Targeted Nanoswitchable Inhibitors of Protein–Protein Interactions Involved in Apoptosis

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein–protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL–Bak and MDM2–p53), but can be also applied to a large pool of therapeutically relevant protein–protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein–protein interface of interest.     

Visualizing the Adenylation Activities and Protein–Protein Interactions of Aryl Acid Adenylating Enzymes

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein–protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein–protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein–protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein–protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.     

Multivalent Ligands with Tailor-Made Anion Binding Motif as Stabilizers of Protein–Protein Interactions

Modulation of protein–protein interactions (PPIs) is essential for understanding and tuning biologically relevant processes. Although inhibitors for PPIs are widely used, the field still lacks the targeted design of stabilizers. Here, we report unnatural stabilizers based on the combination of multivalency effects and the artificial building block guanidiniocarbonylpyrrol (GCP), an arginine mimetic. Unlike other GCP-based ligands that modulate PPIs in different protein targets, only a tetrameric design shows potent activity as stabilizer of the 14-3-3ζ/C-Raf and 14-3-3ζ/Tau complexes in the low-micromolar range. This evidences the role of multivalency for achieving higher specificity in the modulation of PPIs.     

Modulating Protein–Protein Interactions with Visible-Light-Responsive Peptide Backbone Photoswitches

Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of this complex crosstalk has recently attracted attention, even as a novel therapeutic strategy. Herein, we describe the synthesis and characterization of two visible-light-responsive peptide backbone photoswitches based on azobenzene derivatives, to exert optical control over protein–protein interactions (PPI). The novel peptidomimetics undergo fast and reversible isomerization with low photochemical fatigue under alternatively blue-/green-light irradiation cycles. Both bind in the nanomolar range to the protein of interest. Importantly, the best peptidomimetic displays a clear difference between isomers in its protein-binding capacity and, in turn, in its potential to inhibit enzymatic activity through PPI disruption. In addition, crystal structure determination, docking and molecular dynamics calculations allow a molecular interpretation and open up new avenues in the design and synthesis of future photoswitchable PPI modulators.     

Stress-Responsive Expression,Subcellular Localization and Protein–Protein Interactions of the Rice Metacaspase Family

Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.     

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure–activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein–protein interactions.     

Discovery of Small-Molecule Modulators of Protein–RNA Interactions by Fluorescence Intensity-Based Binding Assay

Protein–RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein–RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein–RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin–Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein–microRNA interactions. We clearly showed the structure–activity relationships of this new class of Lin28–let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein–RNA interactions.     

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively.     

Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate–Protein Interactions of Gal-3

The quest for novel natural-like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well-defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin-3 as a model lectin is conducted.     

Prediction of Protein–Protein Interaction with Pairwise Kernel Support Vector Machine

Protein–protein interactions (PPIs) play a key role in many cellular processes. Unfortunately, the experimental methods currently used to identify PPIs are both time-consuming and expensive. These obstacles could be overcome by developing computational approaches to predict PPIs. Here, we report two methods of amino acids feature extraction: (i) distance frequency with PCA reducing the dimension (DFPCA) and (ii) amino acid index distribution (AAID) representing the protein sequences. In order to obtain the most robust and reliable results for PPI prediction, pairwise kernel function and support vector machines (SVM) were employed to avoid the concatenation order of two feature vectors generated with two proteins. The highest prediction accuracies of AAID and DFPCA were 94% and 93.96%, respectively, using the 10 CV test, and the results of pairwise radial basis kernel function are considerably improved over those based on radial basis kernel function. Overall, the PPI prediction tool, termed PPI-PKSVM, which is freely available at http://159.226.118.31/PPI/index.html, promises to become useful in such areas as bio-analysis and drug development.     

Atomic Force Microscopy Study of Protein–Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System

Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 μM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 ± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 ± 0.2 nm, 1.0 ± 0.2 nm and 1.8 ± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1_mon complexes with the heights 2.2 ± 0.2 nm and 2.8 ± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1_mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 ± 1 nm were AFM registered.     

