Major Storage Protein Research Articles

PH Effect on the Structure, Rheology, and Electrospinning of Maize Zein.

As a simple and convenient technology to fabricate micron-to-nanoscale fibers with controllable structure, electrostatic spinning has produced fiber films with many natural advantages, including a large specific surface area and high porosity. Maize zein, as a major storage protein in corn, showed high hydrophobicity and has been successfully applied as a promising carrier for encapsulation and controlled release in the pharmaceutical and food areas. Proteins exhibit different physical and chemical properties at different pH values, and it is worth investigating whether this change in physical and chemical properties affects the properties of electrospun fiber films. We studied the pH effects on zein solution rheology, fiber morphology, and film properties. Rotational rheometers were used to test the rheology of the solutions and establish a correlation between solution concentration and fiber morphology. The critical concentrations calculated by the cross-equation fitting model were 17.6%, 20.1%, 20.1%, 17.1%, and 19.5% (w/v) for pH 4, 5, 6, 7, and 8, respectively. The secondary structure of zein changed with the variation in solution pH. Furthermore, we analyzed the physical properties of the zein films. The contact angles of the fiber membranes prepared with different pH spinning solutions were all above 100, while zein films formed by solvent evaporation showed hydrophilic properties. The results indicated that the rheological properties of zein solutions and the surface properties of the film were affected by the pH value. This study showed that zein solutions can be stabilized to form electrospun fibers at a variety of pH levels and offered new opportunities to further enhance the encapsulation activity of zein films for bioactive materials.

Perturbed iron biology in the prefrontal cortex of people with schizophrenia

Despite loss of grey matter volume and emergence of distinct cognitive deficits in young adults diagnosed with schizophrenia, current treatments for schizophrenia do not target disruptions in late maturational reshaping of the prefrontal cortex. Iron, the most abundant transition metal in the brain, is essential to brain development and function, but in excess, it can impair major neurotransmission systems and lead to lipid peroxidation, neuroinflammation and accelerated aging. However, analysis of cortical iron biology in schizophrenia has not been reported in modern literature. Using a combination of inductively coupled plasma-mass spectrometry and western blots, we quantified iron and its major-storage protein, ferritin, in post-mortem prefrontal cortex specimens obtained from three independent, well-characterised brain tissue resources. Compared to matched controls (n = 85), among schizophrenia cases (n = 86) we found elevated tissue iron, unlikely to be confounded by demographic and lifestyle variables, by duration, dose and type of antipsychotic medications used or by copper and zinc levels. We further observed a loss of physiologic age-dependent iron accumulation among people with schizophrenia, in that the iron level among cases was already high in young adulthood. Ferritin, which stores iron in a redox-inactive form, was paradoxically decreased in individuals with the disorder. Such iron-ferritin uncoupling could alter free, chemically reactive, tissue iron in key reasoning and planning areas of the young-adult schizophrenia cortex. Using a prediction model based on iron and ferritin, our data provide a pathophysiologic link between perturbed cortical iron biology and schizophrenia and indicate that achievement of optimal cortical iron homeostasis could offer a new therapeutic target.

Association of Serum Ferritin Level in Early Second Trimester of Pregnancy with Development of Gestational Diabetes Mellitus: A Prospective Observational Study.

Gestational diabetes mellitus (GDM) is associated with various maternal and perinatal morbidities. Serum ferritin is a major storage protein of iron and also acts as acute phase reactant which is increased in inflammatory conditions. GDM is a state of insulin resistance and associated with inflammation. The aim of this study was to find the correlation between serum ferritin and development of GDM. To determine the serum ferritin concentration in nonanemic pregnant women and its correlation with subsequent development of GDM. In this prospective observational study, 302 nonanemic pregnant women with singleton gestation between 14 and 20weeks, attending antenatal OPD, were enrolled. Serum ferritin was measured at the time of enrolment, and they were followed till 24-28weeks of gestation and subjected to blood glucose test by DIPSI method. A total of 92 women had blood glucose level ≥ 140mg/dl and were labeled as GDM, and 210 pregnant women with blood glucose level < 140mg/dl were labeled as non-GDM. Mean serum ferritin level of women with GDM (56.44 ± 19.19ng/ml) was found to be higher as compared to non-GDM (27.62 ± 12.11ng/ml), and this difference was found to be statistically significant (p < 0.001). The cutoff value of serum ferritin > 37.55ng/ml was found to be 85.9% sensitive and 81.9% specific. We can infer that serum ferritin is associated with development of GDM. Based on the findings of the current study, serum ferritin level can be used a predictive marker for the development of GDM.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.

