Search

Purpose: To investigate of cellular biology and physiology of colon cancer tissues in in vitro requires viable dissociation of single cells. The amount of tissue and dissociation methods can affect the amount of single cell viability. Inadequate initial tissue has negative effects on experiment quality by resulting in insufficient quality and the number of cells. Material and Methods: In the context of this study different-weigh and different-textured colon tumor tissues have been evaluated to emphasize the importance of initial tissue properties during the operation of tissue processing and cell isolation success. Effect of the necrotic areas is also evaluated with the isolated viable cells number and the success of three-dimensional (3D) primary culture. Results: Elevated weight of the tissue resulted with more total isolated cells. Necrotic tissues caused low percentage of viable cells. Since resected tissues were bigger than biopsy samples, resected tissues derived primary 3D culture were succesfully maintained the culture. Conclusion: To conclude, isolated cells from the bigger and non-necrotic tumor tissues showed better growth pattern for 3D cultures. On the other hand, size was found as a crucial parameter for obtaining more viable cancer cells. [ABSTRACT FROM AUTHOR]

The objective of this work is to predict the optimal speed of an aerospace vehicle by aerothermochemical analysis of the hypersonic flow during atmospheric re-entry, out of equilibrium vibrational and chemical behind a detached strong shock. This study focuses on the influence of the ionization process that plays a significant role in the absorption of heat, because the characteristics of hypersonic flows are that molecules behind a strong shock wave become vibrationally excited, partially or completely dissociated and ionized depending on their bond energy, and the velocity of flow. On the other hand, we present the mathematical model that governs the flow of reactive gas mixture out of vibrational and chemical equilibrium that is composed of 79% nitrogen N2 and 21% oxygen O2. Conservation and relaxation equations (chemistryvibration) are presented with particular importance to the expression of source terms. The numerical resolution method used is based on physical modeling, governed by the Euler equations, supplemented by the equations of chemical kinetics using the finite difference method. The results obtained are in good agreement with the specialized literature. [ABSTRACT FROM AUTHOR]

Among neurodegenerative diseases, Alzheimer’s disease (AD) is one of the most grievous disease. The oldest cholinergic hypothesis is used to elevate the level of cognitive impairment and acetylcholinesterase (AChE) comprises the major targeted enzyme in AD. Thus, acetylcholinesterase inhibitors (AChEI) constitutes the essential remedy for the treatment of AD. The study aims to evaluate the interactions between natural molecules and AChE by Surface Plasmon Resonance (SPR). The molecules like alkaloids, polyphenols and substrates of AChE have been considered for the study with a major emphasis on affinity and kinetics. To better understand the activity of small molecules, the investigation is supported by both experimental and theoretical approach such as fluorescence, Circular Dichroism (CD) and molecular docking studies. Amongst the screened ones tannic acid showed promising results compared with others. The methodology followed here have highlighted many molecules with a higher affinity towards AChE and these findings may take lead molecules generated in preclinical studies to treat neurodegenerative diseases. Additionally, we suggest a unique signature for the heterogeneous analyte model using competitive experiments for analyzing simultanous interactions of both the analytes. [ABSTRACT FROM AUTHOR]

Allosteric ligands bind to receptors at sites that are distinct from those endogenous agonists and orthosteric pharmacological agents interact with. Both an allosteric and orthosteric ligand bind simultaneously to the receptor to form a ternary complex, where each ligand influences binding affinity of the other to the receptor, either positively or negatively. Allosteric modulators are an intensively studied group of receptor ligands because of their potentially greater selectivity over orthosteric ligands, with the possibility of fine tuning of the effects of endogenous neurotransmitters and hormones. The affinity of an unlabelled allosteric ligand is commonly estimated by measuring its effects on binding of a radio-labelled orthosteric tracer. This scenario is complicated by many folds when one studies the kinetics of interactions of two allosteric agents, added simultaneously, on binding of an orthosteric tracer. In this paper, we provide, for the first time, theoretical basis for analysis of such complex interactions. We have expanded our analysis to include the possibility of having two allosteric modulators interact with the same or different sites on the receptor. An added value of our analysis is to provide a tool to distinguish between the two situations. Finally, we also modelled binding of two molecules of one allosteric modulator to one receptor. [ABSTRACT FROM AUTHOR]

The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications. [ABSTRACT FROM AUTHOR]

During the entry process, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer undergoes a sequence of conformational changes triggered by both CD4 and coreceptor engagement. Resolving the conformation of these transient entry intermediates has proven challenging. Here, we fine-mapped the antigenicity of entry intermediates induced by increasing CD4 engagement of cell surface–expressed Env. Escalating CD4 triggering led to the sequential adoption of different pre-fusion conformational states of the Env trimer, up to the pre-hairpin conformation, that we assessed for antibody epitope presentation. Maximal accessibility of the coreceptor binding site was detected below Env saturation by CD4. Exposure of the fusion peptide and heptad repeat 1 (HR1) required higher CD4 occupancy. Analyzing the diverse antigenic states of the Env trimer, we obtained key insights into the transitions in epitope accessibility of broadly neutralizing antibodies (bnAbs). Several bnAbs preferentially bound CD4-triggered Env, indicating a potential capacity to neutralize both pre- and post-CD4 engagement, which needs to be explored. Assessing binding and neutralization activity of bnAbs, we confirm antibody dissociation rates as a driver of incomplete neutralization. Collectively, our findings highlight a need to resolve Env conformations that are neutralization-relevant to provide guidance for immunogen development. [ABSTRACT FROM AUTHOR]

The physiochemical determinants of drug-target interactions in the microenvironment of the cell are complex and generally not defined by simple diffusion and intrinsic chemical reactivity. Non-specific interactions of drugs and macromolecules in cells are rarely considered formally in assessing pharmacodynamics. Here, we demonstrate that non-specific interactions lead to very slow incorporation kinetics of DNA binding drugs. We observe a rate of drug incorporation in cell nuclei three orders of magnitude slower than in vitro due to anomalous drug diffusion within cells. This slow diffusion, however, has an advantageous consequence: it leads to virtually irreversible binding of the drug to specific DNA targets in cells. We show that non-specific interactions drive slow drug diffusion manifesting as slow reaction front propagation. We study the effect of non-specific interactions in different cellular compartments by permeabilization of plasma and nuclear membranes in order to pinpoint differential compartment effects on variability in intracellular drug kinetics. These results provide the basis for a comprehensive model of the determinants of intracellular diffusion of small-molecule drugs, their target-seeking trajectories, and the consequences of these processes on the apparent kinetics of drug-target interactions. [ABSTRACT FROM AUTHOR]

Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates. All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions. [ABSTRACT FROM AUTHOR]

There is increasing evidence supporting a role for antibodies in protection against tuberculosis (TB), with functional antibodies being described in the latent state of TB infection. Antibody avidity is an important determinant of antibody-mediated protection. This study characterised the avidity of antibodies against Ag85A, an immunodominant Mycobacterium tuberculosis (M.tb) antigen and constituent of several anti-TB vaccine candidates, in individuals of varied M.tb infection status. Avidity of Ag85A specific antibodies was measured in 30 uninfected controls, 34 individuals with latent TB infection (LTBI) and 75 active pulmonary TB (APTB) cases, employing the more commonly used chaotrope-based dissociation assays, and surface plasmon resonance (SPR). Chaotrope-based assays indicated that APTB was associated with a higher antibody avidity index compared to uninfected controls [adjusted geometric mean ratio (GMR): 1.641, 95% confidence interval (CI): 1.153, 2.337, p = 0.006, q = 0.018] and to individuals with LTBI [adjusted GMR: 1.604, 95% CI: 1.282, 2.006, p < 0.001, q <0.001]. SPR assays showed that APTB was associated with slower dissociation rates, an indication of higher avidity, compared to uninfected controls (adjusted GMR: 0.796, 95% CI: 0.681, 0.932, p = 0.004, q = 0.012) and there was also weak evidence of more avid antibodies in the LTBI compared to the uninfected controls (adjusted GMR: 0.871, 95% CI: 0.763, 0.994, p = 0.041, q = 0.123). We found no statistically significant differences in anti-Ag85A antibody avidity between the APTB and LTBI groups. This study shows that antibodies of increased avidity are generated against a principle vaccine antigen in M.tb infected individuals. It would be important to determine whether TB vaccines are able to elicit a similar response. Additionally, more research is needed to determine whether antibody avidity is important in protection against infection and disease. [ABSTRACT FROM AUTHOR]

The mitotic checkpoint (also called spindle assembly checkpoint) is a signaling pathway that ensures faithful chromosome segregation. Mitotic checkpoint proteins inhibit the anaphase-promoting complex (APC/C) and its activator Cdc20 to prevent precocious anaphase. Checkpoint signaling leads to a complex of APC/C, Cdc20, and checkpoint proteins, in which the APC/C is inactive. In principle, this final product of the mitotic checkpoint can be obtained via different pathways, whose relevance still needs to be fully ascertained experimentally. Here, we use mathematical models to compare the implications on checkpoint response of the possible pathways leading to APC/C inhibition. We identify a previously unrecognized funneling effect for Cdc20, which favors Cdc20 incorporation into the inhibitory complex and therefore promotes checkpoint activity. Furthermore, we find that the presence or absence of one specific assembly reaction determines whether the checkpoint remains functional at elevated levels of Cdc20, which can occur in cancer cells. Our results reveal the inhibitory logics behind checkpoint activity, predict checkpoint efficiency in perturbed situations, and could inform molecular strategies to treat malignancies that exhibit Cdc20 overexpression. [ABSTRACT FROM AUTHOR]

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter’s activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of ‘early’, ‘middle’, and ‘late’ genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene’s transcription as a function of stimulus dose. [ABSTRACT FROM AUTHOR]

Across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. All of these modes of activation rely on naturally evolved binding sites that trigger global conformational changes. Among the enzymes that are of greatest interest for small molecule activation are the seven sirtuin enzymes, nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Here, we introduce a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds. [ABSTRACT FROM AUTHOR]

Within an isogenic population, even in the same extracellular environment, individual cells can exhibit various phenotypic states. The exact role of stochastic gene-state switching regulating the transition among these phenotypic states in a single cell is not fully understood, especially in the presence of positive feedback. Recent high-precision single-cell measurements showed that, at least in bacteria, switching in gene states is slow relative to the typical rates of active transcription and translation. Hence using the lac operon as an archetype, in such a region of operon-state switching, we present a fluctuating-rate model for this classical gene regulation module, incorporating the more realistic operon-state switching mechanism that was recently elucidated. We found that the positive feedback mechanism induces bistability (referred to as deterministic bistability), and that the parameter range for its occurrence is significantly broadened by stochastic operon-state switching. We further show that in the absence of positive feedback, operon-state switching must be extremely slow to trigger bistability by itself. However, in the presence of positive feedback, which stabilizes the induced state, the relatively slow operon-state switching kinetics within the physiological region are sufficient to stabilize the uninduced state, together generating a broadened parameter region of bistability (referred to as stochastic bistability). We illustrate the opposite phenotype-transition rate dependence upon the operon-state switching rates in the two types of bistability, with the aid of a recently proposed rate formula for fluctuating-rate models. The rate formula also predicts a maximal transition rate in the intermediate region of operon-state switching, which is validated by numerical simulations in our model. Overall, our findings suggest a biological function of transcriptional “variations” among genetically identical cells, for the emergence of bistability and transition between phenotypic states. [ABSTRACT FROM AUTHOR]

Single-stranded DNA (ssDNA) is a product of many cellular processes that involve double-stranded DNA, for example during DNA replication and repair, and is formed transiently in many others. Measurement of ssDNA formation is fundamental for understanding many such processes. The availability of a fluorescent biosensor for the determination of single-stranded DNA provides an important route to achieve this. Single-stranded DNA binding proteins (SSBs) protect ssDNA from degradation, but can be displaced to allow processing of the ssDNA. Their tight binding of ssDNA means that they are very good candidates for the development of a biosensor. Previously, the single stranded DNA binding protein from Escherichia coli, labeled with a fluorophore, (DCC-EcSSB) was developed and used for this purpose. However, the multiple binding modes of this protein meant that interpretation of DCC-EcSSB fluorescence was potentially complex in terms of determining the amount of ssDNA. Here, we present an improved biosensor, developed using the tetrameric SSB from Plasmodium falciparum as a new scaffold for fluorophore attachment. Each subunit of this tetrameric SSB was labeled with a diethylaminocoumarin fluorophore at a single site on its surface, such that there is a very large, 20-fold, fluorescence increase when it binds to ssDNA. This adduct can be used as a biosensor to report ssDNA formation. Because SSB from this organism has a single mode of binding ssDNA, namely 65–70 bases per tetramer, over a wide range of conditions, the fluorescent SSB allows simple quantitation of ssDNA. The binding is fast, possibly diffusion controlled, and tight (dissociation constant for DCC-PfSSB <5 pM). Its suitability for real-time assays of ssDNA formation was demonstrated by measurement of AddAB helicase activity, unwinding double-stranded DNA. [ABSTRACT FROM AUTHOR]

Cullin-RING ubiquitin ligases (CRLs) catalyze the ubiquitylation of substrates many of which are degraded by the 26S proteasome. Their modular architecture enables recognition of numerous substrates via exchangeable substrate receptors that competitively bind to a cullin scaffold with high affinity. Due to the plasticity of these interactions there is ongoing uncertainty how cells maintain a flexible CRL repertoire in view of changing substrate loads. Based on a series of in vivo and in vitro studies, different groups proposed that the exchange of substrate receptors is mediated by a protein exchange factor named Cand1. Here, we have performed mathematical modeling to provide a quantitative underpinning of this hypothesis. First we show that the exchange activity of Cand1 necessarily leads to a trade-off between high ligase activity and fast receptor exchange. Supported by measurements we argue that this trade-off yields an optimal Cand1 concentration in cells where the time scale for substrate degradation becomes minimal. In a second step we show through simulations that (i) substrates bias the CRL repertoire leading to preferential assembly of ligases for which substrates are available and (ii) differences in binding affinities or substrate receptor abundances create a temporal hierarchy for the degradation of substrates. Finally, we compare the Cand1-mediated exchange cycle with an alternative architecture lacking Cand1 which indicates superiority of a system with exchange factor if substrate receptors bind substrates and the cullin scaffold in a random order. Together, our results provide general constraints for the operating regimes of molecular exchange systems and suggest that Cand1 endows the CRL network with the properties of an “on demand” system allowing cells to dynamically adjust their CRL repertoire to fluctuating substrate abundances. [ABSTRACT FROM AUTHOR]

A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca2+) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca2+/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca2+ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca2+/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca2+/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca2+, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca2+ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the in vivo frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca2+/CaM-dependent protein kinase II in knockout models of neurogranin. We conclude that competitive tuning could be an important dynamic process underlying synaptic plasticity. [ABSTRACT FROM AUTHOR]

Background: Despite considerable investigational efforts, no method to overcome the pathogenesis caused by loss of function (LoF) mutations in tumor suppressor genes has been successfully translated to the clinic. The most frequent LoF mutation in human cancers is Adenomatous polyposis coli (APC), causing aberrant activation of the Wnt pathway. In nearly all colon cancer tumors, the APC protein is truncated, but still retains partial binding abilities. Objective & methods: Here, we tested the hypothesis that extracellular inhibitors of the Wnt pathway, although acting upstream of the APC mutation, can restore normal levels of pathway activity in colon cancer cells. To this end, we developed and simulated a mathematical model for the Wnt pathway in different APC mutants, with or without the effects of the extracellular inhibitors, Secreted Frizzled-Related Protein1 (sFRP1) and Dickhopf1 (Dkk1). We compared our model predictions to experimental data in the literature. Results: Our model accurately predicts T-cell factor (TCF) activity in mutant cells that vary in APC mutation. Model simulations suggest that both sFRP1 and DKK1 can reduce TCF activity in APC1638N/1572T and Apcmin/min mutants, but restoration of normal activity levels is possible only in the former. When applied in combination, synergism between the two inhibitors can reduce their effective doses to one-fourth of the doses required under single inhibitor application. Overall, re-establishment of normal Wnt pathway activity is predicted for every APC mutant in whom TCF activity is increased by up to 11 fold. Conclusions: Our work suggests that extracellular inhibitors can effectively restore normal Wnt pathway activity in APC-truncated cancer cells, even though these LoF mutations occur downstream of the inhibitory action. The insufficient activity of the truncated APC can be quantitatively balanced by the upstream intervention. This new concept of upstream intervention to control the effects of downstream mutations may be considered also for other partial LoF mutations in other signaling pathways. [ABSTRACT FROM AUTHOR]

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action. [ABSTRACT FROM AUTHOR]

In developing tissues, cell polarization and proliferation are regulated by morphogens and signaling pathways. Cells throughout the Drosophila wing primordium typically show subcellular localization of the unconventional myosin Dachs on the distal side of cells (nearest the center of the disc). Dachs localization depends on the spatial distribution of bonds between the protocadherins Fat (Ft) and Dachsous (Ds), which form heterodimers between adjacent cells; and the Golgi kinase Four-jointed (Fj), which affects the binding affinities of Ft and Ds. The Fj concentration forms a linear gradient while the Ds concentration is roughly uniform throughout most of the wing pouch with a steep transition region that propagates from the center to the edge of the pouch during the third larval instar. Although the Fj gradient is an important cue for polarization, it is unclear how the polarization is affected by cell division and the expanding Ds transition region, both of which can alter the distribution of Ft-Ds heterodimers around the cell periphery. We have developed a computational model to address these questions. In our model, the binding affinity of Ft and Ds depends on phosphorylation by Fj. We assume that the asymmetry of the Ft-Ds bond distribution around the cell periphery defines the polarization, with greater asymmetry promoting cell proliferation. Our model predicts that this asymmetry is greatest in the radially-expanding transition region that leaves polarized cells in its wake. These cells naturally retain their bond distribution asymmetry after division by rapidly replenishing Ft-Ds bonds at new cell-cell interfaces. Thus we predict that the distal localization of Dachs in cells throughout the pouch requires the movement of the Ds transition region and the simple presence, rather than any specific spatial pattern, of Fj. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanism of antibiotics that are currently in use is important for the development of new antimicrobials. The tetracyclines, discovered in the 1940s, are a well-established class of antibiotics that still have a role in treating microbial infections in humans. It is generally accepted that the main target of their action is the ribosome. The estimated affinity for tetracycline binding to the ribosome is relatively low compared to the actual potency of the drug in vivo. Therefore, additional inhibitory effects of tetracycline on the translation machinery have been discussed. Structural evidence suggests that tetracycline inhibits the function of the essential bacterial GTPase Elongation Factor (EF)-Tu through interaction with the bound nucleotide. Based on this, tetracycline has been predicted to impede the nucleotide-binding properties of EF-Tu. However, detailed kinetic studies addressing the effect of tetracycline on nucleotide binding have been prevented by the fluorescence properties of the antibiotic. Here, we report a fluorescence-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically relevant conditions, we demonstrate that tetracycline does not affect EF-Tu’s intrinsic or ribosome-stimulated GTPase activity, nor the stability of the EF-Tu•GTP•Phe-tRNAPhe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) has its ability to bind drug molecules and influence their efficacies. Although anticancer leads NSC48693 and NSC290956 functioned at the same mechanism, the drug efficacies were obviously distinct. To gain insight into the distinct drug efficacy, the molecular and physiological processes of anticancer leads binding HSA have been investigated via a combined experimental and theoretical approach. The binding site, as characterized by fluorescence quenching and molecular modeling, is found to be located at site II in subdomain III A for NSC48693 with tight binding and at site FA1 in subdomain I B for NSC290956 with negatively cooperative binding, respectively. As indicated by the thermodynamic analysis, NSC48693 binds to HSA with an enthalpy driven mechanism, while NSC290956 binding with HSA is entropically driven. The further kinetic analysis indicates that the association rates appear to be similar to these two anticancer leads, however, the dissociation rate of NSC48693 is approximately 5-fold slower than that of NSC290956. For NSC48693, the pharmacodynamic efficacy is less than that of NSC290956, while its pharmacokinetic behavior is better than that of NSC290956. These parameters influence the pharmacodynamic efficacy and pharmacokinetic behavior, which will give further impacts on drug efficacy in vivo. [ABSTRACT FROM AUTHOR]

A stochastic version of the Barkai-Leibler model of chemotaxis receptors in Escherichia coli is studied here with the goal of elucidating the effects of intrinsic network noise in their conformational dynamics. The model was originally proposed to explain the robust and near-perfect adaptation of E. coli observed across a wide range of spatially uniform attractant/repellent (ligand) concentrations. In the model, a receptor is either active or inactive and can stochastically switch between the two states. The enzyme CheR methylates inactive receptors while CheB demethylates active receptors and the probability for a receptor to be active depends on its level of methylation and ligand occupation. In a simple version of the model with two methylation sites per receptor (M = 2), we show rigorously, under a quasi-steady state approximation, that the mean active fraction of receptors is an ultrasensitive function of [CheR]/[CheB] in the limit of saturating receptor concentration. Hence the model shows zero-order ultrasensitivity (ZOU), similar to the classical two-state model of covalent modification studied by Goldbeter and Koshland (GK). We also find that in the limits of extremely small and extremely large ligand concentrations, the system reduces to two different two-state GK modules. A quantitative measure of the spontaneous fluctuations in activity is provided by the variance in the active fraction, which is estimated mathematically under linear noise approximation (LNA). It is found that peaks near the ZOU transition. The variance is a non-monotonic, but weak function of ligand concentration and a decreasing function of receptor concentration. Gillespie simulations are also performed in models with M = 2, 3 and 4. For M = 2, simulations show excellent agreement with analytical results obtained under LNA. Numerical results for M = 3 and M = 4 are qualitatively similar to our mathematical results in M = 2; while all the models show ZOU in mean activity, the variance is found to be smaller for larger M. The magnitude of receptor noise deduced from available experimental data is consistent with our predictions. A simple analysis of the downstream signaling pathway shows that this noise is large enough to affect the motility of the organism, and may have a beneficial effect on it. The response of mean receptor activity to small time-dependent changes in the external ligand concentration is computed within linear response theory, and found to have a bilobe form, in agreement with earlier experimental observations. [ABSTRACT FROM AUTHOR]

Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence–specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3) and the two-finger segment (TZD23) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD–DNA complex. [ABSTRACT FROM AUTHOR]

The principal pacemaker of the circadian clock of the cyanobacterium S. elongatus is a protein phosphorylation cycle consisting of three proteins, KaiA, KaiB and KaiC. KaiC forms a homohexamer, with each monomer consisting of two domains, CI and CII. Both domains can bind and hydrolyze ATP, but only the CII domain can be phosphorylated, at two residues, in a well-defined sequence. While this system has been studied extensively, how the clock is driven thermodynamically has remained elusive. Inspired by recent experimental observations and building on ideas from previous mathematical models, we present a new, thermodynamically consistent, statistical-mechanical model of the clock. At its heart are two main ideas: i) ATP hydrolysis in the CI domain provides the thermodynamic driving force for the clock, switching KaiC between an active conformational state in which its phosphorylation level tends to rise and an inactive one in which it tends to fall; ii) phosphorylation of the CII domain provides the timer for the hydrolysis in the CI domain. The model also naturally explains how KaiA, by acting as a nucleotide exchange factor, can stimulate phosphorylation of KaiC, and how the differential affinity of KaiA for the different KaiC phosphoforms generates the characteristic temporal order of KaiC phosphorylation. As the phosphorylation level in the CII domain rises, the release of ADP from CI slows down, making the inactive conformational state of KaiC more stable. In the inactive state, KaiC binds KaiB, which not only stabilizes this state further, but also leads to the sequestration of KaiA, and hence to KaiC dephosphorylation. Using a dedicated kinetic Monte Carlo algorithm, which makes it possible to efficiently simulate this system consisting of more than a billion reactions, we show that the model can describe a wealth of experimental data. [ABSTRACT FROM AUTHOR]

Metabolite or substrate channeling is a direct transfer of metabolites from one enzyme to the next enzyme in a cascade. Among many potential advantages of substrate channeling, acceleration of the total reaction rate is considered as one of the most important and self-evident. However, using a simple model, supported by stochastic simulations, we show that it is not always the case; particularly at long times (i.e. in steady state) and high substrate concentrations, a channeled reaction cannot be faster, and can even be slower, than the original non-channeled cascade reaction. In addition we show that increasing the degree of channeling may lead to an increase of the metabolite pool size. We substantiate that the main advantage of channeling likely lies in protecting metabolites from degradation or competing side reactions. [ABSTRACT FROM AUTHOR]

Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found–the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT. [ABSTRACT FROM AUTHOR]

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. [ABSTRACT FROM AUTHOR]

Inflammatory cytokines are key drivers of cartilage degradation in post-traumatic osteoarthritis. Cartilage degradation mediated by these inflammatory cytokines has been extensively investigated using in vitro experimental systems. Based on one such study, we have developed a computational model to quantitatively assess the impact of charged small molecules intended to inhibit IL-1 mediated cartilage degradation. We primarily focus on the simplest possible computational model of small molecular interaction with the IL-1 system—direct binding of the small molecule to the active site on the IL-1 molecule itself. We first use the model to explore the uptake and release kinetics of the small molecule inhibitor by cartilage tissue. Our results show that negatively charged small molecules are excluded from the negatively charged cartilage tissue and have uptake kinetics in the order of hours. In contrast, the positively charged small molecules are drawn into the cartilage with uptake and release timescales ranging from hours to days. Using our calibrated computational model, we subsequently explore the effect of small molecule charge and binding constant on the rate of cartilage degradation. The results from this analysis indicate that the small molecules are most effective in inhibiting cartilage degradation if they are either positively charged and/or bind strongly to IL-1α, or both. Furthermore, our results showed that the cartilage structural homeostasis can be restored by the small molecule if administered within six days following initial tissue exposure to IL-1α. We finally extended the scope of the computational model by simulating the competitive inhibition of cartilage degradation by the small molecule. Results from this model show that small molecules are more efficient in inhibiting cartilage degradation by binding directly to IL-1α rather than binding to IL-1α receptors. The results from this study can be used as a template for the design and development of more pharmacologically effective osteoarthritis drugs, and to investigate possible therapeutic options. [ABSTRACT FROM AUTHOR]

The stochastic nature of gene regulatory networks described by Chemical Master Equation (CME) leads to the distribution of proteins. A deterministic bistability is usually reflected as a bimodal distribution in stochastic simulations. Within a certain range of the parameter space, a bistable system exhibits two stable steady states, one at the low end and the other at the high end. Consequently, it appears to have a bimodal distribution with one sub-population (mode) around the low end and the other around the high end. In most cases, only one mode is favorable, and guiding cells to the desired state is valuable. Traditionally, the population was redistributed simply by adjusting the concentration of the inducer or the stimulator. However, this method has limitations; for example, the addition of stimulator cannot drive cells to the desired state in a common bistable system studied in this work. In fact, it pushes cells only to the undesired state. In addition, it causes a position shift of the modes, and this shift could be as large as the value of the mode itself. Such a side effect might damage coordination, and this problem can be avoided by applying a new method presented in this work. We illustrated how to manipulate the intensity of internal noise by using biologically practicable methods and utilized it to prompt the population to the desired mode. As we kept the deterministic behavior untouched, the aforementioned drawback was overcome. Remarkably, more than 96% of cells has been driven to the desired state. This method is genetically applicable to biological systems exhibiting a bimodal distribution resulting from bistability. Moreover, the reaction network studied in this work can easily be extended and applied to many other systems. [ABSTRACT FROM AUTHOR]

HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage. [ABSTRACT FROM AUTHOR]

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. [ABSTRACT FROM AUTHOR]

Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation. [ABSTRACT FROM AUTHOR]

The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification. [ABSTRACT FROM AUTHOR]

Understanding acid-base regulation is often reduced to pigeonholing clinical states into categories of disorders based on arterial blood sampling. An earlier ambition to quantitatively explain disorders by measuring production and elimination of acid has not become standard clinical practice. Seeking back to classical physical chemistry we propose that in any compartment, the requirement of electroneutrality leads to a strong relationship between charged moieties. This relationship is derived in the form of a general equation stating charge balance, making it possible to calculate [H+] and pH based on all other charged moieties. Therefore, to validate this construct we investigated a large number of blood samples from intensive care patients, where both data and pathology is plentiful, by comparing the measured pH to the modeled pH. We were able to predict both the mean pattern and the individual fluctuation in pH based on all other measured charges with a correlation of approximately 90% in individual patient series. However, there was a shift in pH so that fitted pH in general is overestimated (95% confidence interval -0.072–0.210) and we examine some explanations for this shift. Having confirmed the relationship between charged species we then examine some of the classical and recent literature concerning the importance of charge balance. We conclude that focusing on the charges which are predictable such as strong ions and total concentrations of weak acids leads to new insights with important implications for medicine and physiology. Importantly this construct should pave the way for quantitative acid-base models looking into the underlying mechanisms of disorders rather than just classifying them. [ABSTRACT FROM AUTHOR]

Protein binding often involves conformational changes. Important questions are whether a conformational change occurs prior to a binding event (‘conformational selection’) or after a binding event (‘induced fit’), and how conformational transition rates can be obtained from experiments. In this article, we present general results for the chemical relaxation rates of conformational-selection and induced-fit binding processes that hold for all concentrations of proteins and ligands and, thus, go beyond the standard pseudo-first-order approximation of large ligand concentration. These results allow to distinguish conformational-selection from induced-fit processes—also in cases in which such a distinction is not possible under pseudo-first-order conditions—and to extract conformational transition rates of proteins from chemical relaxation data. [ABSTRACT FROM AUTHOR]

Understanding acid-base regulation is often reduced to pigeonholing clinical states into categories of disorders based on arterial blood sampling. An earlier ambition to quantitatively explain disorders by measuring production and elimination of acid has not become standard clinical practice. Seeking back to classical physical chemistry we propose that in any compartment, the requirement of electroneutrality leads to a strong relationship between charged moieties. This relationship is derived in the form of a general equation stating charge balance, making it possible to calculate [H+] and pH based on all other charged moieties. Therefore, to validate this construct we investigated a large number of blood samples from intensive care patients, where both data and pathology is plentiful, by comparing the measured pH to the modeled pH. We were able to predict both the mean pattern and the individual fluctuation in pH based on all other measured charges with a correlation of approximately 90% in individual patient series. However, there was a shift in pH so that fitted pH in general is overestimated (95% confidence interval -0.072–0.210) and we examine some explanations for this shift. Having confirmed the relationship between charged species we then examine some of the classical and recent literature concerning the importance of charge balance. We conclude that focusing on the charges which are predictable such as strong ions and total concentrations of weak acids leads to new insights with important implications for medicine and physiology. Importantly this construct should pave the way for quantitative acid-base models looking into the underlying mechanisms of disorders rather than just classifying them. [ABSTRACT FROM AUTHOR]

Contact dependent growth inhibition (CDI) is the phenomenon where CDI+ bacterial strain (inhibitor) inhibits the growth of CDI−strain (target) by direct cell to cell contact. CDI is mediated by cdiBAI gene cluster where CdiB facilitates the export of CdiA, an exotoxin, on the cell surface and CdiI acts as an immunity protein to protect CDI+ cells from autoinhibition. CdiA-CT, the C-terminal region of the toxin CdiA, from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires binding of a biosynthetic enzyme CysK (O-acetylserine sulfyhydrylase) for activation in the target cells. CdiA-CT can also interact simultaneously with CysK and immunity protein, CdiI, to form a ternary complex in UPEC536. But the role of CysK in the ternary complex is not clear. We studied the hydrodynamic, thermodynamic and kinetic parameters of binary and ternary complexes using AUC, ITC and SPR respectively, to investigate the role of CysK in UPEC536. We report that CdiA-CT binds CdiI and CysK with nanomolar range affinity. We further report that binding of CysK to CdiA-CT improves its affinity towards CdiI by ~40 fold resulting in the formation of a more stable complex with over ~130 fold decrease in dissociation rate. Thermal melting experiments also suggest the role of CysK in stabilizing CdiA-CT/CdiI complex as Tm of the binary complex shifts ~10°C upon binding CysK. Hence, CysK acts a modulator of CdiA-CT/CdiI interactions by stabilizing CdiA-CT/CdiI complex and may play a crucial role in preventing autoinhibition in UPEC536. This study reports a new moonlighting function of a biosynthetic enzyme, CysK, as a modulator of toxin/immunity interactions in UPEC536 inhibitor cells. [ABSTRACT FROM AUTHOR]

Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin. [ABSTRACT FROM AUTHOR]

XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER. [ABSTRACT FROM AUTHOR]

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL2) is a growth factor for several immune cells and its function depends on its binding to IL2Rs in the cell membrane. The most accepted model for the assembling of IL2-IL2R complexes in the cell membrane is the Affinity Conversion Model (ACM). This model postulates that IL2R receptor association is sequential and dependent on ligand binding. Most likely free IL2 binds first to IL2Rα, and then this complex binds to IL2Rβ, and finally to IL2Rγ (γc). However, in previous mathematical models representing this process, the binding of γc has not been taken into account. In this work, the quantitative contribution of the number of IL2Rγ chain to the IL2-IL2R apparent binding affinity and signaling is studied. A mathematical model of the affinity conversion process including the γ chain in the dynamic, has been formulated. The model was calibrated by fitting it to experimental data, specifically, Scatchard plots obtained using human cell lines. This paper demonstrates how the model correctly explains available experimental observations. It was estimated, for the first time, the value of the kinetic coefficients of IL2-IL2R complexes interaction in the cell membrane. Moreover, the number of IL2R components in different cell lines was also estimated. It was obtained a variable distribution in the number of IL2R components depending on the cell type and the activation state. Of most significance, the study predicts that not only the number of IL2Rα and IL2Rβ, but also the number of γc determine the capacity of the cell to capture and retain IL2 in signalling complexes. Moreover, it is also showed that different cells might use different pathways to bind IL2 as consequence of its IL2R components distribution in the membrane. [ABSTRACT FROM AUTHOR]

DNA threading intercalators are a unique class of intercalating agents, albeit little biophysical information is available on their intercalative actions. Herein, the intercalative effects of nogalamycin, which is a naturally-occurring DNA threading intercalator, have been investigated by high-resolution atomic force microscopy (AFM) and spectroscopy (AFS). The results have been compared with those of the well-known chemotherapeutic drug daunomycin, which is a non-threading classical intercalator bearing structural similarity to nogalamycin. A comparative AFM assessment revealed a greater increase in DNA contour length over the entire incubation period of 48 h for nogalamycin treatment, whereas the contour length increase manifested faster in case of daunomycin. The elastic response of single DNA molecules to an externally applied force was investigated by the single molecule AFS approach. Characteristic mechanical fingerprints in the overstretching behaviour clearly distinguished the nogalamycin/daunomycin-treated dsDNA from untreated dsDNA—the former appearing less elastic than the latter, and the nogalamycin-treated DNA distinguished from the daunomycin-treated DNA—the classically intercalated dsDNA appearing the least elastic. A single molecule AFS-based discrimination of threading intercalation from the classical type is being reported for the first time. [ABSTRACT FROM AUTHOR]

Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR’s native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed. [ABSTRACT FROM AUTHOR]

Renaturation of the complementary single strands of DNA is one of the important processes that requires better understanding in the view of molecular biology and biological physics. Here we develop a stochastic dynamical model on the DNA renaturation. According to our model there are at least three steps in the renaturation process viz. nonspecific-contact formation, correct-contact formation and nucleation, and zipping. Most of the earlier two-state models combined nucleation with nonspecific-contact formation step. In our model we suggest that it is considerably meaningful when we combine the nucleation with the zipping since nucleation is the initial step of zipping and nucleated and zipping molecules are indistinguishable. Nonspecific contact formation step is a pure three-dimensional diffusion controlled collision process. Whereas nucleation involves several rounds of one-dimensional slithering and internal displacement dynamics of one single strand of DNA on the other complementary strand in the process of searching for the correct-contact and then initiate nucleation. Upon nucleation, the stochastic zipping follows to generate a fully renatured double stranded DNA. It seems that the square-root dependency of the overall renaturation rate constant on the length of reacting single strands originates mainly from the geometric constraints in the diffusion controlled nonspecific-contact formation step. Further the inverse scaling of the renaturation rate on the viscosity of reaction medium also originates from nonspecific contact formation step. On the other hand the inverse scaling of the renaturation rate with the sequence complexity originates from the stochastic zipping which involves several rounds of crossing over the free-energy barrier at microscopic levels. When the sequence of renaturing single strands of DNA is repetitive with less complexity then the cooperative effects will not be noticeable since the parallel zipping will be a dominant enhancing factor. However for DNA strands with high sequence complexity and length one needs to consider the underlying cooperative effects both at microscopic and macroscopic levels to explain various scaling behaviours of the overall renaturation rate. [ABSTRACT FROM AUTHOR]

Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M-1s-1 and an even slower dissociation rate (kd) in the range of 10−4 s-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not entirely clear from the published literature whether OCTs function as major prostaglandin transporters, the tight binding of the naturally occurring PGE2, as well as the slow dissociation rate, could conceivably affect the transport of lower affinity substrates such as drugs and metabolites by SLC22 transporters. More research is necessary to establish the extent to which individual SLC22 family members actually function as PG transporters in vitro and in vivo and to investigate whether PGs can, independent of being directly transported, alter the ability of SLC22 transporters to handle drugs and other substrates. [ABSTRACT FROM AUTHOR]

The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin. [ABSTRACT FROM AUTHOR]