ABSTRACT
Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Ferritins , Salmonella , Single-Domain Antibodies , Ferritins/immunology , Ferritins/chemistry , Ferritins/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Single-Domain Antibodies/immunology , Salmonella/immunology , Salmonella/genetics , Enzyme-Linked Immunosorbent Assay/methods , Limit of Detection , Antibody Affinity , Antibodies, Bacterial/immunology , Immunoassay/methodsABSTRACT
Despite ferritin's critical role in regulating cellular and systemic iron levels, our understanding of the structure and assembly mechanism of isoferritins, discovered over eight decades ago, remains limited. Unveiling how the composition and molecular architecture of hetero-oligomeric ferritins confer distinct functionality to isoferritins is essential to understanding how the structural intricacies of H and L subunits influence their interactions with cellular machinery. In this study, ferritin heteropolymers with specific H to L subunit ratios were synthesized using a uniquely engineered plasmid design, followed by high-resolution cryo-electron microscopy analysis and deep learning-based amino acid modeling. Our structural examination revealed unique architectural features during the self-assembly mechanism of heteropolymer ferritins and demonstrated a significant preference for H-L heterodimer formation over H-H or L-L homodimers. Unexpectedly, while dimers seem essential building blocks in the protein self-assembly process, the overall mechanism of ferritin self-assembly is observed to proceed randomly through diverse pathways. The physiological significance of these findings is discussed including how ferritin microheterogeneity could represent a tissue-specific adaptation process that imparts distinctive tissue-specific functions to isoferritins.
Subject(s)
Ferritins , Protein Multimerization , Humans , Ferritins/chemistry , Ferritins/metabolism , Ferritins/genetics , Models, Molecular , Cryoelectron MicroscopyABSTRACT
Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.
Subject(s)
Amino Acid Sequence , Ferritins , Nuclear Receptor Coactivators , Phylogeny , Sequence Alignment , Stichopus , Vibrio , Animals , Vibrio/physiology , Stichopus/immunology , Stichopus/genetics , Stichopus/microbiology , Nuclear Receptor Coactivators/genetics , Nuclear Receptor Coactivators/immunology , Ferritins/genetics , Ferritins/immunology , Ferritins/metabolism , Immunity, Innate/genetics , Gene Expression Regulation/immunology , Gene Expression Profiling , Autophagy , Base SequenceABSTRACT
Fluoride is an environmental pollutant that severely injures various organisms in ecosystems. Herein, the non-target organism, fall webworm (Hyphantria cunea), was used to determine the toxicological mechanism of NaF exposure. In this study, H. cunea exposed to NaF showed significant declines in growth and reproduction. The authors conducted RNA sequencing on adipose bodies and midgut tissues from NaF-exposed H. cunea larvae to uncover the toxicological mechanisms. The results showed that extracellular matrix-receptor interaction, pentose and glucuronate interconversions, fatty acid biosynthesis, and ferroptosis might contribute to NaF stress. NaF significantly decreased the antioxidant level, nitrous oxide synthase activity, and NO content, while significantly increasing lipid peroxidation. NaF induced significant changes in the expression of energy metabolism genes. However, the triglyceride content was significantly decreased and the lipase enzyme activity was significantly increased. Moreover, the expression levels of light and heavy chains of ferritin were inhibited in NaF-exposed H. cunea. NaF caused ferritin Fe2+overload in FerHCH1 and FerLCH knockdown H. cunea larvae, activated reactive oxygen species, and reduced the total iron content, eventually increasing the mortality H. cunea larvae. This study identified the toxicological mechanisms of NaF in lipid synthesis and energy metabolism in H. cunea, providing a basis for understanding the molecular mechanisms of NaF toxicity and developing pollution control strategies.
Subject(s)
Larva , Sodium Fluoride , Transcriptome , Animals , Transcriptome/drug effects , Larva/drug effects , Sodium Fluoride/toxicity , Energy Metabolism/drug effects , Lipid Peroxidation/drug effects , Coleoptera/drug effects , Reactive Oxygen Species/metabolism , Ferroptosis/drug effects , Ferritins/metabolism , Ferritins/genetics , Environmental Pollutants/toxicityABSTRACT
Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe2+ and peroxidated lipids. Notably, the Fe2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.