Application of Pharmacological Approaches to Plant–Mammal Interactions

The dominant theory in the field of mammalian herbivore–plant interactions is that intake, and therefore tolerance, of plant secondary metabolites (PSMs) is regulated by mechanisms that reduce absorption and increase detoxification of PSMs. Methods designed by pharmacologists to measure detoxification enzyme activity, metabolite excretion, and most recently, drug absorption, have been successfully applied by ecologists to study PSM intake in a variety of mammalian study systems. Here, we describe several pharmacological and molecular techniques used to investigate the fate of drugs in human that have potential to further advance knowledge of mammalian herbivore–plant interactions.     

Structural characterization of plasma sprayed basalt–SiC glass–ceramic coatings

In the present study, the effect of SiC addition on properties of basalt base glass–ceramic coating was investigated. SiC reinforced glass–ceramic coating was realized by atmospheric air plasma spray coating technique on AISI 1040 steel pre-coated with Ni + 5 wt.%Al bond coat. Composite powder mixture consisted of 10%, 20% and 30% SiC by weight were used for coating treatment. Controlled heat treatment for crystallization was realized on pre-coated samples in argon atmosphere at 800 °C, 900 °C and 1000 °C which determined by differential thermal analysis for 1–4 h in order to obtain to the glass–ceramic structure. Microstructural examination showed that the coating performed by plasma spray coating treatment and crystallized was crack free, homogeneous in macro-scale and good bonded. The hardness of the coated samples changed between 666 ± 27 and 873 ± 32 HV_0.01 depending on SiC addition and crystallization temperature. The more the SiC addition and the higher the treatment temperature, the harder the basalt base SiC reinforced glass–ceramic coating became. X-ray diffraction analysis showed that the coatings include augeite [(CaFeMg)–SiO₃], diopside [Ca(Mg_0.15Fe_0.85)(SiO₃)₂], albite [(Na,Ca)Al(Si,Al)₃O₈], andesine [Na_0.499Ca_0.492(Al_1.488Si_2.506O₈] and moissanite (SiC) phases. EDX analyses support the X-ray diffraction analysis.     

Simulation of Aggregates with Point-Contacting Monomers in the Cluster–Dilute Regime. Part 2: Comparison of Two- and Three-Dimensional Structural Properties as a Function of Fractal Dimension

In the past two decades, several experimental and simulation studies have proposed simple empirical relations between projected two-dimensional (2-D) and three-dimensional (3-D) structural properties of fractal-like aggregates in the cluster–dilute regime. These empirical relations have found extensive use in inferring the 3-D structural properties of aggregates from their projected (i.e., 2-D) properties—measurable from aggregate electron microscopy images. This study probes the limitations and nongeneralizability of these simple and straightforward empirical relationships and proposes replacing them with new empirical formulas. A straightforward empirical relationship for directly determining the 3-D fractal dimension (D_f ) of an aggregate from the knowledge of its 2-D aspect ratio is also identified. These new relationships were derived by comparing the ratios of several 2-D and 3-D structural properties of a statistically significant number of simulated aggregates with point-contacting monomers as a function of their 3-D D_f ranging from 1.0 to 3.0 in increments of 0.1.     

Multi-level Modeling of Silica–Template Interactions During Initial Stages of Zeolite Synthesis

Zeolite synthesis is driven by structure-directing agents, such as tetrapropyl ammonium ions (TPA⁺) for Silicalite-1 and ZSM-5. However, the guiding role of these organic templates in the complex assembly to highly ordered frameworks remains unclear, limiting the prospects for advanced material synthesis. In this work, both static ab initio and dynamic classical modeling techniques are employed to provide insight into the interactions between TPA⁺ and Silicalite-1 precursors. We find that as soon as the typical straight 10-ring channel of Silicalite-1 or ZSM-5 is formed from smaller oligomers, the TPA⁺ template is partially squeezed out of the resulting cavity. Partial retention of the template in the cavity is, however, indispensable to prevent collapse of the channel and subsequent hydrolysis.

