Iron Deposition and Ferroptosis in the Spleen in a Murine Model of Acute Radiation Syndrome

Total body radiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated the impact of radiation-induced iron release on the spleen of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed ~20-60-fold increased Fe3+ in the spleen 7-14 days post-irradiation, also associated with altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not prevent iron deposition in the spleen, and did not significantly modulate most iron-binding proteins. Spleen volumes were markedly decreased 7-14 days, correlating with high Fe3+. At these time points, caspase-3 was activated and we identified four markers of ferroptosis, iron-dependent programmed cell death. Interestingly, p21/Waf1, a marker of accelerated senescence, was not upregulated in vivo. Macrophage inflammation is an important effect of TBI. We investigated the effects of radiation and Fe3+ on the cultured J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, within 24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron binding proteins and triggers ferroptosis.

Adaptation of Dinoroseobacter shibae to oxidative stress and the specific role of RirA

Dinoroseobacter shibae living in the photic zone of marine ecosystems is frequently exposed to oxygen that forms highly reactive species. Here, we analysed the adaptation of D. shibae to different kinds of oxidative stress using a GeLC-MS/MS approach. D. shibae was grown in artificial seawater medium in the dark with succinate as sole carbon source and exposed to hydrogen peroxide, paraquat or diamide. We quantified 2580 D. shibae proteins. 75 proteins changed significantly in response to peroxide stress, while 220 and 207 proteins were differently regulated by superoxide stress and thiol stress. As expected, proteins like thioredoxin and peroxiredoxin were among these proteins. In addition, proteins involved in bacteriochlophyll biosynthesis were repressed under disulfide and superoxide stress but not under peroxide stress. In contrast, proteins associated with iron transport accumulated in response to peroxide and superoxide stress. Interestingly, the iron-responsive regulator RirA in D. shibae was downregulated by all stressors. A rirA deletion mutant showed an improved adaptation to peroxide stress suggesting that RirA dependent proteins are associated with oxidative stress resistance. Altogether, 139 proteins were upregulated in the mutant strain. Among them are proteins associated with protection and repair of DNA and proteins (e. g. ClpB, Hsp20, RecA, and a thioredoxin like protein). Strikingly, most of the proteins involved in iron metabolism such as iron binding proteins and transporters were not part of the upregulated proteins. In fact, rirA deficient cells were lacking a peroxide dependent induction of these proteins that may also contribute to a higher cell viability under these conditions.

Iron Metabolism in the Tumor Microenvironment: Contributions of Innate Immune Cells

Cells of the innate immune system are a major component of the tumor microenvironment. They play complex and multifaceted roles in the regulation of cancer initiation, growth, metastasis and responses to therapeutics. Innate immune cells like neutrophils and macrophages are recruited to cancerous tissues by chemotactic molecules released by cancer cells and cancer-associated stromal cells. Once they reach the tumor, they can be instructed by a network of proteins, nucleic acids and metabolites to exert protumoral or antitumoral functions. Altered iron metabolism is a feature of cancer. Epidemiological studies suggest that increased presence of iron and/or iron binding proteins is associated with increased risks of cancer development. It has been shown that iron metabolism is involved in shaping the immune landscapes in inflammatory/infectious diseases and cancer-associated inflammation. In this article, we will dissect the contribution of macrophages and neutrophils to dysregulated iron metabolism in malignant cells and its impact on cancer growth and metastasis. The mechanisms involved in regulating the actions of macrophages and neutrophils will also be discussed. Moreover, we will examine the effects of iron metabolism on the phenotypes of innate immune cells. Both iron chelating and overloading agents are being explored in cancer treatment. This review highlights alternative strategies for management of iron content in cancer cells by targeting the iron donation and modulation properties of macrophages and neutrophils in the tumor microenvironment.