Subject(s)
Enterovirus A, Human , Enterovirus Infections , Ferroptosis , Motor Neurons , Ferroptosis/drug effects , Humans , Enterovirus A, Human/physiology , Enterovirus A, Human/genetics , Enterovirus A, Human/drug effects , Motor Neurons/virology , Motor Neurons/metabolism , Enterovirus Infections/virology , Enterovirus Infections/metabolism , Virus Replication , Mitochondria/metabolism , Deferoxamine/pharmacology , Viral Load , Iron/metabolism , Ferritins/metabolism , Ferritins/geneticsABSTRACT
Thymol has efficient bactericidal activity against a variety of pathogenic bacteria, but the bactericidal mechanism against Vibrio parahemolyticus (V. parahemolyticus) has rarely been reported. In the current study, we investigated the bactericidal mechanism of thymol against V. parahemolyticus. The Results revealed that 150 µg/mL of thymol had 99.9% bactericidal activity on V. parahemolyticus. Intracellular bursts of reactive oxygen species (ROS), Fe2+accumulation, lipid peroxidation, and DNA breakage were checked by cell staining. The exogenous addition of H2O2 and catalase promoted and alleviated thymol-induced cell death to a certain extent, respectively, and the addition of the ferroptosis inhibitor Liproxstatin-1 also alleviated thymol-induced cell death, confirming that thymol induced Fenton-reaction-dependent ferroptosis in V. parahemolyticus. Proteomic analysis revealed that relevant proteins involved in ROS production, lipid peroxidation accumulation, and DNA repair were significantly upregulated after thymol treatment. Molecular docking revealed two potential binding sites (amino acids 46H and 42F) between thymol and ferritin, and thymol could promote the release of Fe2+ from ferritin proteins through in vitro interactions analyzed. Therefore, we hypothesized that ferritin as a potential target may mediate thymol-induced ferroptosis in V. parahemolyticus. This study provides new ideas for the development of natural inhibitors for controlling V. parahemolyticus in aquatic products.
Subject(s)
Anti-Bacterial Agents , Ferroptosis , Hydrogen Peroxide , Reactive Oxygen Species , Thymol , Vibrio parahaemolyticus , Ferroptosis/drug effects , Thymol/pharmacology , Thymol/chemistry , Reactive Oxygen Species/metabolism , Vibrio parahaemolyticus/drug effects , Hydrogen Peroxide/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Lipid Peroxidation/drug effects , Iron/metabolism , Molecular Docking Simulation , Ferritins/genetics , Ferritins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/geneticsABSTRACT
Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug delivery and biomedical applications. Our research investigated the genetic link of a specific anti-HER2 peptide (LTVSPWY) to the exposed N-terminal region of the maize (Zea mays) ferritin 1 (ZmFer1) protein nanocage, employing either a 7-amino acid (for LTVS-ZmFer1) or 16-amino acid (for LTVS-L-ZmFer1) linker. We utilized a heat treatment method to load the chemotherapeutic drug doxorubicin into the protein nanocage. The construct with the longer linker (LTVS-L) produced a greater amount of soluble protein nanocage and was selected for further experiments. The average size, polydispersity index, and zeta potential of the engineered protein nanocage were 19.01 nm, 0.168, and - 2.13 mV, respectively. The LTVS-L-ZmFer1 protein nanocage exhibited excellent thermal stability, withstanding temperatures up to 100 °C with only partial denaturation. Furthermore, we observed that cellular uptake of the LTVS-L-ZmFer1 protein nanocages in HER2-positive breast cancer cells was significantly higher compared to ZmFer1 after labeling with FITC (fluorescein isothiocyanate) (P-value = 0.0001). In addition, we observed a significant decrease in the viability of SKBR3 cells when treated with DOX-loaded LTVS-L-ZmFer1 protein nanocages compared to cells treated with DOX-loaded ZmFer1 protein nanocages. Therefore, this new treatment strategy may prove to be an effective way to reduce both the side effects and toxicity associated with conventional cancer treatments in patients with HER2-positive breast cancer.
