Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/-MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

The AJ072 antibody against the human transferrin receptor labels HeLa cells by surface immunofluorescence

The AJ072 antibody against the human transferrin receptor labels the cell membrane of HeLa cells by surface immunofluorescence; AM236 does not.

Epigenetic upregulation by valproic acid of transferrin receptor 1, and not glucose transporter 1 or CD98hc, at the blood-brain barrier

BackgroundThe objectives of the present study were to investigate whether the expression of transferrin receptor 1 (TfR1), glucose transporter 1 (Glut1), or Cluster of Differentiation 98 Heavy Chain (CD98hc) is epigenetically regulated in brain capillary endothelial cells (BCECs) denoting the blood-brain barrier (BBB).MethodsThe expression of these targets was investigated both in vitro and in vivo following treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Mice were injected intraperitoneally with VPA followed by analysis of isolated brain capillaries, and the capillary depleted brain samples. Brain tissue, isolated brain capillaries, and cultured primary endothelial cells were analyzed by RT-qPCR, immunolabeling and ELISA for expression of TfR1, Glut1 and CD98hc. We also studied the vascular targeting in VPA-treated mice injected with monoclonal anti-transferrin receptor (Ri7) conjugated with 1.4 nm gold nanoparticles. ResultsValidating the effects of VPA on gene transcription in BCECs, transcriptomic analysis identified 24,371 expressed genes, of which 305 were differentially expressed with 192 upregulated and 113 downregulated genes. In vitro using BCECs co-cultured with glial cells, the mRNA expression of Tfrc was significantly higher after VPA treatment for 6 h with its expression returning to baseline after 24 h. Conversely, the mRNA expression of Glut1 and Cd98hc was unaffected by VPA treatment. In vivo, the TfR1 protein expression in brain capillaries increased significantly after treatment with both 100 mg/kg and 400 mg/kg VPA. Conversely, VPA treatment did not increase GLUT1 or CD98hc. Using ICP-MS-based quantification, the brain uptake of nanogold conjugated anti-TfR1 antibodies was non-significant in spite of increased expression of TfR1. ConclusionsWe report that VPA treatment upregulates TfR1 at the BBB both in vivo and in vitro in isolated primary endothelial cells. In contrast, VPA treatment does not influence the expression of GLUT1 and CD98hc. The increase in the overall TfR1 protein expression however does not increase transport of TfR-targeted monoclonal antibody and indicates that targeted delivery using the transferrin receptor should aim for increased mobilization of already available transferrin receptor molecules to improve trafficking through the BBB.

DIAGNOSTIC ACCURACY OF SERUM FERRITIN AND SOLUBLE SERUM TRANSFERRIN RECEPTOR, TAKING BONE MARROW IRON STAIN AS A GOLD STANDARD FOR IRON DEFICIENCY ANEMIA IN HETEROGENOUS GROUP OF PATIENTS

Objective: To determine the diagnostic accuracy of serum ferritin and soluble serum transferrin receptor (sTfR), taking bone marrow iron stain as a gold standard for iron deficiency anaemia in heterogeneous group of patients. Study Design: Cross-sectional diagnostic accuracy study. Place and Duration of Study: Department of Diagnostic, Combined Military Hospital Lahore, from Mar to Aug 2020. Methodology: A total of 55 adult patients, of both genders, undergoing bone marrow examination for any reason were enrolled. Patients with known hemolytic condition (sickle cell anemia, megaloblastic anemia), taking erythropoietin/iron supplements, transfused red cell concentrate (RCC) recently or undergoing chemotherapy were excluded. Age, gender, clinical history and results of bone marrow examination, complete blood count (CBC), serum Ferritin and C-reactive protein (CRP) were recorded. Results: Serum ferritin was found to be less sensitive (28%) but more specific (100%) for reflecting reduced bone marrow iron stores as compared to sTfR (sensitivity: 60%, specificity: 96.6%). sTfR had highest likelihood ratio (15) and diagnostic accuracy (80%). On Receiver Operator Characteristic (ROC) graph Transferrin index (AUC=0.908) showed maximum accuracy, followed by Ferritin (AUC=0.884) and sTfR (AUC=0.879). Conclusion: Serum soluble transferring receptor (sTfR) and transferrin index has advantage over serum ferritin alone in predicting the bone marrow iron stores and differentiating iron deficiency anemia from anemia of chronic disease.

Kinetics of Blood–Brain Barrier Transport of Monoclonal Antibodies Targeting the Insulin Receptor and the Transferrin Receptor

Biologic drugs are large molecule pharmaceuticals that do not cross the blood–brain barrier (BBB), which is formed by the brain capillary endothelium. Biologics can be re-engineered for BBB transport as IgG fusion proteins, where the IgG domain is a monoclonal antibody (MAb) that targets an endogenous BBB transporter, such as the insulin receptor (IR) or transferrin receptor (TfR). The IR and TfR at the BBB transport the receptor-specific MAb in parallel with the transport of the endogenous ligand, insulin or transferrin. The kinetics of BBB transport of insulin or transferrin, or an IRMAb or TfRMAb, can be quantified with separate mathematical models. Mathematical models to estimate the half-time of receptor endocytosis, MAb or ligand exocytosis into brain extracellular space, or receptor recycling back to the endothelial luminal membrane were fit to the brain uptake of a TfRMAb or a IRMAb fusion protein in the Rhesus monkey. Model fits to the data also allow for estimates of the rates of association of the MAb in plasma with the IR or TfR that is embedded within the endothelial luminal membrane in vivo. The parameters generated from the model fits can be used to estimate the brain concentration profile of the MAb over time, and this brain exposure is shown to be a function of the rate of clearance of the antibody fusion protein from the plasma compartment.

Dysregulated Transferrin Receptor Disrupts T Cell Iron Homeostasis to Drive Inflammation in Systemic Lupus Erythematosus

T cells in systemic lupus erythematosus (SLE) exhibit mitochondrial abnormalities including elevated oxidative stress. Because excess iron can promote these phenotypes, we tested iron regulation of SLE T cells. A CRISPR screen identified Transferrin Receptor (CD71) as important for Th1 cells but detrimental for induced regulatory T cells (iTreg). Activated T cells induce CD71 to increase iron uptake, but this was exaggerated in T cells from SLE-prone mice which accumulated iron. Treatment of T cells from SLE-prone mice with CD71 blocking antibody reduced intracellular iron and mTORC1 signaling and restored mitochondrial physiology. While Th1 cells were inhibited, CD71 blockade enhanced iTreg. In vivo this treatment reduced pathology and increased IL-10 in SLE-prone mice. Importantly, disease severity correlated with CD71 expression on SLE patient T cells and blocking CD71 enhanced IL-10 secretion. Excess T cell iron uptake thus contributes to T cell dysfunction and can be targeted to correct SLE-associated pathology.

Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review

Protein nanocages have been studied extensively, due to their unique architecture, exceptional biocompatibility and highly customization capabilities. In particular, ferritin nanocages (FNs) have been employed for the delivery of a vast array of molecules, ranging from chemotherapeutics to imaging agents, among others. One of the main favorable characteristics of FNs is their intrinsic targeting efficiency toward the Transferrin Receptor 1, which is overexpressed in many tumors. Furthermore, genetic manipulation can be employed to introduce novel variants that are able to improve the loading capacity, targeting capabilities and bio-availability of this versatile drug delivery system. In this review, we discuss the main characteristics of FN and the most recent applications of this promising nanotechnology in the field of oncology with a particular emphasis on the imaging and treatment of solid tumors.