Generation of multi-layered protein bodies in N. benthamiana for the encapsulation of vaccine antigens.

The ability of plants to assemble particulate structures such as virus-like particles and protein storage organelles allows the direct bioencapsulation of recombinant proteins during the manufacturing process, which holds promise for the development of new drug delivery vehicles. Storage organelles found in plants such as protein bodies (PBs) have been successfully used as tools for accumulation and encapsulation of recombinant proteins. The fusion of sequences derived from 27-kDa-γ-zein, a major storage protein of maize, with a protein of interest leads to the incorporation of the chimeric protein into the stable and protected environment inside newly induced PBs. While this procedure has proven successful for several, but not all recombinant proteins, the aim of this study was to refine the technology by using a combination of PB-forming proteins, thereby generating multi-layered protein assemblies in N. benthamiana. We used fluorescent proteins to demonstrate that up to three proteinaceous components can be incorporated into different layers. In addition to 27-kDa-γ-zein, which is essential for PB initiation, 16-kDa-γ-zein was identified as a key element to promote the incorporation of a third zein-component into the core of the PBs. We show that a vaccine antigen could be incorporated into the matrix of multi-layered PBs, and the protein microparticles were characterized by confocal and electron microscopy as well as flow cytometry. In future, this approach will enable the generation of designer PBs that serve as drug carriers and integrate multiple components that can be functionalized in different ways.

Bio-rafiniranje sačme pamučnog semena iz naftne industrije za izolaciju ekološki prihvatljivog prirodnog proteinskog polimera

Cottonseed meal (CSM) is a by-product of the oil industry. Globulins (salt soluble, vicilin, and legumin families) are the major dominant storage proteins in cottonseed and account for 60%-70% of seed proteins. However, these are unsuitable for human consumption due to their gossypol content. In this work, protein was extracted from defatted cottonseed flour with alkaline solution and its precipitation at different pH value was investigated. The crude protein was quantified using standard micro-Kjeldahl nitrogen method. Central composite design expert was conducted and the effect of variables in the extraction of protein were studied, namely, extraction solvent concentration, temperature and time. The optimum scheme of extraction was obtained at 0.1M NaOH with material to liquor ratio (MLR) 1:20 at pH 12 using temperature of 67℃ and a time of 90 min. Under these conditions, the response gives a flour dissolution percentage of 80.52% and protein purity percentage 80.40%. In addition, protein solubility and water absorption capacity were also determined and the chemical structure of protein was characterized by means of Fourier Transform Infrared Spectroscopy.

In-depth magnetometry and EPR analysis of the spin structure of human-liver ferritin: from DC to 9 GHz.