Subject(s)
Doxorubicin , Drug Delivery Systems , Receptor, ErbB-2 , Humans , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Doxorubicin/pharmacology , Drug Delivery Systems/methods , Cell Line, Tumor , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ferritins/chemistry , Ferritins/metabolism , Ferritins/genetics , Zea mays/genetics , Protein Engineering/methods , Female , Drug Carriers/chemistry , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Quantitative susceptibility mapping (QSM) is an MRI modality used to non-invasively measure iron content in the brain. Iron exhibits a specific anatomically varying pattern of accumulation in the brain across individuals. The highest regions of accumulation are the deep grey nuclei, where iron is stored in paramagnetic molecule ferritin. This form of iron is considered to be what largely contributes to the signal measured by QSM in the deep grey nuclei. It is also known that QSM is affected by diamagnetic myelin contents. Here, we investigate spatial gene expression of iron and myelin related genes, as measured by the Allen Human Brain Atlas, in relation to QSM images of age-matched subjects. We performed multiple linear regressions between gene expression and the average QSM signal within 34 distinct deep grey nuclei regions. Our results show a positive correlation (p < .05, corrected) between expression of ferritin and the QSM signal in deep grey nuclei regions. We repeated the analysis for other genes that encode proteins thought to be involved in the transport and storage of iron in the brain, as well as myelination. In addition to ferritin, our findings demonstrate a positive correlation (p < .05, corrected) between the expression of ferroportin, transferrin, divalent metal transporter 1, several gene markers of myelinating oligodendrocytes, and the QSM signal in deep grey nuclei regions. Our results suggest that the QSM signal reflects both the storage and active transport of iron in the deep grey nuclei regions of the brain.
Subject(s)
Ferritins , Homeostasis , Iron , Magnetic Resonance Imaging , Myelin Sheath , Humans , Iron/metabolism , Male , Female , Myelin Sheath/metabolism , Myelin Sheath/genetics , Adult , Homeostasis/physiology , Ferritins/metabolism , Ferritins/genetics , Brain/metabolism , Brain/diagnostic imaging , Gene Expression , Middle Aged , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Young Adult , Brain Mapping/methodsABSTRACT
Parkinson's disease (PD) poses a significant challenge in neurodegenerative disorders, characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). The intricate mechanisms orchestrating DA neurodegeneration in PD are not fully understood, necessitating the exploration of innovative therapeutic approaches. Recent studies have implicated ferroptosis as a major contributor to the loss of DA neurons, revealing a complex interplay between iron accumulation and neurodegeneration. However, the sophisticated nature of this process challenges the conventional belief that mere iron removal could effectively prevent DA neuronal ferroptosis. Here, we report JWA, alternatively referred to as ARL6IP5, as a negative regulator of ferroptosis, capable of ameliorating DA neuronal loss in the context of PD. In this study, synchronized expression patterns of JWA and tyrosine hydroxylase (TH) in PD patients and mice were observed, underscoring the importance of JWA for DA neuronal survival. Screening of ferroptosis-related genes unraveled the engagement of iron metabolism in the JWA-dependent inhibition of DA neuronal ferroptosis. Genetic manipulation of JWA provided compelling evidence linking its neuroprotective effects to the attenuation of NCOA4-mediated ferritinophagy. Molecular docking, co-immunoprecipitation, and immunofluorescence studies confirmed that JWA mitigated DA neuronal ferroptosis by occupying the ferritin binding site of NCOA4. Moreover, the JWA-activating compound, JAC4, demonstrated promising neuroprotective effects in cellular and animal PD models by elevating JWA expression, offering a potential avenue for neuroprotection in PD. Collectively, our work establishes JWA as a novel regulator of ferritinophagy, presenting a promising therapeutic target for addressing DA neuronal ferroptosis in PD.