Structural and functional characterisation of KNNS–BNKZ lead-free piezoceramics

ABSTRACT

Promising piezoelectric properties have been reported recently for lead-free 0.96(K_0.48Na_0.52Nb_0.95Sb_0.05)–0.04Bi_0.5(Na_0.82K_0.18)_0.5ZrO₃ (KNNS–BNKZ) ceramics. The presence of coexisting rhombohedral and tetragonal phases is thought to play a key role in their functional properties, but a thorough understanding is currently lacking. In this experiment, (1?x)KNNS–(x)BNKZ ceramics with x = 0–0.05 were prepared by the mixed-oxide method. High-resolution synchrotron X-ray powder diffraction (SXPD) measurements reveal that the addition of BNKZ into KNNS ceramics leads to an increase in the rhombohedral–orthorhombic phase transition temperature (T_R?O) and a decrease in the orthorhombic–tetragonal phase transition temperature (T_O?T) leading to orthorhombic–tetragonal and rhombohedral–tetragonal phase coexistence at room temperature for compositions with x = 0.02 and 0.04, respectively. By combining the SXPD results with microstructure, evidence is also found for the occurrence of chemical heterogeneity, which could provide an additional means to control the functional properties. The structural observations are correlated with changes in the dielectric and ferroelectric properties.     

The Shear Stress–Strain Behaviour of Low-modulus Structural Adhesives

The shear stress–strain behaviour of two low-modulus structural adhesives has been measured using the butt-torsion test. The Nadai correction for non-linear shear behaviour is explained as it is necessary to understand how this correction can be applied to butt joints. The results for one adhesive were accurately used to predict the strength of a lap joint, and it was shown that the strength of such a joint can approach that of a conventional, modern, structural epoxy. Structural adhesives are usually reckoned to be those with a high strength (50 MPa and upwards) and (these days), a strain to failure of at least 10% in tension, and which usually have a tensile modulus of 2 GPa or so. However, adhesives which are significantly less stiff, less strong, but much more ductile are entering the ‘structural’ arena. In order to evaluate their effectiveness and use in design, it is necessary to be able to measure accurately their stress–strain behaviour. Two such materials are 3M 9245 Structural Bonding Tape (SBT) and 3M 7838 B/A.     

Interactions between Extractant Molecules: Organic-Phase Thermodynamics of TALSPEAK–MME

To reduce the cost and complexity of separations for closed nuclear fuel cycles, solvent extraction processes based on combined solvating and cation exchange extractants are being considered. One such process, Trivalent Actinide Lanthanide Separations using Phosphorus Extractants and Aqueous Komplexes–Mixed Monofunctional Extractants (TALSPEAK–MME), that combines the neutral extractant Cyanex-923 and cation exchanging extractant 2-ethyl(hexyl)phosphonic acid mono-2-ethyl(hexyl) ester (HEH[EHP]) has shown considerable promise. However, little knowledge of the underlying chemistry of this process has been reported. In this report, ³¹P NMR and FT-IR spectroscopies have been used to investigate organic-phase extractant interactions. A 1:1 adduct between Cyanex-923 and HEH[EHP] has been identified. The equilibrium constant describing the formation of this adduct (log K) has been determined to be between 2.04 and 2.21, signifying relatively weak interactions between the extractants. In parallel, it has been determined that the presence of this adduct does not change the nitric acid extraction mechanism observed by Cyanex-923; its presence merely reduces the free concentration of Cyanex-923 available to extract nitric acid thus slightly reducing the total acid partitioned to the organic phase. These findings were used to calculate an extractant speciation diagram for TALSPEAK–MME, the results of which were used to improve understanding of the metal ion extraction behavior observed in this system.     

The Application of an Emerging Technique for Protein–Protein Interaction Interface Mapping: The Combination of Photo-Initiated Cross-Linking Protein Nanoprobes with Mass Spectrometry

Protein–protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8–Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.