Biomimetic peroxidase MOF-Fe promotes bone defect repair by inhibiting TfR2 and activating the BMP2 pathway.

BACKGROUND: Large bone defects pose a clinical treatment challenge; inhibiting transferrin receptor 2 (TfR2), which is involved in iron metabolism, can promote osteogenesis. Iron-based metal-organic frameworks (MOF-Fe) particles not only inhibit TfR2 but also serve as biomimetic catalysts to remove hydrogen peroxide in reactive oxygen species (ROS); excess ROS can disrupt the normal functions of osteoblasts, thereby hindering bone regeneration. This study explored the potential effects of MOF-Fe in increasing osteogenic activity and clearing ROS. METHODS: In vitro experiments were performed to investigate the osteogenic effects of MOF-Fe particles and assess their impact on cellular ROS levels. To further validate the role of MOF-Fe in promoting bone defect repair, we injected MOF-Fe suspensions into the femoral defects of SD rats and implanted MOF-Fe-containing hydrogel scaffolds in rabbit cranial defect models and observed their effects on bone healing. RESULTS: In vitro, the presence of MOF-Fe significantly increased the expression levels of osteogenesis-related genes and proteins compared to those in the control group. Additionally, compared to those in the untreated control group, the cells treated with MOF-Fe exhibited a significantly increased ability to remove hydrogen peroxide from ROS and generate oxygen and water within the physiological pH range. In vivo experiments further confirmed the positive effect of MOF-Fe in promoting bone defect repair. CONCLUSION: This study supports the application of MOF-Fe as an agent for bone regeneration, particularly for mitigating ROS and activating the bone morphogenetic protein (BMP) pathway, demonstrating its potential value.

TFR1-Mediated Iron Metabolism Orchestrates Tumor Ferroptosis and Immunity in Non-Small Cell Lung Cancer.

This study aimed to investigate the underlying molecular mechanisms of transferrin receptor (TFR1) in non-small cell lung cancer (NSCLC). Histological analysis was performed using hematoxylin-eosin (HE) staining. The number of CD8+ T cell were determined by flow cytometry and immunofluorescence assays. mRNA levels were analyzed by qRT-PCR. Protein expression was detected by western blot. Ferroptosis was detected by using propidium iodide (PI) staining. Xenograft experiment was applied for determining tumor growth. The results showed that interferon (IFN)-γ plus iron dextran (FeDx) induced iron overload and the ferroptosis of NSCLC cells. Moreover, IFN-γ-mediated upregulation of TFR1 promoted ferritinophagy and tumor cell ferroptosis via blocking via blocking ferritin heavy chain 1 (FTH1)/ ferritin light chain (FTL) signaling. However, TFR1 knockout suppressed the ferroptosis of tumor cells. Furthermore, FeDx-mediated iron overload promoted the sensitivity of anti-programmed death ligand 1 (PD-L1) therapies. Clinically, TFR1 was downregulated in NSCLC patients. Low levels of TFR1 predicted decreased CD8+ T cells. Taken together, IFN-γ combined with iron metabolism therapies may provide a novel alternative for NSCLC.

TFR1 knockdown alleviates iron overload and mitochondrial dysfunction during neural differentiation of Alzheimer's disease-derived induced pluripotent stem cells by interacting with GSK3B.

Iron metabolism disorders are implicated in the pathogenesis of Alzheimer's disease (AD). It was previously reported that transferrin receptor (TFR1) expression was upregulated in AD mouse model. However, the precise biological functions of TFR1 in AD progression remains unclear. Herein, we observed a gradual increase in TFR1 protein expression during the differentiation of AD patient-derived induced pluripotent stem cells (AD-iPS). TFR1 knockdown inhibited the protein expression of ferritin and ferritin heavy chain 1 (FTH1), enhanced the expression of ferroportin 1 (FPN1), and decreased intracellular levels of total iron, labile iron, and reactive oxygen species (ROS). Moreover, TFR1 knockdown improved mitochondrial membrane potential (MMP), increased adenosine triphosphate (ATP) content, downregulated mitochondrial fission proteins, and upregulated mitochondrial fusion proteins. TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS, while TFR1 overexpression showed the opposite results. Additionally, TFR1interacted with glycogen synthase kinase 3 beta (GSK3B) and promoted GSK3B expression. GSK3B overexpression reversed the inhibitory effects of TFR1 knockdown on iron overload and mitochondrial dysfunction in AD-iPS differentiated neural cells. In conclusion, TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS by promoting GSK3B expression. Our findings provide a potential therapeutic target for the treatment of AD.

