Iron-binding Activity Research Articles

Arabidopsis Dph4 is an Hsp70 Cochaperone with Iron-Binding Properties.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s with a wide range of functions in protein homeostasis. Although the J-domain is required for the stimulation of Hsp70s ATPase activity, the functional specificity of JDPs is governed by domains or regions other than the J-domain. Jjj3/Dph4, a class III JDP, is required for diphthamide (DPH) biosynthesis in eukaryotes, including yeast and mammals. Dph4 has a conserved N-terminal J-domain and an uncharacterized C-terminal domain containing a signature CSL zinc finger motif. Previously, we showed that the Dph4 ortholog in Arabidopsis thaliana (atDjC13/AtJjj3/AtDph4) could restore DPH biosynthesis in yeast jjj3Δ mutant in a J-domain-dependent manner. Here, we characterize the C-terminal CSL motif of AtDph4 using yeast genetic and biochemical approaches. The CSL motif of AtDph4 is essential for DPH biosynthesis, and like human Dph4, AtDph4 showed distinct iron-binding activity, which is not present in its yeast counterpart. ScDph4 and AtDph4 proteins exhibit distinct iron-binding capabilities, as evidenced by UV-vis spectrophotometry, SEM-EDS (energy-dispersive spectroscopy function on the scanning electron microscope) and electron paramagnetic resonance (EPR) spectra analyses. Collectively, our data suggests that beyond their role as an Hsp70 cochaperone, Dph4 homologues in complex eukaryotes may have iron-binding abilities, indicating a potential role in iron-sulfur cluster assembly and iron homeostasis.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein

BackgroundGenetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood.ResultsTo investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes.ConclusionsThis study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Comparative Transcriptomic Analysis of Insecticide-Resistant Aedes aegypti from Puerto Rico Reveals Insecticide-Specific Patterns of Gene Expression.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

Discovery of New Siderophores from a Marine Streptomycetes sp. via Combined Metabolomics and Analysis of Iron-Chelating Activity

The marine-derived Streptomyces sp. FIMYZ-003 strain was found to produce novel siderophores with yields negatively correlated with the iron concentration in the medium. Mass spectrometry (MS)-based metabolomics coupled with metallophore assays identified two novel α-hydroxycarboxylate-type siderophores, fradiamines C and D (3 and 4), together with two related known siderophores, fradiamines A and B (1 and 2). Their chemical structures were elucidated by nuclear magnetic resonance (NMR) and MS experiments. The annotation of a putative fra biosynthetic gene cluster enabled us to propose the biosynthetic pathway of fradiamines A-D. Furthermore, the solution-phase iron-binding activity of fradiamines was evaluated using metabolomics, confirming them as general iron scavengers. Fradiamines A-D exhibited Fe(III) binding activity equivalent to that of deferoxamine B mesylate. Growth analysis of pathogenic microbes demonstrated that fradiamine C promoted the growth of Escherichia coli and Staphylococcus aureus, but fradiamines A, B, and D did not. The results indicate that fradiamine C may serve as a novel iron carrier applicable to antibiotic delivery strategies to treat and prevent foodborne pathogens.

Functional Divergence of the N-Lobe and C-Lobe of Transferrin Gene in Pungitius sinensis (Amur Stickleback).

Transferrin is an important iron-binding glycosylated protein and plays key roles in iron-binding and immune response. Here, a 2037-bp open reading frame was obtained from our previous transcriptome sequencing data of Amur stickleback, which encoded a 679 amino acid putative transferrin protein harbored obvious N-lobe and C-lobe domains. The tissue-specific expression pattern showed that the transcript was detected in a variety of tissues, with the highest signal in liver. Moreover, Streptococcus iniae pathogen stimulation can increase the expression level of this transcript, implying important immune properties for organisms. Next, N-lobes and C-lobes were obtained from 45 fish species. The phylogenetic tree showed that N-lobes and C-lobes were in two different evolutionary branches, and they had different motif composition. Functional divergence indicated a higher evolutionary rate or site-specific alteration among the N-lobe and C-lobe groups. Ka/Ks value of C-lobe group was relatively higher than that of N-lobe group, indicating a faster change rate of C-lobe sequences in evolution. Moreover, some sites experiencing positive selection were also found, which may be involved in the iron- or anion-binding, pathogen resistance and diversification of transferrin protein. Differential iron-binding activity was also detected between N-lobe and C-lobe of Amur stickleback transferrin protein with Chrome Azurol S assay. Compared with the C-lobe, the N-lobe showed stronger growth inhibitory activity of Escherichia coli, implying their potential antibacterial properties. This study will give a reference for subsequent research of transferrin proteins.

