Quantitative Analysis Of Interaction Research Articles

Quantitative Analysis of Interaction Between CADM1 and Its Binding Cell-Surface Proteins Using Surface Plasmon Resonance Imaging.

The cell adhesion molecule (CADM) family of the immunoglobulin superfamily (IgSF) comprises four members, CADM1–CADM4, and participates in the formation of epithelial and synaptic adhesion through cell–cell homophilic and heterophilic interactions. To identify the partners that interact with each member of the CADM family proteins, we set up a platform for multiple detection of the extracellular protein–protein interactions using surface plasmon resonance imaging (SPRi) and analyzed the interactions between the CADM family proteins and 10 IgSF of their structurally related cell adhesion molecules. SPRi analysis identified a new interaction between CADM1 and CADM4, where this heterophilic interaction was shown to be involved in morphological spreading of adult T-cell leukemia (ATL) cells expressing CADM1 when incubated on CADM4-coated glass. Moreover, class-I MHC-restricted T-cell-associated molecule (CRTAM) was identified to show the highest affinity to CADM1 among its binding partners by comparing the dissociation constants calculated from the SPR sensorgrams. These results suggest that the SPRi platform would provide a novel screening tool to characterize extracellular protein–protein interactions among cell-surface and secreted proteins, including IgSF molecules.

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases.

Poxvirus subversion of B7-mediated T cell co-stimulation

Abstract Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Our group has been investigating a large sequence-diverse family of secreted poxvirus proteins that appear to share a conserved beta-sandwich fold, but differ in their binding specificities. Host proteins discovered to interact with this viral protein family include: chemokines, GM-CSF, IL-2, MHC class I, and GAGs. We have termed members of this superfamily; Poxvirus Immune Evasion (PIE) proteins, and there appears to be at least 20 distinct subfamilies. As it turns out, cowpox virus (CPXV BR) contains 10 PIE proteins, one of which is secreted from virally infected cells and specifically binds B7.1 (CD80) and B7.2 (CD86), two membrane proteins expressed on professional antigen presenting cells and activated T cells. We have termed this viral protein B7 Response Modifier (BRM) and demonstrated that it competes with the CD28 as well as CTLA4 receptors expressed on T-cells for binding to B7 proteins. However, at low concentration, BRM competes primarily with CD28 for binding to B7.2, consistent with the idea that cowpox uses BRM to undermine T cell activation but not checkpoint control. Quantitative interaction analysis reveals that BRM binds the ectodomain of B7 proteins with higher affinity than that reported for CD28 or CTLA-4. Functionally, BRM can potently disrupt T-cell proliferation and IL2 production co-stimulated by B7 proteins. Furthermore, supernatants of wild-type but not BRM deleted CPXV are capable of suppressing B7.2-mediated T-cell activation. In sum, our findings define a novel mechanism of viral immune evasion that highlights the role of CD28 co-stimulation in host defense of poxvirus infections.

Biophysical Analyses for Probing Glycan-Protein Interactions.

Glycan-protein interactions occur at many physiological events, and the analyses are of considerable importance for understanding glycan-dependent mechanisms. Biophysical approaches including 3D structural analysis are essential for revealing glycan-protein interactions at the atomic level. The inherent diversity of glycans suits them to function as identification tags, e.g., distinguish self from the nonself components of pathogens. However, the complexity of glycans and poor affinities for interaction partners limit the usefulness of conventional analyses. To cope with such troublesome glycans, a logical sequence of biophysical analyses need to be developed. In this chapter, we introduce a workflow of glycan-protein interaction analysis consisting of six steps: preparation of lectin and glycan, screening of glycan ligand, determination of binding epitope, quantitative interaction analysis, 3D structural analysis, and molecular dynamics simulation. Our increasing knowledge and understanding of lectin-glycan interactions will hopefully lead to the design of glyco-based medicines and vaccines.