Human transferrin receptor can mediate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Comparing in vitro affinity measurements of antibodies to TfR1: Surface plasmon resonance versus on-cell affinity.

Despite years of utilizing the transferrin receptor 1 (TfR1) to transport large biomolecules into the brain, there is no consensus on how to optimally measure affinity to it. The aim of this study was to compare different methods for measuring the affinities of anti-TfR1 antibodies. Antibodies 15G11, OX26 and 8D3 are known to successfully carry large biologics across the blood-brain barrier in humans, rats, and mice, respectively. The affinity to their respective species of TfR1 was measured with different surface plasmon resonance setups in Biacore and an on-cell assay. When the antibody was captured and TfR1 was the analyte, the dissociation in Biacore was very slow. The dissociation was faster when the antibody was the analyte and TfR1 was the ligand. The Biacore setup with capture of N-terminal FLAG-tag TfR1 yielded the most similar apparent affinities as the cell assay. In conclusion, it is important to evaluate assay parameters including assay orientation, surface capture method, and antibody-format when comparing binding kinetics for TfR1 antibodies. Although it seems possible to determine relative affinities of TfR1 antibodies using the methods described here, both the FLAG-tag TfR1 capture setup and cell assays likely yield apparent affinities that are most translatable in vivo.

Exploring Lactoferrin as a novel marker for disease pathology and ferroptosis regulation in gestational diabetes.

Iron overload is linked to heightened susceptibility to ferroptosis, a process increasingly implicated in diabetes pathogenesis. This present study aims to assess the utility of Lactoferrin in predicting different stages of GDM and explore its association with disease pathology and ferroptosis. In this observational study, 72 pregnant women were recruited and categorized into three groups: healthy pregnant women without diabetes (NGDM, n = 24), early gestational diabetes (eGDM, n = 24), and established gestational diabetes (GDM, n = 24), all receiving standard antenatal care at 12 weeks of gestation. Circulating levels of ferritin, soluble transferrin receptor (sTFR), and Lactoferrin using multiplexed bead-based cytokine immunoassay. Gene expression analysis focused on analyzing crucial ferroptosis regulators, SLC7A11 and GPX4, in isolated peripheral blood mononuclear cells (PBMCs). A significant elevation in ferritin levels and a decrease in the sTFR: Ferritin ratio supported iron overload and disrupted iron homeostasis in GDM subjects. Notably, Lactoferrin levels were significantly lower in women with GDM than in the control group and those with eGDM. This decline in Lactoferrin correlated with increased hyperglycemia indicators and reduced expression of ferroptosis regulators among GDM patients. Furthermore ROC curve analysis demonstrated that Lactoferrin shows promise as a valuable marker for distinguishing individuals with GDM from those with eGDM. Lactoferrin shows promise as a biomarker for detecting GDM. These findings indicate its role as a potential biomarker and highlight Lactoferrin as a critical regulator of hyperglycemia and ferroptosis in women with GDM.

Targeted transport of biotherapeutics at the blood-brain barrier.

INTRODUCTION: The treatment of neurological diseases is significantly hampered by the lack of available therapeutics. A major restraint for the development of drugs is denoted by the presence of the blood-brain barrier (BBB), which precludes the transfer of biotherapeutics to the brain due to size restraints. AREAS COVERED: Novel optimism for transfer of biotherapeutics to the brain has been generated via development of targeted therapeutics to nutrient transporters expressed by brain capillary endothelial cells (BCECs). Targeting approaches with antibodies acting as biological drug carriers allow for proteins and genetic material to enter the brain, and qualified therapy using targeted proteins for protein replacement has been observed in preclinical models and now emerging in the clinic. Viral vectors denote an alternative for protein delivery to the brain by uptake and transduction of BCECs, or by transport through the BBB leading to neuronal transduction. EXPERT OPINION: The breaching of the BBB to large molecules has opened for treatment of diseases in the brain. A sturdier understanding of how biotherapeutics undergo transport through the BBB and how successful transport into the brain can be monitored is required to further improve the translation from successful preclinical studies to the clinic.

Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting.

BACKGROUND: Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models. METHODS: Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin. RESULTS: TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice. CONCLUSIONS: TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.

Iron Biology - An Overview for Laboratorians.

Iron serves a critical role in many metabolic processes, including oxygen delivery (e.g., hemoglobin) and oxygen utilization for the generation of ATP (e.g., cytochromes). Disorders of iron metabolism are best recognized and evaluated in the context of iron's absorption, transportation, monitoring, cellular uptake, and recycling. This review highlights these processes so that disorders of iron deficiency and iron excess can be better understood. Key players in iron metabolism will be highlighted, such as hepcidin, ferroportin, erythroferrone, transferrin, ferritin, HFE, and the transferrin receptors.

Transferrin Receptor-Targeted Nonspherical Microbubbles for Blood-Brain Barrier Sonopermeation.

Microbubbles (MB) are widely used for ultrasound (US) imaging and drug delivery. MB are typically spherically shaped, due to surface tension. When heated above their glass transition temperature, polymer-based MB can be mechanically stretched to obtain an anisotropic shape, endowing them with unique features for US-mediated blood-brain barrier (BBB) permeation. It is here shown that nonspherical MB can be surface-modified with BBB-specific targeting ligands, thereby promoting binding to and sonopermeation of blood vessels in the brain. Actively targeted rod-shaped MB are generated via 1D stretching of spherical poly(butyl cyanoacrylate) MB and via subsequently functionalizing their shell with antitransferrin receptor (TfR) antibodies. Using US and optical imaging, it is demonstrated that nonspherical anti-TfR-MB bind more efficiently to BBB endothelium than spherical anti-TfR-MB, both in vitro and in vivo. BBB-associated anisotropic MB produce stronger cavitation signals and markedly enhance BBB permeation and delivery of a model drug as compared to spherical BBB-targeted MB. These findings exemplify the potential of antibody-modified nonspherical MB for targeted and triggered drug delivery to the brain.

Subcellular trafficking and transcytosis efficacy of different receptor types for therapeutic antibody delivery at the blood‒brain barrier.

Here, we report an experimental setup to benchmark different receptors for targeted therapeutic antibody delivery at the blood-brain barrier. We used brain capillary endothelial-like cells derived from induced pluripotent stem cells (hiPSC-BECs) as a model system and compared them to colon epithelial Caco-2 cells. This approach helped to identify favourable receptors for transport into the cell layer itself or for directing transport for transcytosis across the cell layer. The sorting receptors transferrin receptor and sortilin were shown to be efficient as antibody cargo receptors for intracellular delivery to the cell layer. In contrast, the cell surface receptors CD133 and podocalyxin were identified as static and inefficient receptors for delivering cargo antibodies. Similar to in vivo studies, the hiPSC-BECs maintained detectable transcytotic transport via transferrin receptor, while transcytosis was restricted using sortilin as a cargo receptor. Based on these findings, we propose the application of sortilin as a cargo receptor for delivering therapeutic antibodies into the brain microvascular endothelium.

TFEB regulates cellular labile iron and prevents ferroptosis in a TfR1-dependent manner.