Genetic Engineering of Talaromyces marneffei to Enhance Siderophore Production and Preliminary Testing for Medical Application Potential.

Siderophores are compounds with low molecular weight with a high affinity and specificity for ferric iron, which is produced by bacteria and fungi. Fungal siderophores have been characterized and their feasibility for clinical applications has been investigated. Fungi may be limited in slow growth and low siderophore production; however, they have advantages of high diversity and affinity. Hence, the purpose of this study was to generate a genetically modified strain in Talaromyces marneffei that enhanced siderophore production and to identify the characteristics of siderophore to guide its medical application. SreA is a transcription factor that negatively controls iron acquisition mechanisms. Therefore, we deleted the sreA gene to enhance the siderophore production and found that the null mutant of sreA (ΔsreA) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1H- and 13C-NMR techniques and identified as a coprogen B. The compound showed a powerful iron-binding activity and could reduce labile iron pool levels in iron-loaded hepatocellular carcinoma (Huh7) cells. In addition, the coprogen B showed no toxicity to the Huh7 cells, demonstrating its potential to serve as an ideal iron chelator. Moreover, it inhibits the growth of Candida albicans and Escherichia coli in a dose-dependent manner. Thus, we have generated the siderophore-enhancing strain of T. marneffei, and the coprogen B isolated from this strain could be useful in the development of a new iron-chelating agent or other medical applications.

Engineered Bacteria with Genetic Circuits Accumulating Nanomagnets as MRI Contrast Agents.

The demand for highly efficient cancer diagnostic tools increases alongside the high cancer incidence nowadays. Moreover, there is an imperative need for novel cancer treatment therapies that lack the side effects of conventional treatment options. Developments in this aspect employ magnetic nanoparticles (MNPs) for biomedical applications due to their stability, biocompatibility, and magnetic properties. Certain organisms, including many bacteria, can synthesize magnetic nanocrystals, which help their spatial orientation and survival by sensing the earth's geomagnetic field. This work aims to convert Escherichia coli to accumulate magnetite, which can further be coupled with drug delivery modules. The authors design magnetite accumulating bacterial machines using genetic circuitries hiring Mms6 with iron-binding activity and essential in magnetite crystal formation. The work demonstrates that the combinatorial effect of Mms6 with ferroxidase, iron transporter protein, and material binding peptide enhances the paramagnetic behavior of the cells in magnetic resonance imaging (MRI) measurements. Cellular machines are also engineered to display Mms6 peptide on the cell surface via an autotransporter protein that shows augmented MRI performance. The findings are promising for endowing a probiotic bacterium, able to accumulate magnetite intracellularly or extracellularly, serving as a theranostics agent for cancer diagnostics via MRI scanning and hyperthermia treatment.

Extradiol cleavage of L‐DOPA as strategy for natural product biosynthesis

L‐DOPA dioxygenase was first discovered as part of a biosynthetic gene cluster to the natural product antibiotic, lincomycin. Within the larger biosynthetic context, L‐DOPA dioxygenase is part of a mini‐pathway to the synthon 3‐vinyl‐2,3‐pyrroline‐5‐carboxylic acid (VPCA). This synthon is elaborated and embedded within the final product structure of lincomycin, and also within the natural product structures of anthramycin, sibiromycin, tomaymycin and hormaomycin. Using the VPCA mini‐pathway as a starting point, we searched sequence space to identify 1) novel natural product pathways containing a VPCA synthon and 2) instances of L‐DOPA dioxygenase homologs that were not associated with a known VPCA containing natural product pathway, and that might provide new sources of natural product diversity and answer questions about the evolutionary origins of L‐DOPA dioxygenase. Using bioinformatic methods, including PSI‐BLAST, genome context analysis and structurally informed multiple‐sequence alignment, we identified a variety of known and novel natural product pathways that utilize an L‐DOPA dioxygenase ‐ many with the context of a VPCA synthon. Representative examples from these pathways were isolated and tested for iron binding and L‐DOPA dioxygenase activity.

