FerryCalin: an Engineered Lipocalin Protein Directed against the Transferrin Receptor with Potential for Brain Drug Delivery

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin variants were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin ⋅ Fe³⁺), among those an Anticalin – dubbed FerryCalin – exhibiting a dissociation constant (K_D) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to the fast k_on rate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.     

Chorioamnionitis Precipitates Perinatal Alterations of Heme-Oxygenase-1 (HO-1) Homeostasis in the Developing Rat Brain

Chorioamnionitis (CHORIO), placental insufficiency, and preterm birth are well-known antecedents of perinatal brain injury (PBI). Heme-oxygenase-1 (HO-1) is an important inducible enzyme in oxidative and inflammatory conditions. In the brain, HO-1 and the iron regulatory receptor, transferrin receptor-1 (TfR1), are known to be involved in iron homeostasis, oxidative stress, and cellular adaptive mechanisms. However, the role of HO pathway in the pathophysiology of PBI has not been previously studied. In this study, we set out to define the ontogeny of the HO pathway in the brain and determine if CHORIO changed its normal developmental regulation. We also aimed to determine the role of HO-1/TfR1 in CHORIO-induced neuroinflammation and peripheral inflammation in a clinically relevant rat model of PBI. We show that HO-1, HO-2, and TfR1 expression are developmentally regulated in the brain during the perinatal period. CHORIO elevates HO-1 and TfR1 mRNA expression in utero and in the early postnatal period and results in sustained increase in HO-1/TfR1 ratios in the brain. This is associated with neuroinflammatory and peripheral immune phenotype supported by a significant increase in brain mononuclear cells and peripheral blood double negative T cells suggesting a role of HO-1/TfR1 pathway dysregulation in CHORIO-induced neuroinflammation.     

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.     

Magnetospirillum magneticum as a Living Iron Chelator Induces TfR1 Upregulation and Decreases Cell Viability in Cancer Cells

Interest has grown in harnessing biological agents for cancer treatment as dynamic vectors with enhanced tumor targeting. While bacterial traits such as proliferation in tumors, modulation of an immune response, and local secretion of toxins have been well studied, less is known about bacteria as competitors for nutrients. Here, we investigated the use of a bacterial strain as a living iron chelator, competing for this nutrient vital to tumor growth and progression. We established an in vitro co-culture system consisting of the magnetotactic strain Magnetospirillum magneticum AMB-1 incubated under hypoxic conditions with human melanoma cells. Siderophore production by 10⁸ AMB-1/mL in human transferrin (Tf)-supplemented media was quantified and found to be equivalent to a concentration of 3.78 µM ± 0.117 µM deferoxamine (DFO), a potent drug used in iron chelation therapy. Our experiments revealed an increased expression of transferrin receptor 1 (TfR1) and a significant decrease of cancer cell viability, indicating the bacteria’s ability to alter iron homeostasis in human melanoma cells. Our results show the potential of a bacterial strain acting as a self-replicating iron-chelating agent, which could serve as an additional mechanism reinforcing current bacterial cancer therapies.     

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.     

Multivalent Display and Receptor‐Mediated Endocytosis of Transferrin on Virus‐Like Particles

The structurally regular and stable self‐assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell‐ and tissue‐targeting molecules and therapeutic agents in a convenient and well‐defined manner. The human iron‐transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qβ. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide‐functionalized Qβ capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qβ particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin‐mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP‐labeled clathrin light chains. By testing Qβ particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.     

Mechanism‐Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches

After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism‐guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo‐Shine‐Dalgarno (SD) sequence located near the 3′‐end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamer's P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions.     

Theranostic Polyaminocarboxylate–Cyanine–Transferrin Conjugate for Anticancer Therapy and Near‐Infrared Optical Imaging

Iron chelation therapy has been recognized as a promising antitumor therapeutic strategy. Herein we report a novel theranostic agent for targeted iron chelation therapy and near‐infrared (NIR) optical imaging of cancers. The theranostic agent was prepared by incorporation of a polyaminocarboxylate‐based cytotoxic chelating agent (N‐NE3TA; 7‐[2‐[(carboxymethyl)amino]ethyl]‐1,4,7‐triazacyclononane‐1,4‐diacetic acid) and a NIR fluorescent cyanine dye (Cy5.5) onto a tumor‐targeting transferrin (Tf). The N‐NE3TA–Tf conjugate (without Cy5.5) was characterized and evaluated for antiproliferative activity in HeLa, HT29, and PC3 cancer cells, which have elevated expression levels of the transferrin receptor (TfR). The N‐NE3TA–Tf conjugate displayed significant inhibitory activity against all three cancer cell lines. The NIR dye Cy5.5 was then incorporated into N‐NE3TA–Tf, and the resulting cytotoxic and fluorescent transferrin conjugate N‐NE3TA–Tf–Cy5.5 was shown by microscopy to enter TfR‐overexpressing cancer cells. This theranostic conjugate has potential application for dual use in targeted iron chelation cancer therapy and NIR fluorescence imaging.     

In vitro and in vivo ligand binding to the 5HT(3) serotonin receptor characterised by time-resolved fluorescence spectroscopy

The binding of the fluorescein-labelled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilised ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT(3)R), was characterised by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was observed for the GR-flu-receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by determining the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation experiments indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, we succeeded in measuring the binding of GR-flu to 5HT(3) receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomolecular interactions in live cells.     

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a β chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX ( S ystematic E volution of L igands by Ex ponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (K_D, 80–510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381–393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524–543) and C-Met (557–568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.     