Lactoferrin and hematoma detoxification after intracerebral hemorrhage

This review discusses the role of lactoferrin (LTF) in detoxifying hematoma after intracerebral hemorrhage (ICH). Subsequent to ICH, neutrophils enter the ICH-affected brain, where they release various granule content, including LTF. LTF is an iron-binding glycoprotein that binds Fe3+ with high affinity. Unlike other iron binding proteins, LTF can retain Fe3+ at the low pH associated with inflamed tissue. LTF’s ability to sequester Fe3+ is of particular importance to ICH pathogenesis, as large quantities of free iron, which is pro-oxidative and pro-inflammatory are generated in the ICH-affected brain due to blood hemolysis. LTF delivered to ICH-affected brain, either as therapeutic agent or through infiltrated PMNs (cells containing high levels of LTF), could benefit ICH pathogenesis. LTF is a protein with a high isoelectric point (8.7), property that enables it to binding to negatively charged apoptotic cells or proteins. Here, LTF could act as a bridging molecule that couples the apoptotic cells to LTF receptors on the cellular membranes of microglia/macrophages to facilitates the efferocytosis/erythrophagocytosis of apoptotic cells and damaged red blood cells. Thus, by virtue of sequestrating iron and facilitating efferocytosis, LTF may contribute to hematoma detoxification and hematoma/inflammation resolution after ICH.

Regulation of Iron Homeostasis Through Parkin-mediated Lactoferrin Ubiquitylation

ABSTRACTSomatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson’s disease. Lactotransferrin is a member of the transferrin iron binding proteins that regulate iron homeostasis and increased levels of Lactotransferrin and its receptor have been observed in neurodegenerative disorders like Parkinson’s disease. Here, we report that Parkin binds to Lactotransferrin and ubiquitylates it to regulate iron homeostasis.

A secreted metal-binding protein protects necrotrophic phytopathogens from reactive oxygen species

Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.

Transferrin Identification in Sterlet (Acipenser ruthenus) Reproductive System

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Vascular Expression of Hemoglobin Alpha in Antarctic Icefish Supports Iron Limitation as Novel Evolutionary Driver

Frigid temperatures of the Southern Ocean are known to be an evolutionary driver in Antarctic fish. For example, many fish have reduced red blood cell (RBC) concentration to minimize vascular resistance. Via the oxygen-carrying protein hemoglobin, RBCs contain the vast majority of the body’s iron, which is known to be a limiting nutrient in marine ecosystems. Since lower RBC levels also lead to reduced iron requirements, we hypothesized that low iron availability was an additional evolutionary driver of Antarctic fish speciation. Antarctic Icefish of the family Channichthyidae are known to have extreme alteration of iron metabolism due to loss of two iron-binding proteins, hemoglobin and myoglobin, and no RBCs. Loss of hemoglobin is considered a maladaptive trait allowed by relaxation of predator selection, since extreme adaptations are required to compensate for the loss of oxygen-carrying capacity. However, iron dependency minimization may have driven hemoglobin loss instead of a random evolutionary event. Given the variety of functions that hemoglobin serves in the endothelium, we suspected the protein corresponding to the 3’ truncated Hbα fragment (Hbα-3’f) that was not genetically excluded by icefish, may still be expressed as a protein. Using whole mount confocal microscopy, we show that Hbα-3’f is expressed in the vascular endothelium of icefish retina, suggesting this Hbα fragment may still serve an important role in the endothelium. These observations support a novel hypothesis that iron minimization could have influenced icefish speciation with the loss of the iron-binding portion of Hbα in Hbα-3’f, as well as hemoglobin β and myoglobin.

Metal cofactors trafficking and assembly in the cell: a molecular view

Metal ions are essential cofactors required by the proteome of organisms from any kingdom of life to correctly exert their functions. Dedicated cellular import, transport and homeostasis systems assure that the needed metal ion is correctly delivered and inserted into the target proteins and avoid the presence of free metal ions in the cell, preventing oxidative damaging. Among metal ions, in eukaryotic organisms copper and iron are required by proteins involved in absolutely essential functions, such as respiration, oxidative stress protection, catalysis, gene expression regulation. Copper and iron binding proteins are localized in essentially all cellular compartments. Copper is physiologically present mainly as individual metal ion. Iron can be present both as individual metal ion or as part of cofactors, such as hemes and iron-sulfur (Fe-S) clusters. Both metal ions are characterized by the ability to cycle between different oxidation states, which enable them to catalyze redox reactions and to participate in electron transfer processes. Here we describe in detail the main processes responsible for the trafficking of copper and iron sulfur clusters, with particular interest for the structural aspects of the maturation of copper and iron-sulfur-binding proteins.