Comparison of polypeptides that bind the transferrin receptor for targeting gold nanocarriers

The ability to target therapeutic agents to specific tissues is an important element in the development of new disease treatments. The transferrin receptor (TfR) is one potential target for drug delivery, as it expressed on many dividing cells and on brain endothelium, the key cellular component of the blood-brain barrier. The aim of this study was to compare a set of new and previously-described polypeptides for their ability to bind to brain endothelium, and investigate their potential for targeting therapeutic agents to the CNS. Six polypeptides were ranked for their rate of endocytosis by the human brain endothelial cell line hCMEC/D3 and the murine line bEnd.3. One linear polypeptide and two cyclic polypeptides showed high rates of uptake. These peptides were investigated to determine whether serum components, including transferrin itself affected uptake by the endothelium. One of the cyclic peptides was strongly inhibited by transferrin and the other cyclic peptide weakly inhibited. As proof of principle the linear peptide was attached to 2nm glucose coated gold-nanoparticles, and the rate of uptake of the nanoparticles measured in a hydrogel model of the blood-brain barrier. Attachment of the TfR-targeting polypeptide significantly increased the rates of endocytosis by brain endothelium and increased movement of nanoparticles across the cells

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of FeNhTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system

Crystal Structure of DNA Polymerase β with DNA Containing the Base Lesion Spiroiminodihydantoin in a Templating Position

The first high-resolution crystal structure of spiroiminodihydantoin (dSp1) was obtained in the context of the DNA polymerase β active site and reveals two areas of significance. First, the structure verifies the recently determined <i>S</i> configuration at the spirocyclic carbon. Second, the distortion of the DNA duplex is similar to that of the single-oxidation product 8-oxoguanine. For both oxidized lesions, adaptation of the <i>syn</i> conformation results in similar backbone distortions in the DNA duplex. The resulting conformation positions the dSp1 A-ring as the base-pairing face whereas the B-ring of dSp1 protrudes into the major groove

Remote Mutations Induce Functional Changes in Active Site Residues of Human DNA Polymerase β

With the formidable growth in the volume of genetic information, it has become essential to identify and characterize mutations in macromolecules not only to predict contributions to disease processes but also to guide the design of therapeutic strategies. While mutations of certain residues have a predictable phenotype based on their chemical nature and known structural position, many types of mutations evade prediction based on current information. Described in this work are the crystal structures of two cancer variants located in the palm domain of DNA polymerase β (pol β), S229L and G231D, whose biological phenotype was not readily linked to a predictable structural implication. Structural results demonstrate that the mutations elicit their effect through subtle influences on secondary interactions with a residue neighboring the active site. Residues 229 and 231 are 7.5 and 12.5 Å, respectively, from the nearest active site residue, with a β-strand between them. A residue on this intervening strand, M236, appears to transmit fine structural perturbations to the catalytic metal-coordinating residue D256, affecting its conformational stability

Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase β (Pol β) functions in base excision repair to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. To probe the mechanism that may underlie E288K’s loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT because of an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure

Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase β (Pol β) functions in base excision repair to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. To probe the mechanism that may underlie E288K’s loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT because of an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure