Systematic Modeling, Prediction, and Comparison of Domain–Peptide Affinities: Does it Work Effectively With the Peptide QSAR Methodology?

The protein–protein association in cellular signaling networks (CSNs) often acts as weak, transient, and reversible domain–peptide interaction (DPI), in which a flexible peptide segment on the surface of one protein is recognized and bound by a rigid peptide-recognition domain from another. Reliable modeling and accurate prediction of DPI binding affinities would help to ascertain the diverse biological events involved in CSNs and benefit our understanding of various biological implications underlying DPIs. Traditionally, peptide quantitative structure-activity relationship (pQSAR) has been widely used to model and predict the biological activity of oligopeptides, which employs amino acid descriptors (AADs) to characterize peptide structures at sequence level and then statistically correlate the resulting descriptor vector with observed activity data via regression. However, the QSAR has not yet been widely applied to treat the direct binding behavior of large-scale peptide ligands to their protein receptors. In this work, we attempted to clarify whether the pQSAR methodology can work effectively for modeling and predicting DPI affinities in a high-throughput manner? Over twenty thousand short linear motif (SLiM)-containing peptide segments involved in SH3, PDZ and 14-3-3 domain-medicated CSNs were compiled to define a comprehensive sequence-based data set of DPI affinities, which were represented by the Boehringer light units (BLUs) derived from previous arbitrary light intensity assays following SPOT peptide synthesis. Four sophisticated MLMs (MLMs) were then utilized to perform pQSAR modeling on the set described with different AADs to systematically create a variety of linear and nonlinear predictors, and then verified by rigorous statistical test. It is revealed that the genome-wide DPI events can only be modeled qualitatively or semiquantitatively with traditional pQSAR strategy due to the intrinsic disorder of peptide conformation and the potential interplay between different peptide residues. In addition, the arbitrary BLUs used to characterize DPI affinity values were measured via an indirect approach, which may not very reliable and may involve strong noise, thus leading to a considerable bias in the modeling. The Rprd2 = 0.7 can be considered as the upper limit of external generalization ability of the pQSAR methodology working on large-scale DPI affinity data.

Boar spermadhesin AWN: Novel insights in its binding behavior and localization on sperm

As a major spermadhesin first found in the seminal plasma of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN in/on epididymal, ejaculated, capacitated and acrosome-reacted boar sperm using epifluorescence and electron microscopy, as well as an analysis of potential lipid binding partners of native and recombinant AWN. By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment of ejaculated, capacitated and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane, and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the equatorial segment during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine and in line with our previous study describing an interaction with phosphatidic acid, the current results show a rather electrostatically-driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the equatorial segment that suggest a possible role in sperm-oocyte fusion.

Repositioning of Etravirine as a Potential CK1ε Inhibitor by Virtual Screening

CK1ε is a key regulator of WNT/β-catenin and other pathways that are linked to tumor progression; thus, CK1ε is considered a target for the development of antineoplastic therapies. In this study, we performed a virtual screening to search for potential CK1ε inhibitors. First, we characterized the dynamic noncovalent interactions profiles for a set of reported CK1ε inhibitors to generate a pharmacophore model, which was used to identify new potential inhibitors among FDA-approved drugs. We found that etravirine and abacavir, two drugs that are approved for HIV infections, can be repurposed as CK1ε inhibitors. The interaction of these drugs with CK1ε was further examined by molecular docking and molecular dynamics. Etravirine and abacavir formed stable complexes with the target, emulating the binding behavior of known inhibitors. However, only etravirine showed high theoretical binding affinity to CK1ε. Our findings provide a new pharmacophore for targeting CK1ε and implicate etravirine as a CK1ε inhibitor and antineoplastic agent.