Subject(s)
Dopaminergic Neurons , Ferritins , Ferroptosis , Nuclear Receptor Coactivators , Parkinson Disease , Parkinson Disease/metabolism , Parkinson Disease/pathology , Parkinson Disease/genetics , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Animals , Mice , Humans , Nuclear Receptor Coactivators/metabolism , Nuclear Receptor Coactivators/genetics , Ferritins/metabolism , Ferritins/genetics , Iron/metabolism , Disease Models, Animal , Protein Binding , Autophagy , MaleABSTRACT
Porcine epidemic diarrhea virus (PEDV), a highly virulent enteropathogenic coronavirus, is a significant threat to the pig industry. High frequency mutations in the PEDV genome have limited the effectiveness of current vaccines in providing immune protection. Developing efficient vaccines that can quickly adapt to mutant strains is a challenging but crucial task. In this study, we chose the pivotal protein heptad repeat (HR) responsible for coronavirus entry into host cells, as the vaccine antigen. HR-Fer nanoparticles prepared using ferritin were evaluated them as PEDV vaccine candidates. Nanoparticle vaccines elicited stronger neutralizing antibody responses in mice compared to monomer vaccines. Additionally, HR protein delivered via nanoparticles increased antigen uptake by antigen-presenting cells in vitro by 2.75-fold. The collective results suggest that HR can be used as antigens for vaccines, and the HR vaccine based on ferritin nanoparticles significantly enhances immunogenicity.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , Coronavirus Infections , Nanoparticles , Porcine epidemic diarrhea virus , Spike Glycoprotein, Coronavirus , Swine Diseases , Viral Vaccines , Animals , Porcine epidemic diarrhea virus/immunology , Porcine epidemic diarrhea virus/genetics , Nanoparticles/chemistry , Viral Vaccines/immunology , Viral Vaccines/genetics , Viral Vaccines/administration & dosage , Antibodies, Viral/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Coronavirus Infections/immunology , Mice , Antibodies, Neutralizing/immunology , Swine Diseases/prevention & control , Swine Diseases/virology , Swine Diseases/immunology , Swine , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Mice, Inbred BALB C , Ferritins/immunology , Ferritins/genetics , Ferritins/metabolism , Female , Chlorocebus aethiops , NanovaccinesABSTRACT
The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.
Subject(s)
Cryoelectron Microscopy , Ferritins , Nuclear Receptor Coactivators , Ferritins/metabolism , Ferritins/chemistry , Ferritins/genetics , Humans , Nuclear Receptor Coactivators/metabolism , Nuclear Receptor Coactivators/chemistry , Nuclear Receptor Coactivators/genetics , Protein Binding , Binding Sites , Iron/metabolism , Autophagy , Models, Molecular , HEK293 Cells , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Oxidoreductases/genetics , Proteolysis , MutationABSTRACT
Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy of the digestive system. Hypoxia is a crucial player in tumor ferroptosis resistance. However, the molecular mechanism of hypoxia-mediated ferroptosis resistance in ESCC remains unclear. Here, USP2 expression was decreased in ESCC cell lines subjected to hypoxia treatment and was lowly expressed in clinical ESCC specimens. Ubiquitin-specific protease 2 (USP2) depletion facilitated cell growth, which was blocked in USP2-overexpressing cells. Moreover, USP2 silencing enhanced the iron ion concentration and lipid peroxidation accumulation as well as suppressed ferroptosis, while upregulating USP2 promoted ferroptotic cell death in ESCC cells. Furthermore, knockout of USP2 in ESCC models discloses the essential role of USP2 in promoting ESCC tumorigenesis and inhibiting ferroptosis. In contrast, overexpression of USP2 contributes to antitumor effect and ferroptosis events in vivo. Specifically, USP2 stably bound to and suppressed the degradation of nuclear receptor coactivator 4 (NCOA4) by eliminating the Lys48-linked chain, which in turn triggered ferritinophagy and ferroptosis in ESCC cells. Our findings suggest that USP2 plays a crucial role in iron metabolism and ferroptosis and that the USP2/NCOA4 axis is a promising therapeutic target for the management of ESCC.
Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Ferroptosis , Ubiquitin Thiolesterase , Humans , Ferroptosis/genetics , Esophageal Squamous Cell Carcinoma/pathology , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Neoplasms/pathology , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/genetics , Animals , Mice , Cell Line, Tumor , Nuclear Receptor Coactivators/metabolism , Nuclear Receptor Coactivators/genetics , Gene Expression Regulation, Neoplastic , Ferritins/metabolism , Ferritins/genetics , Mice, Nude , Autophagy/genetics , Hypoxia/metabolism , Cell Proliferation/genetics , MaleABSTRACT
Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Ferritins , GTPase-Activating Proteins , Iron , Transferrin , Animals , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Endosomes/metabolism , Ferritins/metabolism , Ferritins/genetics , Iron/metabolism , Protein Transport , Transferrin/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolismABSTRACT
Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.