Influence of new antimicrobial peptides of the medicinal leech <i>Hirudo medicinalis</i> on the functional activity of neutrophil granule proteins

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms.
 AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins the main effector cells of the immune system.
 MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis.
 RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity.
 CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the bodys fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit if it necessary to protect against damage to the bodys own tissues.

Aspergillus fumigatus pan-genome analysis identifies genetic variants associated with human infection.

Aspergillus fumigatus is an environmental saprobe and opportunistic human fungal pathogen. Despite an estimated annual occurrence of more than 300,000 cases of invasive disease worldwide, a comprehensive survey of the genomic diversity present in A. fumigatus-including the relationship between clinical and environmental isolates and how this genetic diversity contributes to virulence and antifungal drug resistance-has been lacking. In this study we define the pan-genome of A. fumigatus using a collection of 300 globally sampled genomes (83 clinical and 217 environmental isolates). We found that 7,563 of the 10,907 unique orthogroups (69%) are core and present in all isolates and the remaining 3,344 show presence/absence of variation, representing 16-22% of the genome of each isolate. Using this large genomic dataset of environmental and clinical samples, we found an enrichment for clinical isolates in a genetic cluster whose genomes also contain more accessory genes, including genes coding for transmembrane transporters and proteins with iron-binding activity, and genes involved in both carbohydrate and amino-acid metabolism. Finally, we leverage the power of genome-wide association studies to identify genomic variation associated with clinical isolates and triazole resistance as well as characterize genetic variation in known virulence factors. This characterization of the genomic diversity of A. fumigatus allows us to move away from a single reference genome that does not necessarily represent the species as a whole and better understand its pathogenic versatility, ultimately leading to better management of these infections.

Intracellular Iron Binding and Antioxidant Activity of Phytochelators.

Phytochelators have been studied as templates for designing new drugs for chelation therapy. This work evaluated key chemical and biological properties of five candidate phytochelators for iron overload diseases: maltol, mimosine, morin, tropolone, and esculetin. Intra- and extracellular iron affinity and antioxidant activity, as well as the ability to scavenge iron from holo-transferrin, were studied in physiologically relevant settings. Tropolone and mimosine (and, to a lesser extent, maltol) presented good binding capacity for iron, removing it from calcein, a high-affinity fluorescent probe. Tropolone and mimosine arrested iron-mediated oxidation of ascorbate with the same efficiency as the standard iron chelator DFO. Also, both were cell permeant and able to access labile pools of iron in HeLa and HepG2 cells. Mimosine was an effective antioxidant in cells stressed by iron and peroxide, being as efficient as the cell-permeant iron chelator deferiprone. These results reinforce the potential of those molecules, especially mimosine, as adjuvants in treatments for iron overload.

The Attenuation of TNF-α-mediated Inflammatory Responses in Human Lung Adenocarcinoma Cell Line by Perilla Seed and Seed Meal Extract

Abstract Thai perilla (Perilla frutescens) extracts, which contain a substantial quantity of bioactive substances including phenolics and flavonoids, have shown marked anti-inflammatory activities in several investigated models. In the present study, the effect of perilla seed extract (PSE) and seed meal extract (PSME) on TNF-α-induced inflammatory response in human lung adenocarcinoma A549 cells was investigated. The total phenolic and flavonoid contents in PSME was lower than PSE. Markedly, rosmarinic acid was identified as the main constituent in both extracts. However, the DPPH and ABTS assays indicated that the antioxidant capacity of PSME was equal to PSE. Moreover, the iron-binding activity of PSE and PSME were exhibited by complex formation with Fe3+-NTA, indicating that the extracts may inhibit hydroxyl radical production via Fenton reaction. In vitro cytotoxicity analysis showed that both PSE or PSME co-treated with TNF-α, at 24 h exposure, were not toxic to the A549 cells. Interestingly, PSE and PSME dramatically exhibited an anti-inflammatory activity by inhibiting the mRNA expression of pro-inflammatory cytokines, IL-1β, IL-6, IL-8, and TNF-α, but did not influence iNOS and COX-2 mRNA expressions. Moreover, both extracts significantly reduced reactive oxygen species (ROS) production in TNF-α-induced A549 cells. The findings presented in this paper suggest that PSE and PSME could mitigate TNF-α-mediated inflammatory responses via limiting pro-inflammatory cytokine expressions and decreasing ROS production. Thus, perilla seed and seed meal, the by-product of a perilla seed oil cold-pressed extraction process, could be developed as food supplements or functional foods for the prevention of inflammation-induced lung carcinogenesis development. Keywords: Human lung adenocarcinoma cell line, Inflammation, Perilla seed, Perilla seed meal, Tumor necrosis factor-alpha (TNF-α)

The iron chaperone and nucleic acid–binding activities of poly(rC)-binding protein 1 are separable and independently essential

Poly(rC)-binding protein (PCBP1) is a multifunctional adaptor protein that can coordinate single-stranded nucleic acids and iron-glutathione complexes, altering the processing and transfer of these ligands through interactions with other proteins. Multiple phenotypes are ascribed to cells lacking PCBP1, but the relative contribution of RNA, DNA, or iron chaperone activity is not consistently clear. Here, we report the identification of amino acid residues required for iron coordination on each structural domain of PCBP1 and confirm the requirement of iron coordination for binding target proteins BolA2 and ferritin. We further construct PCBP1 variants that lack either nucleic acid- or iron-binding activity and examine their functions in human cells and mouse tissues depleted of endogenous PCBP1. We find that these activities are separable and independently confer essential functions. While iron chaperone activity controls cell cycle progression and suppression of DNA damage, RNA/DNA-binding activity maintains cell viability in both cultured cell and mouse models. The coevolution of RNA/DNA binding and iron chaperone activities on a single protein may prove advantageous for nucleic acid processing that depends on enzymes with iron cofactors.

Bioactive Properties of Fish Protein Hydrolysates from Amazon Sailfin Catfish (Pterygoplichthys pardalis) from the Victoria Reservoir in Sri Lanka

<em>Pterygoplichthys pardalis </em>is an underutilized by-catch fish species considered as an invasive fish, which is a potential threat to Sri Lankan freshwater fishery industry. This study attempted to produce bioactive fish protein hydrolysates (FPH) using <em>P.</em> <em>pardalis</em>. Acid-aided aqueous extract of fish protein was produced and the best protein extraction (PE) was selected. The lyophilized extracts were hydrolyzed using pepsin, papain and trypsin enzymes (1:100), and were subjected to analyze antioxidant properties by DPPH scavenging assay and TBARS assay, metal chelation activity and antimicrobial activity by agar well diffusion method. The 15% citric acid sample showed the highest (p&lt;0.05) PE yield (1.95%±0.04 w/w). The pepsin (0 hour) treated hydrolysate showed the highest (p&lt;0.05) radical scavenging activity (79.71%±5.19). None of the hydrolysates exhibited antioxidant properties against TBARS and showed no significant difference (p&gt;0.05) in the ferrous chelating activity (pepsin (0 hour): 52.31%±0.93, papain (9 hours): 50.95%±1.17). Antibacterial activity against <em>E. coli </em>was positive in all hydrolysates, where pepsin (0 hour) hydrolysate showed the highest activity against <em>E. coli</em>. This concludes, FPHs of acidsoluble protein of <em>P. pardalis </em>contain antioxidant properties, iron-binding activity and antibacterial activity.

PEAT HUMIC ACIDS – PROSPECTIVE BIOLOGICALLY ACTIVE SUBSTANCES WITH ANTIOXIDANT ACTIVITY FOR THE DEVELOPMENT OF PROTECTIVE AGENTS

A present study was carried out to investigate the antioxidant activity (AOA) of humic acids (HA) isolated by sodium hydroxide and sodium pyrophosphate from nine peat species of the Tomsk region different by botanical composition, degree of decomposition and ash content. All HA samples had shown the AOA in the study. As the results of four research methods, it was found that HAs are highly effective in inhibiting the free ABTS•+ radical cation, superoxide anion radical O2-•, hydroxyl radical HO•, and are able to bind Fe2+ in a wide range of concentrations. The activity of HAs in different tests was not equal between samples; HAs isolated by sodium hydroxide have had a higher AOA in inhibition of the superoxide anion–radical O2-• in the model reaction compared to HAs isolated by sodium pyrophosphate within the same peat species. In the study of iron binding activity, an inverse relationship was observed. In the model reactions of the free ABTS•+ radical cation and the hydroxyl radical HO• inhibition, a comparable level of activity was registered between the HA samples isolated by different solutions. Such an uneven distribution of the activity between various HA samples can be explained by the unequal chemical parameters of their structure, which depend on their origin and method of isolation. Using of such classical antioxidants with an established mechanism of action as comparison drugs, as a water-soluble analogue of tocopherol - "Trolox", a water-soluble antioxidant – ascorbic acid, a classic chelator – ethylenediaminetetraacetic acid (EDTA), a classic trap of a hydroxyl radical – mannitol, it is possible to conclude that the investigated HAs are effective antioxidants belonging to the groups of proton donors and complexing agents.

Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli.

Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system.IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

Zagreb topological indices for hexadentate 3-hydroxypyridinones-terminated dendrimers used in iron binding and anti-microbial activities

Among the degree based topological indices which are mostly used in the study of chemical graph theory, the Zagreb index plays a prominent role in the study of therapies and treatments. There were different versions of the Zagreb indices defined in the literature and used for different purposes. In this paper, we calculated the different versions of the Zagreb indices for the hexadentate 3-hydroxypyridinones-terminated dendrimers which are used in iron binding and anti-microbial activities. These indices may play an important role in determining properties of these compounds. Keywords: Anti-microbial activities, dendrimers, iron binding, topological index, Zagreb index.

Antarctic krill derived peptide as a nanocarrier of iron through the gastrointestinal tract

Antarctic krill (Euphausia superba) might have specific peptides that chelate iron ions and are iron carriers. In this study, iron-binding peptides were isolated from Antarctic krill using enzymatic (trypsin) hydrolysis and immobilized metal affinity chromatography (IMAC); the binding mode and surface morphology of Antarctic krill peptides (AKP)-iron complexes as well as their iron delivery behavior in the gastrointestinal tract were investigated. Results showed that the AKP bound 79.3% of available iron from the enzymatic hydrolysis for 180 min. The contents of negatively-charged amino acids (Asp and Glu) on the AKP showed a positive correlation (r = 0.80) with iron-binding activities (the data includes Asn and Gln), while positively-charged amino acids showed a negative correlation (r = −0.83). Moreover, His, Ser and Thr on the AKP were also involved in the iron-binding activities. Iron-binding peptides from Antarctic krill hydrolysates, isolated using IMAC-Fe3+ and identified using HPLC-MS/MS, showed a mass of 712–2451 Da, and were enriched in Asp (Asn), Glu (Gln), His, Ser or Thr. The iron ions mainly bound to carboxyl groups of AKP, thus forming nano-scale, spherical particles. The iron nanocarriers formed by AKP showed a significant iron transport activity after simulated gastrointestinal digestion, as compared to FeSO4 (P < 0.05). The results suggested that the AKP-iron nanocomposite has the potential to be a nutraceutical supplement for improving iron absorption.

Transcriptomic changes associated with husk scald incidence on pomegranate fruit peel during cold storage

Pomegranate fruit is valued for its social, economic, aesthetic and health benefits. The fruit rapidly loses quality after harvest due to continued metabolic responses and physiological disorders under sub-optimal conditions. The incidence of physiological disorder such as husk scald manifests during storage and commercial shipping, which affects the appearance and limits marketability. Despite the importance of pomegranate husk scald, little information is available about the origin and molecular mechanisms. Therefore, the aim of this study was to investigate the scald incidence of pomegranate fruit at molecular level using RNA-Seq (Ion Proton™ Next Generation Sequencing) by analyzing peel transcriptomic changes. The RNA-seq analysis generated 98,441,278 raw reads. 652 Differentially Expressed Genes (DEGs) with a fold change of > |2|, a p value ≤ 0.05 and a false discovery rate (FDR) of <0.05 were identified between healthy and scald fruit peels. An analysis of the gene ontologies of these DEGs revealed the 432 genes were assigned with molecular functions, 272 as cellular components and 205 as part of biological processes. In this analysis, genes (Pgr023188 and Pgr025081) that encode uncharacterized protein and gene (Pgr007593) that encodes glycosyltransferase showed significantly highest fold changes. Genes (Pgr003448, Pgr006024 and Pgr023696) involved in various iron binding and oxidoreductase activities were significantly suppressed. This is the first transcriptome analysis of pomegranate fruit peel related to husk scald development. Results obtained from this study will add valuable information on husk scald related changes on pomegranate fruit at genomic level and provide insight on other related physiological disorders.