Ferritin, the major iron storage protein in organisms, stores iron in the form of iron oxyhydroxide most likely involving phosphorous as a constituent, the mineral form of which is not well understood. Therefore, the question of how the ca. 2000 iron atoms in the ferritin core are magnetically coupled is still largely open. The ferritin core, with a diameter of 5-8 nm, is encapsulated in a protein shell that also catalyzes the uptake of iron and protects the core from outside interactions. Neurodegenerative disease is associated with iron imbalance, generating specific interest in the magnetic properties of ferritin. Here we present 9 GHz continuous wave EPR and a comprehensive set of magnetometry techniques including isothermal remanent magnetization (IRM) and AC susceptibility to elucidate the magnetic properties of the core of human liver ferritin. For the analysis of the magnetometry data, a new microscopic model of the ferritin-core spin structure is derived, showing that magnetic moment is generated by surface-spin canting, rather than defects. The analysis explicitly includes the distribution of magnetic parameters, such as the distribution of the magnetic moment. This microscopic model explains some of the inconsistencies resulting from previous analysis approaches. The main findings are a mean magnetic moment of 337μB with a standard deviation of 0.947μB. In contrast to previous reports, only a relatively small contribution of paramagnetic and ferrimagnetic phases is found, in the order of maximally 3%. For EPR, the over 30 mT wide signal of the ferritin core is analyzed using the model of the giant spin system [Fittipaldi et al., Phys. Chem. Chem. Phys., 2016, 18, 3591-3597]. Two components are needed minimally, and the broadening of these components suggests a broad distribution of the magnetic resonance parameters, the zero-field splitting, D, and the spin quantum number, S. We compare parameters from EPR and magnetometry and find that EPR is particularly sensitive to the surface spins of the core, revealing the potential to use EPR as a diagnostic for surface-spin disorder.

Lipid Peroxidation and Iron Metabolism: Two Corner Stones in the Homeostasis Control of Ferroptosis

Regulated cell death (RCD) has a significant impact on development, tissue homeostasis, and the occurrence of various diseases. Among different forms of RCD, ferroptosis is considered as a type of reactive oxygen species (ROS)-dependent regulated necrosis. ROS can react with polyunsaturated fatty acids (PUFAs) of the lipid (L) membrane via the formation of a lipid radical L• and induce lipid peroxidation to form L-ROS. Ferroptosis is triggered by an imbalance between lipid hydroperoxide (LOOH) detoxification and iron-dependent L-ROS accumulation. Intracellular iron accumulation and lipid peroxidation are two central biochemical events leading to ferroptosis. Organelles, including mitochondria and lysosomes are involved in the regulation of iron metabolism and redox imbalance in ferroptosis. In this review, we will provide an overview of lipid peroxidation, as well as key components involved in the ferroptotic cascade. The main mechanism that reduces ROS is the redox ability of glutathione (GSH). GSH, a tripeptide that includes glutamic acid, cysteine, and glycine, acts as an antioxidant and is the substrate of glutathione peroxidase 4 (GPX4), which is then converted into oxidized glutathione (GSSG). Increasing the expression of GSH can inhibit ferroptosis. We highlight the role of the xc- GSH-GPX4 pathway as the main pathway to regulate ferroptosis. The system xc-, composed of subunit solute carrier family members (SLC7A11 and SLC3A2), mediates the exchange of cystine and glutamate across the plasma membrane to synthesize GSH. Accumulating evidence indicates that ferroptosis requires the autophagy machinery for its execution. Ferritinophagy is used to describe the removal of the major iron storage protein ferritin by the autophagy machinery. Nuclear receptor coactivator 4 (NCOA4) is a cytosolic autophagy receptor used to bind ferritin for subsequent degradation by ferritinophagy. During ferritinophagy, stored iron released becomes available for biosynthetic pathways. The dysfunctional ferroptotic response is implicated in a variety of pathological conditions. Ferroptosis inducers or inhibitors targeting redox- or iron metabolism-related proteins and signal transduction have been developed. The simultaneous detection of intracellular and extracellular markers may help diagnose and treat diseases related to ferroptotic damage.

Transcriptome Analysis Reveals Potential Mechanism in Storage Protein Trafficking within Developing Grains of Common Wheat