Subject(s)
Butylated Hydroxyanisole , Human Umbilical Vein Endothelial Cells , Phospholipid Hydroperoxide Glutathione Peroxidase , Animals , Humans , Mice , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Butylated Hydroxyanisole/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Ferroptosis/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Cell Movement/drug effects , Ferritins/metabolism , Ferritins/genetics , Cyclohexylamines , Oxidoreductases , PhenylenediaminesABSTRACT
The anemia of inflammation (AI) is characterized by the presence of inflammation and abnormal elevation of hepcidin. Accumulating evidence has proved that Rocaglamide (RocA) was involved in inflammation regulation. Nevertheless, the role of RocA in AI, especially in iron metabolism, has not been investigated, and its underlying mechanism remains elusive. Here, we demonstrated that RocA dramatically suppressed the elevation of hepcidin and ferritin in LPS-treated mice cell line RAW264.7 and peritoneal macrophages. In vivo study showed that RocA can restrain the depletion of serum iron (SI) and transferrin (Tf) saturation caused by LPS. Further investigation showed that RocA suppressed the upregulation of hepcidin mRNA and downregulation of Fpn1 protein expression in the spleen and liver of LPS-treated mice. Mechanistically, this effect was attributed to RocA's ability to inhibit the IL-6/STAT3 pathway, resulting in the suppression of hepcidin mRNA and subsequent increase in Fpn1 and TfR1 expression in LPS-treated macrophages. Moreover, RocA inhibited the elevation of the cellular labile iron pool (LIP) and reactive oxygen species (ROS) induced by LPS in RAW264.7 cells. These findings reveal a pivotal mechanism underlying the roles of RocA in modulating iron homeostasis and also provide a candidate natural product on alleviating AI.
Subject(s)
Benzofurans , Hepcidins , Homeostasis , Interleukin-6 , Iron , Animals , Mice , Anemia/metabolism , Anemia/genetics , Anemia/drug therapy , Anemia/pathology , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Ferritins/metabolism , Ferritins/genetics , Gene Expression Regulation/drug effects , Hepcidins/drug effects , Hepcidins/genetics , Hepcidins/metabolism , Homeostasis/drug effects , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Interleukin-6/metabolism , Interleukin-6/genetics , Iron/metabolism , Lipopolysaccharides/pharmacology , Liver/metabolism , Liver/pathology , Macrophages/metabolism , Macrophages/drug effects , RAW 264.7 Cells , Reactive Oxygen Species/metabolism , Receptors, Transferrin/metabolism , Receptors, Transferrin/genetics , Signal Transduction/drug effects , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Benzofurans/pharmacologyABSTRACT
Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic expression system to produce recombinant human heavy-chain ferritin nanoparticles and investigate their targeting ability in brain tissue. The human heavy-chain ferritin gene was cloned into the prokaryotic expression vector pET28a and transformed into Escherichia coli BL21 (DE3) competent cells to explore optimal expression conditions. The recombinant protein was then purified to evaluate its immunoreactivity and characteristics. Additionally, the distribution of the administered protein in normal mice and its permeability in an in vitro blood-brain barrier (BBB) model were measured. The results demonstrate that the purified protein can self-assemble extracellularly into nano-cage structures of approximately 10 nm and is recognized by corresponding antibodies. The protein effectively penetrates the blood-brain barrier and exhibits slow clearance in mouse brain tissue, showing excellent permeability in the in vitro BBB model. This study highlights the stable expression of recombinant human heavy-chain ferritin using the Escherichia coli prokaryotic expression system, characterized by favorable nano-cage structures and biological activity. Its exceptional brain tissue targeting and slow metabolism lay an experimental foundation for its application in neuropharmaceutical delivery and vaccine development fields.
