas que de por sí son anemizantes. En este trabajose revisarán las principales pruebas de laboratorio utilizadas para la identificación de deficiencias de hierro, sensibilidad, especificidad, ventajas y limitaciones para su uso.

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins.

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5 degrees C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100-110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-kb monocistronic mRNA in exponential growing cells and an important increase inflp mRNA amount after a downshift but also an upshift in temperature.

Evidence for two types of subunits in the bacterioferritin of Magnetospirillum magnetotacticum.

In order to investigate the role of bacterioferritin (Bfr) in the biomineralization of magnetite by microorganisms, we have cloned and sequenced the bfr genes from M. magnetotacticum. The organism has two bfr genes that overlap by one nucleotide. Both encode putative protein products of 18 kDa, the expected size for Bfr subunits, and show a strong similarity to other Bfr subunit proteins. By scanning the DNA sequence databases, we found that a limited number of other organisms, including N. gonorrhea, P. aeruginosa, and Synechocystis PCC6803, also have two bfr genes. When the sequences of a number of microbial Bfrs are compared with each other, they fall into two distinct types with the organisms mentioned above having one of each type. Differences in heme- and metal-binding sites and ferroxidase activities of the two types of subunits are discussed.

Ferritin mRNAs in Schistosoma mansoni do not have iron-responsive elements for post-transcriptional regulation.

Schistosoma mansoni possesses two isoforms of ferritin, soma and yolk ferritin. The soma ferritin occurs at a low level in most cells of both genders, whereas the yolk ferritin is a female-specific gene product that is expressed at high level in the vitellarium. In higher animals, ferritin mRNA is regulated by iron via the interaction of cytoplasmic binding proteins (IRPs) with a specific sequence element in the 5' untranslated region (UTR) referred to as the iron-responsive element (IRE). Sequence studies of the 5' UTRs, gel retardation assays, and hybridization experiments show that neither ferritin mRNAs of S. mansoni is regulated by an IRE/IRP mechanism. It is suggested that ferritins in schistosomes are controlled only at the transcriptional level.

Characteristics and expression of binding sites specific for ferritin H-chain on human cell lines.

Purified recombinant human ferritin composed solely of H subunit was radiolabeled and incubated with proerythroleukemic K562 human cells. A specific binding was detected, and it could be displaced only by ferritins, natural or recombinant, containing large proportion of the H subunit. The specific ferritin H-chain binding was saturable, and cells showed 17,000 to 23,000 binding sites per cell. The affinity constant measured at 37 degrees C was of 3 x 10(8) M-1. Treatment with pronase eliminated the specific binding. The binding sites were expressed in a high number during the cellular exponential phase of growth and progressively decreased to disappear when cells reached the plateau phase. Treatment of the cells with desferrioxamine increased recombinant H-ferritin binding, while iron had little effect. K562 cells induced to differentiate by hemin failed to bind ferritin H. Ferritin H-chain binding capacity is present on various cell lines such as HL60, lung cancer, and hepatoma cells. Analysis of the binding sites by western blotting showed a peptide with apparent mol wt of about 100 kd.

Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin.

The two subunit types of human liver ferritin were purified to homogeneity. Both subunits reassembled in a well-defined manner and formed spherical particles that resembled natural apoferritin in electron micrographs. Affinity chromatography methods were employed to obtain preparations of antibodies that interacted exclusively either with the H or with the L polypeptides, demonstrating that distinct immunological properties may be ascribed to each subunit of ferritin. The amino acid compositions of the subunits were similar, but the larger H subunit had fewer leucine, phenylalanine, and arginine residues. It is therefore improbable that H subunits undergo proteolytic processing and are precursors for L subunits. Circular dichroism data indicated that homopolymers assembled from L-type subunits had substantially more ordered secondary structures and greater alpha-helical contents than their H counterparts. Small differences in the environment of tryptophan residues were evident from fluorescence spectra of each homopolymer. In isoelectric focusing experiments reassembled H or L homopolymers migrated as families of proteins within discrete pI ranges which are probably representative of subpopulations of each subunit type. The H homopolymer focused at lower pI's than the L component. These data substantiate the contention that both subunits are authentic polypeptide moieties of ferritin with some common structural features, but the results also underscore prominent dissimilarities in their properties.