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which defines the viscoelastic properties of wheat dough. The synthesis of these storage proteins is controlled by the endoplasmic reticulum (ER) and is directed into the vacuole via the Golgi apparatus. In the present study, transcriptome analysis was used to explore the potential mechanism within critical stages of grain development of wheat cultivar "Shaannong 33" and its sister line used as the control (CK). Samples were collected at 10 DPA (days after anthesis), 14 DPA, 20 DPA, and 30 DPA for transcriptomic analysis. The comparative transcriptome analysis identified that a total of 18,875 genes were differentially expressed genes (DEGs) between grains of four groups "T10 vs. CK10, T14 vs. CK14, T20 vs. CK20, and T30 vs. CK30", including 2824 up-regulated and 5423 down-regulated genes in T30 vs. CK30. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment highlighted the maximum number of genes regulating protein processing in the endoplasmic reticulum (ER) during grain enlargement stages (10-20 DPA). In addition, KEGG database analysis reported 1362 and 788 DEGs involved in translation, ribosomal structure, biogenesis, flavonoid biosynthesis pathway and intracellular trafficking, secretion, and vesicular transport through protein processing within ER pathway (ko04141). Notably, consistent with the higher expression of intercellular storage protein trafficking genes at the initial 10 DPA, there was relatively low expression at later stages. Expression levels of nine randomly selected genes were verified by qRT-PCR, which were consistent with the transcriptome data. These data suggested that the initial stages of "cell division" played a significant role in protein quality control within the ER, thus maintaining the protein quality characteristics at grain maturity. Furthermore, our data suggested that the protein synthesis, folding, and trafficking pathways directed by a different number of genes during the grain enlargement stage contributed to the observed high-quality characteristics of gluten protein in Shaannong 33 (Triticum aestivum L.).

Cryptococcus neoformans can utilize ferritin as an iron source.

Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening pulmonary disease and cryptococcal meningitis, mainly in immunocompromised patients. In this study, we found that C. neoformans can use ferritin, a major iron storage protein in vertebrates, as a sole iron source.

Zein Microparticles and Nanoparticles as Drug Delivery Systems.

Zein is a natural, biocompatible, and biodegradable polymer widely used in the pharmaceutical, biomedical, and packaging fields because of its low water vapor permeability, antibacterial activity, and hydrophobicity. It is a vegetal protein extracted from renewable resources (it is the major storage protein from corn). There has been growing attention to producing zein-based drug delivery systems in the recent years. Being a hydrophobic biopolymer, it is used in the controlled and targeted delivery of active principles. This review examines the present-day landscape of zein-based microparticles and nanoparticles, focusing on the different techniques used to obtain particles, the optimization of process parameters, advantages, disadvantages, and final applications.

Staphylococcus aureus ftnA 3'-Untranslated Region Modulates Ferritin Production Facilitating Growth Under Iron Starvation Conditions.

Iron acquisition and modulation of its intracellular concentration are critical for the development of all living organisms. So far, several proteins have been described to be involved in iron homeostasis. Among them, ferritins act as the major iron storage proteins, sequestering internalized iron and modulating its concentration inside bacterial cells. We previously described that the deletion of the 3’-untranslated region (3’UTR) of the ftnA gene, which codes for ferritin in Staphylococcus aureus, increased the ftnA mRNA and ferritin levels. Here, we show that the ferritin levels are affected by RNase III and PNPase, which target the ftnA 3’UTR. Rifampicin mRNA stability experiments revealed that the half-life of the ftnA mRNA is affected by both RNase III and the ftnA 3’UTR. A transcriptional fusion of the ftnA 3’UTR to the gfp reporter gene decreased green fluorescent protein (GFP) expression, indicating that the ftnA 3’UTR could work as an independent module. Additionally, a chromosomal deletion of the ftnA 3’UTR impaired S. aureus growth under conditions of iron starvation. Overall, this work highlights the biological relevance of the ftnA 3’UTR for iron homeostasis in S. aureus.

Effect of the Post-Harvest Processing on Protein Modification in Green Coffee Beans by Phenolic Compounds.