Autophagy is a major clearance pathway for misfolded α-synuclein which promotes ferroptosis through NCOA4-mediated ferritin degradation. The regulation of these two processes to achieve improved neuroprotection in Parkinson's disease (PD) must be elucidated. Transcription factor EB (TFEB) is a master regulator of both autophagy and lysosome biogenesis, and lysosomes are important cellular iron storage organelles; however, the role of TFEB in ferroptosis and iron metabolism remains unclear. In this study, TFEB overexpression promoted the clearance of misfolded α-synuclein and prevented ferroptosis and iron overload. TFEB overexpression up-regulated transferrin receptor 1 (TfR1) synthesis and increased the localization of TfR1 in the lysosome, facilitating lysosomal iron import and transient lysosomal iron storage. TFEB overexpression increased the levels of cellular iron-safe storage proteins (both ferritin light and heavy chains). These functions in iron metabolism maintain the cellular labile iron at a low level and electrical activity, even under iron overload conditions. Notably, lower levels of cellular labile iron and the upregulation of ferritin light and heavy chains were reversed after TfR1 knockdown in cells overexpressing TFEB, indicating that TFEB regulates cellular labile iron and suppresses ferroptosis in a TfR1 dependent manner. Taken together, this evidence of the regulation of iron metabolism enriches our understanding of the function of TFEB. In addition, TFEB overexpression protects against ferroptosis and iron overload and provides a new direction and perspective for autophagy regulation in PD.

Characterization of fifteen key genes involved in iron metabolism and their responses to dietary iron sources in yellow catfish Pelteobagrus fulvidraco.

BACKGROUND: Iron is an essential metal element for organisms, whose metabolism is regulated by many genes and also dietary iron sources. However, the characterization, distribution and the responses of iron metabolism-related genes to different iron sources were not clear in fish. METHODS: The full-length cDNA sequences of fifteen iron metabolism-relevant genes (tf, tfr1, hp, fpn1, ho1, ho2, tfr2, hjv, hepcidin, fth, ftl, ftm, irp1, irp2 and hif2α.) were obtained via 3' and 5' RACE PCR from yellow catfish, a widely distributed freshwater teleost in China and other Asian countries. Their molecular characterizations were analyzed via the bioinformatic methods. Real-time quantitative PCR was used to explore their mRNA distribution in nine tissues. Their mRNA expression responses in four tissues (heart, brain, kidney and gill) were explored in yellow catfish fed diets with five iron sources, including ferrous sulfate (FeSO4), ferrous bisglycinate (Fe-Gly), ferrous chloride (FeCl2), ferric citrate (Fe-CA) and ferric oxide nanoparticles (Fe2O3NPs). RESULTS: Compared with mammals and other teleost, these members shared similar domains. Their mRNAs were expressed in nine tested tissues, but mRNA levels varied. Yellow catfish fed the diets containing Fe-Gly and Fe2O3NPs had higher iron contents in heart, brain, kidney and gill. Meantime, different dietary iron sources addition affected their mRNA expression differentially in brain, heart, kidney and gill. It should be pointed out that only three biological replicate tanks were used in the present feeding treatment, and more biological replicate tanks (more than five) should be emphasized in further researches. CONCLUSION: Taken together, our study identified fifteen iron metabolism-relevant genes, explored their mRNA expression in nine tissues, and their mRNA expression in the responses to different dietary iron sources in four tissues, indicating their important regulatory function in iron metabolism and homeostasis.

A Perfused In Vitro Human iPSC-Derived Blood-Brain Barrier Faithfully Mimics Transferrin Receptor-Mediated Transcytosis of Therapeutic Antibodies.

Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood-brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm2), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.

Cis-monounsaturated fatty acids inhibit ferroptosis through downregulation of transferrin receptor 1.

Ferroptosis, a form of cell death mediated by lipid peroxidation, is implicated in various pathological processes. Although monounsaturated fatty acids (MUFAs) can inhibit ferroptotic lipid peroxidation, the underlying structural mechanism of this antagonistic effect remains poorly understood. We hypothesized that MUFAs with different structures (including chain length, conformation, and double bond position) may affect their regulatory effect on ferroptosis. In this study, 11 MUFAs with varying structures were screened to identify those with an inhibitory effect on ferroptosis. Results from 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide assays indicated that only exogenous MUFAs with cis-conformation and centered double bond could inhibit ferroptosis. Meanwhile, it was found that suppressing the expression of SCD1 and SCD5 genes could sensitize cells to ferroptosis indicating the protective role of endogenous MUFA against ferroptosis. Additionally, western blot analysis revealed that cis-MUFAs with centered double bond downregulated the protein levels of transferrin receptor 1. Flow cytometry confirmed that these MUFAs led to decreases in intracellular iron, reactive oxygen species, and lipid peroxides. It was also found that SCD1 inhibitor could enhance ferroptosis inducer-mediated tumor suppression both in vivo and in vitro. Overall, these findings shed light on the particular structural features of MUFAs that contribute to their ferroptosis-resistant properties and suggest the potential therapeutic relevance of natural MUFAs in a range of ferroptosis-related diseases.

Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody.

BACKGROUND: Transferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood-brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aß-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6. METHODS: Young (3-5 months) and aged (17-20 months) WT and tg-ArcSwe mice (AD model) were injected with 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice. RESULTS: The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6. CONCLUSION: Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aß antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aß-pathology did not influence the early brain distribution of the bispecific antibody. In summary, age and bispecific antibody dose were important factors determining brain delivery, while genotype was not.

A temporal difference in the stabilization of two mRNAs with a 3' iron-responsive element during iron deficiency.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) maintain cellular iron homeostasis and coordinate it with metabolism and possibly cellular behavior. The mRNA encoding transferrin receptor-1 (TFRC, TfR1), which is a major means of iron importation, has five IREs within its 3' UTR, and IRP interactions help maintain cytosolic iron through the protection of the TfR1 mRNA from degradation. An IRE within the 3' UTR of an mRNA splice variant encoding human cell division cycle 14A (CDC14A) has the potential to coordinate the cellular iron status with cellular behavior through a similar IRP-mediated mechanism. However, the stability of the CDC14A splice variant was reported earlier to be unaffected by the cellular iron status, which suggested that the IRE is not functional. We labeled newly synthesized mRNA in HEK293 cells with 5-ethynyl uridine and found that the stability of the CDC14A variant is responsive to iron deprivation, but there are two major differences from the regulation of TfR1 mRNA stability. First, the decay of the CDC14A mRNA does not utilize the Roquin-mediated reaction that acts on the TfR1 mRNA, indicating that there is flexibility in the degradative machinery antagonized by the IRE-IRP interactions. Second, the stabilization of the CDC14A mRNA is delayed relative to the TfR1 mRNA and does not occur until IRP binding activity has been induced. The result is consistent with a hierarchy of IRP interactions in which the maintenance of cellular iron through the stabilization of the TfR1 mRNA is initially prioritized.

Influenza A virus exploits transferrin receptor recycling to enter host cells.

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

Dysregulation of iron homeostasis by TfR-1 renders EZH2 wild type diffuse large B-cell lymphoma resistance to EZH2 inhibition.

EZH2 has been regarded as an efficient target for diffuse large B-cell lymphoma (DLBCL), but the clinical benefits of EZH2 inhibitors (EZH2i) are limited. To date, only EPZ-6438 has been approved by FDA for the treatment of follicular lymphoma and epithelioid sarcoma. We have discovered a novel EZH1/2 inhibitor HH2853 with a better antitumor effect than EPZ-6438 in preclinical studies. In this study we explored the molecular mechanism underlying the primary resistance to EZH2 inhibitors and sought for combination therapy strategy to overcome it. By analyzing EPZ-6438 and HH2853 response profiling, we found that EZH2 inhibition increased intracellular iron through upregulation of transferrin receptor 1 (TfR-1), ultimately triggered resistance to EZH2i in DLBCL cells. We demonstrated that H3K27ac gain by EZH2i enhanced c-Myc transcription, which contributed to TfR-1 overexpression in insensitive U-2932 and WILL-2 cells. On the other hand, EZH2i impaired the occurrence of ferroptosis by upregulating the heat shock protein family A (Hsp70) member 5 (HSPA5) and stabilizing glutathione peroxidase 4 (GPX4), a ferroptosis suppressor; co-treatment with ferroptosis inducer erastin effectively overrode the resistance of DLBCL to EZH2i in vitro and in vivo. Altogether, this study reveals iron-dependent resistance evoked by EZH2i in DLBCL cells, and suggests that combination with ferroptosis inducer may be a promising therapeutic strategy.