Subject(s)
Blood-Brain Barrier , Brain , Escherichia coli , Ferritins , Nanoparticles , Recombinant Proteins , Animals , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Mice , Blood-Brain Barrier/metabolism , Brain/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Nanoparticles/chemistry , Ferritins/metabolism , Ferritins/genetics , Ferritins/chemistry , Apoferritins/metabolism , Apoferritins/genetics , Apoferritins/chemistry , Tissue DistributionABSTRACT
Ferritin, transferrin, and transferrin receptors I and II play a vital role in iron metabolism, health, and indication of iron deficiency anaemia in fish. To evaluate the use of high-iron diets to prevent or reverse channel catfish (Ictalurus punctatus) anaemia of unknown causes, we investigated the expression of these iron-regulatory genes and proteins in channel catfish fed plant-based diets. Catfish fingerlings were fed five diets supplemented with 0 (basal), 125, and 250 mg/kg of either inorganic iron or organic iron for 2 weeks. Ferritin, transferrin, and transferrin receptor I and II mRNA and protein expression levels in fish tissues (liver, intestine, trunk kidney, and head kidney) and plasma were determined. Transferrin (iron transporter) and TfR (I and II) genes were generally highly expressed in fish fed the basal diet compared to those fed the iron-supplemented diets. In contrast, ferritin (iron storage) genes were more expressed in the trunk kidney of fish fed the iron-supplemented diets than in those fed the basal diet. Our results demonstrate that supplementing channel catfish plant-based diets with iron from either organic or inorganic iron sources affected the expression of the iron-regulatory genes and increased body iron status in the fish.
Subject(s)
Animal Feed , Diet , Ferritins , Ictaluridae , Iron , Receptors, Transferrin , Transferrin , Animals , Ictaluridae/genetics , Ferritins/genetics , Ferritins/metabolism , Ferritins/blood , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Transferrin/metabolism , Transferrin/genetics , Diet/veterinary , Animal Feed/analysis , Iron/metabolism , Dietary Supplements/analysis , Gene Expression Regulation/drug effects , Fish Diseases , Iron, Dietary/administration & dosage , Iron, Dietary/metabolism , Gene Expression/drug effectsABSTRACT
Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.
Subject(s)
Inflammasomes , Liver Diseases , Rats , Mice , Animals , Inflammasomes/genetics , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Hepatic Stellate Cells/metabolism , Ferritins/genetics , Ferritins/metabolism , Interleukin-1beta/metabolism , Inflammation/genetics , Inflammation/metabolismABSTRACT
OBJECTIVE: To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy. METHODS: Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe2+) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining. RESULTS: Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe2+ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe2+. CONCLUSIONS: Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.
Subject(s)
Acupuncture Therapy , Brain Ischemia , Reperfusion Injury , Rats , Male , Animals , Rats, Sprague-Dawley , Brain Ischemia/genetics , Brain Ischemia/therapy , Brain Ischemia/metabolism , Cerebral Infarction , Reperfusion Injury/genetics , Reperfusion Injury/therapy , Reperfusion Injury/metabolism , Ferritins/genetics , Nuclear Receptor Coactivators/metabolismABSTRACT
In this study, we evaluated the histomorphology, reactive oxygen species (ROS), protein degradation, and iron metabolism characteristics and differential expression analysis of genes for siderophores synthesis and protease secretion in prepared beef steaks inoculated alone or co-inoculated with P. weihenstephanensis, B. thermotrichothrix and M. caseolyticus at 4 °C for 12 days. The results showed that the P. weihenstephanensis was the key bacteria that degraded protein in the process of prepared beef steaks spoilage, which led to protein oxidation by promoting ferritin degradation to release free iron and inducing ROS accumulation. The highest expression of FpvA and AprE was detected in the P. weihenstephanensis group by comparing qRT-PCR of the different inoculation groups. Both qRT-PCR and Western blot revealed that ferritin heavy polypeptide and ferritin light chain polypeptide gene and protein expressions were significantly higher in the P. weihenstephanensis inoculation group compared to the other inoculation groups. Results suggested that FpvA and AprE might play roles in meat spoilage and were potential positional, physiological and functional candidate genes for improving the quality traits of prepared beef steaks. This work may provide insights on controlling food quality and safety by intervening in spoilage pathways targeting iron carrier biosynthesis or protease secretion genes.