The protein fraction, important for coffee cup quality, is modified during post-harvest treatment prior to roasting. Proteins may interact with phenolic compounds, which constitute the major metabolites of coffee, where the processing affects these interactions. This allows the hypothesis that the proteins are denatured and modified via enzymatic and/or redox activation steps. The present study was initiated to encompass changes in the protein fraction. The investigations were limited to major storage protein of green coffee beans. Fourteen Coffea arabica samples from various processing methods and countries were used. Different extraction protocols were compared to maintain the status quo of the protein modification. The extracts contained about 4–8 µg of chlorogenic acid derivatives per mg of extracted protein. High-resolution chromatography with multiple reaction monitoring was used to detect lysine modifications in the coffee protein. Marker peptides were allocated for the storage protein of the coffee beans. Among these, the modified peptides K.FFLANGPQQGGK.E and R.LGGK.T of the α-chain and R.ITTVNSQK.I and K.VFDDEVK.Q of β-chain were detected. Results showed a significant increase (p < 0.05) of modified peptides from wet processed green beans as compared to the dry ones. The present study contributes to a better understanding of the influence of the different processing methods on protein quality and its role in the scope of coffee cup quality and aroma.

Zein-Based Nanoparticles as Oral Carriers for Insulin Delivery.

Zein, the major storage protein from corn, has a GRAS (Generally Regarded as Safe) status and may be easily transformed into nanoparticles, offering significant payloads for protein materials without affecting their stability. In this work, the capability of bare zein nanoparticles (mucoadhesive) and nanoparticles coated with poly(ethylene glycol) (mucus-permeating) was evaluated as oral carriers of insulin (I-NP and I-NP-PEG, respectively). Both nanocarriers displayed sizes of around 270 nm, insulin payloads close to 80 µg/mg and did not induce cytotoxic effects in Caco-2 and HT29-MTX cell lines. In Caenorhabditis elegans, where insulin decreases fat storage, I-NP-PEG induced a higher reduction in the fat content than I-NP and slightly lower than the control (Orlistat). In diabetic rats, nanoparticles induced a potent hypoglycemic effect and achieved an oral bioavailability of 4.2% for I-NP and 10.2% for I-NP-PEG. This superior effect observed for I-NP-PEG would be related to their capability to diffuse through the mucus layer and reach the surface of enterocytes (where insulin would be released), whereas the mucoadhesive I-NP would remain trapped in the mucus, far away from the absorptive epithelium. In summary, PEG-coated zein nanoparticles may be an interesting device for the effective delivery of proteins through the oral route.

Valorization of Peel-Based Agro-Waste Flour for Food Products: A Systematic Review on Proximate Composition and Functional Properties

With the steadily growing world population, effective methods are needed to alleviate food shortages. One possible strategy could be to utilize agro-waste materials that accumulate in large quantities at every stage of the economic chain during harvesting, food production, and consumption. Peel-based agro-waste consists of promising materials that can be utilized to potentially substitute commonly used raw materials in products traditionally made from wheat, tapioca, and rice flours. In this systematic review, we aim at establishing prospective proximate components as basic nutrients and their valorization potential as substitutes in traditional flour products (bread, biscuits, etc.). Generally, the peel contains high levels of fiber and relatively low digestible carbohydrates, providing a healthier food ingredient. In terms of protein, it should be pointed out that seeds such as wheat utilize insoluble gluten as their major storage protein, while proteins in peel were found in quite high percentage although they were not yet well characterized. However, the general effect of using peel to substitute wheat in food products are the reduction of dough elasticity, increased hardness of the end-products, faster water absorption rate of the products, and in some cases, bitter taste and darker colors. The latter two could have been contributed by the secondary metabolites such as phenolic compounds. On the other hand, substitution of peel into food products can have valuable health benefits, e.g., retention of antioxidant activity due to the phenolic compounds or simply adding fiber. In this review, literature on the composition of promising agro-waste raw materials is being discussed in the relationship with physical properties and appearance of potential end-products. Antinutritional compounds and pretreatment processes are also being considered. It is hoped that a critical discussion will lead to a better understanding and higher acceptance of the incorporation of peel into food products.

Isolation, purification and characterization of the antibacterial, antihypertensive and antioxidative properties of the bioactive peptides in the purified and proteolyzed major storage protein of pigeon pea (Cajanus cajan) seeds

Previous studies in pigeon pea showed health benefits but very few explored peptide bioactivities. This study examined antimicrobial, antioxidant, and antihypertensive activities of peptides in purified and proteolyzed major globulin fraction, cajanin, of pigeon pea (Cajanus cajan) seeds. In-silico analysis showed 41 antihypertensive and nine antioxidant peptides but zero antibacterial peptides from cajanin sequence. The Pepsin-Chymotrypsin-Trypsin (PCT) protein digest has no antibacterial activity against Escherichia coli, Candida albicans, and Staphylococcus aureus but has high % ACE inhibition (87.50%). Two HPLC fractions showed low IC50 values (HPLC Fraction 1: 0.00535 mg/ml; HPLC Fraction 2: 0.00432 mg/ml), comparable to Captopril (0.00379 mg/ml). This fractions also showed H2O2 scavenging activity (HPLC Fraction 1: 1.47%; HPLC Fraction 2: 1.51%) and Total Antioxidative Capacity of 0.00088 mg/mL (HPLC Fraction1) and 0.00110 mg/mL (HPLC Fraction 2) ascorbic acid equivalent. Results from this study serve as reference for further investigations of novel pharmaceutical agents that can be derived from legumes.

Soybean β-Conglycinin: Structure Characteristic, Allergenicity, Plasma Lipid-Controlling, Prevention of Obesity and Non-alcoholic Fatty Liver Disease.

Soybean has become an important world commodity because of its low price, good nutritional value and recognized functional health benefits in recent years. β-conglycinin is one of the major storage proteins in soybean. It has captured a growing interest recently because of its allergenicity and potential health benefits, which continues to drive the research and commercial development of β-conglycinin-based food products and ingredients. In this review, the structure, the amino acid composition, extraction methods and electrophoretic pattern of β-conglycinin are briefly summarized. Studies on β-conglycinin by allergenicity, plasma lipid-controlling, obesity and nonalcoholic fatty liver disease are highlighted, critically discussing their main shortcomings and challenges and identifying the research gaps. Studies to date have demonstrated the cultivation of β-conglycinin with health benefits as functional ingredients and foodstuffs. The current research focuses on proteins, mainly challenging the mechanisms of subunit/peptide effects interaction and identifying and characterizing the hidden biological activities in the polypeptide chains. There is much scope for further exploration into various aspects of β-conglycinin, such as the selection of mutant strains and genetic engineering and prospects on targeted β-conglycinin exploitation in the nutraceutical area. In addition, the safety evaluation of β-conglycinin and its stabilized emulsions deserve more attention to food-related applications.

Trihelix Transcription Factor ZmThx20 Is Required for Kernel Development in Maize

Maize kernels are the harvested portion of the plant and are related to the yield and quality of maize. The endosperm of maize is a large storage organ that constitutes 80–90% of the dry weight of mature kernels. Maize kernels have long been the study of cereal grain development to increase yield. In this study, a natural mutation that causes abnormal kernel development, and displays a shrunken kernel phenotype, was identified and named “shrunken 2008 (sh2008)”. The starch grains in sh2008 are loose and have a less proteinaceous matrix surrounding them. The total storage protein and the major storage protein zeins are ~70% of that in the wild-type control (WT); in particular, the 19 kDa and 22 kDa α-zeins. Map-based cloning revealed that sh2008 encodes a GT-2 trihelix transcription factor, ZmThx20. Using CRISPR/Cas9, two other alleles with mutated ZmThx20 were found to have the same abnormal kernel. Shrunken kernels can be rescued by overexpressing normal ZmThx20. Comparative transcriptome analysis of the kernels from sh2008 and WT showed that the GO terms of translation, ribosome, and nutrient reservoir activity were enriched in the down-regulated genes (sh2008/WT). In short, these changes can lead to defects in endosperm development and storage reserve filling in seeds.

Uncovering the Metabolic and Stress Responses of Human Embryonic Stem Cells to FTH1 Gene Silencing.

Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs’ response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.

CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles

AbstractExtracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism–associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5′ untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.