Oligocarboxylates as useful templates in dynamic combinatorial chemistry

Abstract Dynamic combinatorial chemistry deals with systems (libraries) of multiple compounds formed by reversible reactions. Interactions of all library components with introduced template molecule are reflected in modification of library composition and are analysed simultaneously. In our studies we investigated a single-substrate dynamic combinatorial library of macrocycles, based on dipicolinic acid diamide, with disulphide bond exchange as the reversible reaction. The library components equipped with hydrogen bond donors interact with anionic guests – carboxylates which act as templates inducing amplification of selected library members. We proved that quantitative analysis of interactions with templates is possible, which led us to a novel method of analysis of association constants of static receptors introduced to the system. With a large set of carboxylates differing in number of anionic groups, size, shape, and flexibility of the linker, we proved that the library is very sensitive to structural parameters of the template. We also showed that with mediation of a photoswitchable azobenzene-based template it is possible to change the library composition by light stimulus. Similarly, with mediation of EDTA the library is sensitive to introduction of metal cations.

A surface plasmon resonance-based method for monitoring interactions between G protein-coupled receptors and interacting proteins.

The present protocol describes a method by which interactions between G protein-coupled receptors (GPCR) and intracellular proteins can be monitored in real-time and without the use of exogenous labels. The method is based on surface plasmon resonance (SPR) and uses synthetic peptides as mimics of intracellular GPCR domains. These peptides are covalently immobilized onto sensor chips and brought into contact with putative interacting proteins in the flow cells of the SPR instrument. The method allows flexible experimental designs, rapid testing of hypotheses and quantitative analysis of interactions. Relative to other established methods, it provides both an alternative and a complementary approach with several key advantages. The present protocol describes the method step-by-step, using the interaction between the serotonin 5-HT7 receptor and the calcium-binding protein S100B as an example.

Abstract 86: Live-cell profiling of inhibitors targeting the N/C interaction within the androgen receptor

Abstract Androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render resistance to these treatments. Since intramolecular interactions between the N- and C-terminus of AR are implicated in AR activation, it is essential to understand how AR inhibitors alter the N/C interaction within the AR, both for the wt receptor and for its mutant forms. In this study we applied our recently developed Fluorescent-Two Hybrid (F2H) assay for live-cell analysis of AR N/C interactions. F2H enables real-time visualization and quantitative analysis of interactions between GFP- and RFP- tagged proteins in live mammalian cells. The F2H principle employs a tethering strategy: a GFP-tagged protein is enriched at the protein interaction platform of engineered F2H-BHK cells and serves as bait, whereas an RFP-tagged protein serves as a prey. Here, AR-LBD (wt or mut) was fused to GFP, AR-TAD was fused to RFP, both constructs were co-transfected into the F2H cells, treated with modulatory compounds and the LBD/TAD interactions were evaluated by fluorescence microscopy. With F2H we analyzed the effects of DHT +/- flutamide, abiraterone, bicalutamide and enzalutamide in live cells. As expected, we observed striking differences in the effects of these treatments on the interactions between the AR-TAD and wt or mut AR-LBD (wt vs W741L, F876L, T877A, F876L-T877A). Further, we compared the effects of the selected compounds on AR-TAD and AR-LBD (wt vs mut) interactions in dose-response F2H assays. We also compared kinetics of these inhibitors with real-time imaging in F2H cells. We further validated our F2H findings with cell-lysate-ELISA on GFP-multiTrap plates. The acquired results correlate well with the published proliferation/reporter-assay data and clarify the role of the intramolecular AR N/C interactions in downstream effects. Taken together, our results demonstrate that the fully reversible cellular F2H assay enables side-by-side profiling of new inhibitors and activators of protein-protein interactions with respect to their intracellular activity, cell penetration and kinetics. Citation Format: Larisa Yurlova, Andrea Buchfellner, Jacqueline Gregor, Tina Romer, Ian Hickson. Live-cell profiling of inhibitors targeting the N/C interaction within the androgen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 86. doi:10.1158/1538-7445.AM2015-86

Study on the interaction and relation of society, economy and environment based on PCA–VAR model: As a case study of the Bohai Rim region, China

Ongoing success throughout regional development is contingent on maintaining the function, quality and harmony progress in society, economy and environment domains, so exploring the interaction and relation among them should be considerable significant. The model by coupling principal component analysis and vector autoregressive, which relate the aggregated values and dynamic analysis among factors, is proposed to achieve the qualitative and quantitative analysis of interaction and relation among society, economy and environment subsystem, providing a framework to conceptualize the influences among their changes and simulate the future scenarios in the Bohai Rim region. The impulse response analysis and variance decomposition of vector autoregressive method, in particular, permit dynamic interaction between every two systems and display clear decomposition of contribution for each change, respectively. This study results show that: there is a virtuous circle of promotion between economic growth and social progress no matter which is regarded as the endogenous variable during the study period, and yet the negative effect to environmental changes had been produced; what the improvement of social and environmental situation need most might be advancing its own progress; it is also reflected that the entire complicated system walk on the path of unsustainable development due to the evident disequilibrium of three subsystems; and the scenario analysis results obviously tell that in order to attain coordinated development, the annual growth rate of 12% to 16% of overall environmental level should be anticipated along with 8% of social and economic level improve. The study guide future possibilities for relatively more harmonious interconnections among social, economic and the environmental development.

Design of eye models used in quantitative analysis of interaction between chromatic and higher-order aberrations of eye

Special kinds of eye models are constructed by means of optical system design to quantitatively investigate impacts of longitudinal chromatic aberration (LCA), transverse chromatic aberration (TCA) and LCA+TCA on retina image quality and on depth of focus (DOF), as well as interaction between chromatic and higher-order aberrations. Results show that LCA plays a main role in enhancement of DOF and higher-order aberrations further increase DOF. For most of the eyes the impact of higher-order aberrations on vision is much smaller than that of LCA+TCA and the presence of LCA+TCA further reduces the impact of higher-order aberrations. The impact of LCA approximates to that of LCA+TCA, and the impact of TCA approximates to that of normal level of higher-order aberrations and is negligible.

Quantitative Analysis of Interaction among Employment in China’s Urban Area, Urbanization and Economic Development

Quantitative Analysis of Interaction among Employment in China’s Urban Area, Urbanization and Economic Development

An examination of teacher–student interactions in inclusive classrooms: teacher interviews and classroom observations

Teacher–student interactions in 17 inclusive classrooms were examined using a mixed‐methods approach that involved quantitative analysis of interactions recorded during classroom observations and follow‐up interviews with seven general educators.Observational findings suggest that classrooms were organised along traditional lines with the vast majority of interactions provided by general educators to the whole class, followed by brief intervals of 1 : 1 interactions directed towards individual students, most frequently children with disabilities. Interview findings suggest that teachers were aware of the greater individual attention devoted to students with disabilities and described a number of ways that they adapt their instruction that are consistent with the research on effective teaching. In addition, participants struggled with the dilemma of balancing their attention between students whom they perceived as needing it most and ensuring that the class as a whole made adequate progress. The analysis of both observational and interview data indicates that paraprofessionals were responsible for a large portion of instruction and curricular decision making for children with severe disabilities in these classrooms.

Homogeneous label-free fluorescent assay of small molecule-protein interactions using protein binding-inhibited transcription nanomachine

Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.

Quantitative analysis of dynamic protein–protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.

Quantitative analysis of interaction between domain walls and magnetic nanoparticles

We have studied the potential energy and effective field induced by the presence of a single superparamagnetic particle above a magnetic domain wall in a 5 nm ferromagnetic film (Ms = 800 emu/cm3) with uniaxial crystalline anisotropy (Ku < 107 erg/cm3). The wall width, wall type (head-to-head, Néel, and perpendicular Bloch), film dimensions, particle height, and external applied field are found to affect the performance of particle sensing systems. Results and optimization strategies derived from this model are presented. The calculated change in depinning field (ΔHdp) is compared against experimental data and micromagnetic simulation. This comparison provides justification for further development in terms of integration with micromagnetic simulations.

3P-275 細胞サイズリポソーム内における内封反応システムと脂質膜の相互作用の定量的解析(生命の起源・進化,第46回日本生物物理学会年会)

3P-275 細胞サイズリポソーム内における内封反応システムと脂質膜の相互作用の定量的解析(生命の起源・進化,第46回日本生物物理学会年会)

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.

Kinetics of the interaction of desAABB–fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

ASSESSMENT METHODS FOR IMPACT OF ENVIRONMENTALLY FRIENDLY ENTREPRENEURSHIP AND DRIVING FORCES TO THE QUALITY OF ENVIRONMENT

Environmentally friendly entrepreneurship (further ‐ EFE) is one of the tools for improvement environmental quality. There might be made theoretical approaches, how quantitatively large is the impact of the EFE in changes of the quality of environment. In this field theoretical suggestion is prior to practice, because in the most of countries there is domination of several cases, but it could not be used as overall tendency [8; 10]. Opposite impact to quality of environment might be created by driving forces (driving forces are economical indicators, which stimulate changes in quality of environment) [1] like overcon‐sumption, growth of gross domestic product (further ‐ GDP) etc. The article will highlight three possible approaches: 1) the assessment of EFE and driving forces to environmental quality; 2) quantitative analysis of interaction between environmental pressure (pressure indicators characterized impact of enterprises made to environment) indicators and driving forces indicators. In the second part there will be compared four countries: Baltic states and Sweden and 3) descriptive analysis of interaction between enterprise size and positive arrangements according environmental protection in Latvia in 1999 and 2001. Mainly there will be used the method of mathematical modelling [6] - descriptive, regression and power models and their attributes.

Theoretical analysis of gel electrophoretic data for interaction of lysine rich histone with- supercoiled DNA

We demonstrate the possibility of using gel electrophoresis as a technique for the quantitative analysis of interaction of lysine rich histone with DNA. On the basis of theoretical framework for extended ligand binding to one-dimensional lattices such as DNA we have set up systems of equations which relate the ligand-to-DNA ratio to the observed gel migration distance of the complex. From the analysis of experimental data for gel electrophoresis of supercoiled DNA in the presence of lysine rich histones we have found that the observed variation of electrophoretic mobilities of the histone-DNA complexes at low histone-to-DNA ratios can be described by a non-cooperative binding behaviour. At this limit we have estimated the intrinsic binding constant to be of the order of 10 3 M −1.

A quantitative analysis of interactions between photoreceptors in the salamander (Ambystoma) retina.

A quantitative description of the electrical properties of the photoreceptor layer in the salamander retina was obtained from earlier data on the characteristics of isolated rods and cones and on rod-rod coupling, and from new data on rod-cone and cone-cone coupling and on the rod photocurrent. Injecting -1 nA current into a rod elicits hyperpolarizations of about 20 mV in an adjacent rod and 4 mV in an adjacent cone. Responses of more distant receptors are smaller. Injecting -1 nA into a cone elicits hyperpolarizations of about 4 mV in an adjacent rod and 0.4 mV in a nearby cone. Depolarizing current evokes smaller responses. Assuming, in agreement with anatomical evidence, that each rod is electrically coupled to four rods and to four cones around it, and that there is no direct electrical coupling between cones, we found these results could be predicted from the properties of isolated rods and cones if adjacent rods are coupled by a resistance of 300 M omega and adjacent rods and cones are coupled by a resistance of 5000 M omega. The small cone-cone coupling seen is due to coupling via intervening rods. The two halves of double cones are not electrically coupled. The spectral sensitivity of both halves is a maximum around 620 nm wave-length. The rod photocurrent has been characterized by voltage-clamping rods isolated from the retina. In agreement with Bader, MacLeish & Schwartz (1979) we found the time course of the photocurrent to be approximately independent of voltage between -35 and -85 mV. The voltage responses of rods, single cones and double cones isolated from the retina obey the principle of univariance. Responses of receptors in the retina do not obey univariance. The main deviations from univariance observed can be explained if adjacent rods and cones are coupled by a resistance of 5000 M omega. Our data demonstrate that rod-cone coupling is relatively weak. We simplified our description of the photoreceptor network, by omitting cones, to investigate the spatiotemporal processing that the rod network is capable of. Computer simulations predict, as is found experimentally, that the rod voltage response to a large spot of bright light should show a much more pronounced initial transient hyperpolarization than the response to a small spot of light of the same intensity. This difference is produced by the combination of electrical coupling of the rods with the existence of a voltage-gated current, IA, in the rod membrane.(ABSTRACT TRUNCATED AT 400 WORDS)