Methods and Applications of In Silico Aptamer Design and Modeling

Aptamers are nucleic acid analogues of antibodies with high affinity to different targets, such as cells, viruses, proteins, inorganic materials, and coenzymes. Empirical approaches allow the design of in vitro aptamers that bind particularly to a target molecule with high affinity and selectivity. Theoretical methods allow significant expansion of the possibilities of aptamer design. In this study, we review theoretical and joint theoretical-experimental studies dedicated to aptamer design and modeling. We consider aptamers with different targets, such as proteins, antibiotics, organophosphates, nucleobases, amino acids, and drugs. During nucleic acid modeling and in silico design, a full set of in silico methods can be applied, such as docking, molecular dynamics (MD), and statistical analysis. The typical modeling workflow starts with structure prediction. Then, docking of target and aptamer is performed. Next, MD simulations are performed, which allows for an evaluation of the stability of aptamer/ligand complexes and determination of the binding energies with higher accuracy. Then, aptamer/ligand interactions are analyzed, and mutations of studied aptamers made. Subsequently, the whole procedure of molecular modeling can be reiterated. Thus, the interactions between aptamers and their ligands are complex and difficult to understand using only experimental approaches. Docking and MD are irreplaceable when aptamers are studied in silico.     

Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.     

Mechanistic basis for RNA aptamer-based induction of TetR

The TetR aptamer induces TetR controlled gene expression, and represents an interesting tool for application in synthetic biology. We have analysed the mechanistic basis for RNA aptamer-based induction of TetR. The aptamer binds TetR with a high affinity in the order of 10(7) M(-1), which is similar to operator DNA binding under the used ionic conditions. We identified the binding epitope of the aptamer on TetR, which consists of amino acids T27, N47 and K48 of both monomers, using loss-of-function analysis and electrophoretic mobility shift assays. Tetracycline-induced conformational changes of TetR led to reorientation of the DNA reading head. This movement destroys the composite binding epitope for the aptamer and leads to reduced RNA binding by one order of magnitude. The aptamer can actively displace TetR from the operator DNA; this could be the key factor for its activity in vivo.     

On Iron Metabolism and Its Regulation

Iron is a critical metal for several vital biological processes. Most of the body’s iron is bound to hemoglobin in erythrocytes. Iron from senescent red blood cells is recycled by macrophages in the spleen, liver and bone marrow. Dietary iron is taken up by the divalent metal transporter 1 (DMT1) in enterocytes and transported to portal blood via ferroportin (FPN), where it is bound to transferrin and taken up by hepatocytes, macrophages and bone marrow cells via transferrin receptor 1 (TfR1). While most of the physiologically active iron is bound hemoglobin, the major storage of most iron occurs in the liver in a ferritin-bound fashion. In response to an increased iron load, hepatocytes secrete the peptide hormone hepcidin, which binds to and induces internalization and degradation of the iron transporter FPN, thus controlling the amount of iron released from the cells into the blood. This review summarizes the key mechanisms and players involved in cellular and systemic iron regulation.     

Stimuli-Responsive and Reversible Nanoassemblies of G-Triplexes

G-triplex (G3) structures formed with three consecutive G-tracts have recently been identified as a new emerging guanine-rich DNA fold. There could likely be a wide range of biological functions for G3s as occurring for G-quadruplex (G4) structures formed with four consecutive G-tracts. However, in comparison to the many reports on G4 nanoassemblies that organize monomers together in a controllable manner, G3-favored nanoassemblies have yet to be explored. In this work, we found that a natural alkaloid of sanguinarine can serve as a dynamic ligand glue to reversibly switch the dimeric nanoassemblies of the thrombin binding aptamer G3 (TBA-G3). The glue planarity was considered to be a crucial factor for realizing this switching. More importantly, external stimuli including pH, sulfite, O₂ and H₂O₂ can be employed as common regulators to easily modulate the glue's adhesivity for constructing and destructing the G3 nanoassemblies as a result of the ligand converting between isoforms. However, this assembly behavior does not occur with the counterpart TBA-G4. Our work demonstrates that higher-order G3 nanoassemblies can be reversibly operated by manipulating ligand adhesivity. This provides an alternative understanding of the unique behavior of guanine-rich sequences and focuses attention on the G3 fold since the nanoassembly event investigated herein might occur in living cells.     

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.     

Comparison of Two DNA Aptamers for Dopamine Using Homogeneous Binding Assays

Dopamine is an essential neurotransmitter and its detection is important for bioanalytical chemistry. Two very different DNA aptamers have been reported for dopamine, one derived from an RNA aptamer (named Apt1) and other obtained via direct aptamer selection (named Apt2). In this study, we used four homogeneous binding assays to compare these two DNA dopamine aptamers. Thiazole orange (TO) fluorescence assay indicated that the Apt2 specifically bound with dopamine with a K_d of 2.37 μM, which was consistent with that from the isothermal titration calorimetry (ITC) assay. However, Apt1 had much less TO fluorescence change and also no signal from ITC. By labeling the two ends of the two aptamers by a fluorophore and a quencher, the aptamer beacons showed binding of dopamine only for Apt2. Finally, the label-free AuNP-based colorimetric assay showed no difference between these two aptamer sequences, and even non-binding random DNA showed the same response, indicating that AuNPs were not a good probe for detecting dopamine. According to the data, Apt1 does not appear to be able to bind dopamine specifically, while Apt2 showed specific binding and could be used for developing related biosensors.