Engineered discoidin domain from factor VIII binds αvβ3 integrin with antibody-like affinity

Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to αvβ3 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 Å resolution. We have christened this discoidin domain alternative scaffold the “discobody.”

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Localization of sources of human evoked responses

The evoked response, a signal present in the electro-encephalogram when specific sense modalities are stimulated with brief sensory inputs, has not yet revealed as much about brain function as it apparently promised when first recorded in the late 1940's. One of the problems has been to record the responses at a large number of points on the surface of the head; thus in order to achieve greater spatial resolution than previously attained, a 50-channel recording system was designed to monitor experiments with human visually evoked responses.

Conventional voltage versus time plots of the responses were found inadequate as a means of making qualitative studies of such a large data space. This problem was solved by creating a graphical display of the responses in the form of equipotential maps of the activity at successive instants during the complete response. In order to ascertain the necessary complexity of any models of the responses, factor analytic procedures were used to show that models characterized by only five or six independent parameters could adequately represent the variability in all recording channels.

One type of equivalent source for the responses which meets these specifications is the electrostatic dipole. Two different dipole models were studied: the dipole in a homogeneous sphere and the dipole in a sphere comprised of two spherical shells (of different conductivities) concentric with and enclosing a homogeneous sphere of a third conductivity. These models were used to determine nonlinear least squares fits of dipole parameters to a given potential distribution on the surface of a spherical approximation to the head. Numerous tests of the procedures were conducted with problems having known solutions. After these theoretical studies demonstrated the applicability of the technique, the models were used to determine inverse solutions for the evoked response potentials at various times throughout the responses. It was found that reliable estimates of the location and strength of cortical activity were obtained, and that the two models differed only slightly in their inverse solutions. These techniques enabled information flow in the brain, as indicated by locations and strengths of active sites, to be followed throughout the evoked response.