An Evolutionary Perspective on Hox Binding Site Preferences in Two Different Tissues

Transcription factor (TF) networks define the precise development of multicellular organisms. While many studies focused on TFs expressed in specific cell types to elucidate their contribution to cell specification and differentiation, it is less understood how broadly expressed TFs perform their precise functions in the different cellular contexts. To uncover differences that could explain tissue-specific functions of such TFs, we analyzed here genomic chromatin interactions of the broadly expressed Drosophila Hox TF Ultrabithorax (Ubx) in the mesodermal and neuronal tissues using bioinformatics. Our investigations showed that Ubx preferentially interacts with multiple yet tissue-specific chromatin sites in putative regulatory regions of genes in both tissues. Importantly, we found the classical Hox/Ubx DNA binding motif to be enriched only among the neuronal Ubx chromatin interactions, whereas a novel Ubx-like motif with rather low predicted Hox affinities was identified among the regions bound by Ubx in the mesoderm. Finally, our analysis revealed that tissues-specific Ubx chromatin sites are also different with regards to the distribution of active and repressive histone marks. Based on our data, we propose that the tissue-related differences in Ubx binding behavior could be a result of the emergence of the mesoderm as a new germ layer in triploblastic animals, which might have required the Hox TFs to relax their binding specificity.

Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

<b><i>Introduction:</i></b> As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. <b><i>Methods:</i></b> In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. <b><i>Results:</i></b> We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. <b><i>Conclusions:</i></b> Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens’ antigens.

Interaction of proteins and amino acids with iso-α-acids during wort preparation in the brewhouse

This paper investigates the binding behavior of iso-α-acids from hops on free wort amino acids and proteins concerning the wort production process in breweries. The studies were carried out with different amino acids, bovine serum albumin and wort. To identify the nature of reaction between iso-α-acids and these substances, analyses of free amino nitrogen, HPLC and isothermal titration calorimetry were performed. According to the results, the iso-α-acids do not form covalent bonds with free amino acids of wort. However, iso-α-acids, especially isohumulone and isoadhumulone, form ionic bonds with wort proteins. A distinction must be made between proteins that are present in the hot trub, and those that are still dissolved in the hot wort. Proteins that are already coagulated and precipitated no longer react with iso-α-acids. Future experiments will investigate whether the established ionic bonds between iso-α-acids and proteins from the wort preparation process are maintained during fermentation until the finished beer or beer foam. If this is the case, which is induced by the experiments, there is a measurable loss of iso-α-acids in the hot wort, but at the same time, a gain for the later beer foam retention, as the iso-α-acids will stabilize it.

Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

New Carbonic Anhydrase-II Inhibitors from Marine Macro Brown Alga Dictyopteris hoytii Supported by In Silico Studies

In continuation of phytochemical investigations of the methanolic extract of Dictyopteris hoytii, we have obtained twelve compounds (1–12) through column chromatography. Herein, three compounds, namely, dimethyl 2-bromoterepthalate (3), dimethyl 2,6-dibromoterepthalate (4), and (E)-3-(4-(dimethoxymethyl)phenyl) acrylic acid (5) are isolated for the first time as a natural product, while the rest of the compounds (1, 2, 6–12) are known and isolated for the first time from this source. The structures of the isolated compounds were elucidated by advanced spectroscopic 1D and 2D NMR techniques including 1H, 13C, DEPT, HSQC, HMBC, COSY, NEOSY, and HR-MS and comparison with the reported literature. Furthermore, eight compounds (13–20) previously isolated by our group from the same source along with the currently isolated compounds (1–12) were screened against the CA-II enzyme. All compounds, except 6, 8, 14, and 17, were evaluated for in vitro bovine carbonic anhydrase-II (CA-II) inhibitory activity. Eventually, eleven compounds (1, 4, 5, 7, 9, 10, 12, 13, 15, 18, and 19) exhibited significant inhibitory activity against CA-II with IC50 values ranging from 13.4 to 71.6 μM. Additionally, the active molecules were subjected to molecular docking studies to predict the binding behavior of those compounds. It was observed that the compounds exhibit the inhibitory potential by specifically interacting with the ZN ion present in the active site of CA-II. In addition to ZN ion, two residues (His94 and Thr199) play an important role in binding with the compounds that possess a carboxylate group in their structure.