Kinetic regulation of multi-ligand binding proteins

Background: Second messengers, such as calcium, regulate the activity of multisite binding proteins in a concentration-dependent manner. For example, calcium binding has been shown to induce conformational transitions in the calcium-dependent protein calmodulin, under steady state conditions. However, intracellular concentrations of these second messengers are often subject to rapid change. The mechanisms underlying dynamic ligand-dependent regulation of multisite proteins require further elucidation. Results: In this study, a computational analysis of multisite protein kinetics in response to rapid changes in ligand concentrations is presented. Two major physiological scenarios are investigated: i) Ligand concentration is abundant and the ligand-multisite protein binding does not affect free ligand concentration, ii) Ligand concentration is of the same order of magnitude as the interacting multisite protein concentration and does not change. Therefore, buffering effects significantly influence the amounts of free ligands. For each of these scenarios the influence of the number of binding sites, the temporal effects on intermediate apo- and fully saturated conformations and the multisite regulatory effects on target proteins are investigated. Conclusions: The developed models allow for a novel and accurate interpretation of concentration and pressure jump-dependent kinetic experiments. The presented model makes predictions for the temporal distribution of multisite protein conformations in complex with variable numbers of ligands. Furthermore, it derives the characteristic time and the dynamics for the kinetic responses elicited by a ligand concentration change as a function of ligand concentration and the number of ligand binding sites. Effector proteins regulated by multisite ligand binding are shown to depend on ligand concentration in a highly nonlinear fashion

Drug diffusion along an intact mammalian cochlea

Intratympanic drug administration depends on the ability of drugs to pass through the round window membrane (RW) at the base of the cochlea and diffuse from this location to the apex. While the RW permeability for many different drugs can be promoted, passive diffusion along the narrowing spiral of the cochlea is limited. Earlier measurements of the distribution of marker ions, corticosteroids and antibiotics demonstrated that the concentration of substances applied to the RW was two to three orders of magnitude higher in the base compared to the apex. The measurements, however, involved perforating the cochlear bony wall and, in some cases, sampling perilymph. These manipulations can change the flow rate of perilymph and lead to intake of perilymph through the cochlear aqueduct, thereby disguising concentration gradients of the delivered substances. In this study, the suppressive effect of salicylate on cochlear amplification via block of the outer hair cell (OHC) somatic motility was utilized to assess salicylate diffusion along an intact guinea pig cochlea in vivo. Salicylate solution was applied to the RW and threshold elevation of auditory nerve responses was measured at different times and frequencies after application. Resultant concentrations of salicylate along the cochlea were calculated by fitting the experimental data using a mathematical model of the diffusion and clearing of salicylate in a tube of variable diameter combined with a model describing salicylate action on cochlear amplification. Concentrations reach a steady-state at different times for different cochlear locations and it takes longer to reach the steady-state at more apical locations. Even at the steady state, the predicted concentration at the apex negligible. Model predictions for the geometry of the longer human cochlea show even higher differences in the steady-state concentrations of the drugs between cochlear base and apex. Our findings confirm conclusions that achieving therapeutic drug concentrations throughout the entire cochlear duct is hardly possible when the drugs are applied to the RW and are distributed via passive diffusion. Assisted methods of drug delivery are needed to reach a more uniform distribution of drugs along the cochlea

A systems model of phosphorylation for inflammatory signaling events

Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits

Multisite phosphorylation enables switching between multiple T cell phenotypes.

<p>(A) Schematic diagram of IRF-5 phosphorylation and dephosphorylation by TBK-1 and AP, respectively, represents one of many intracellular multiphosphorylation examples observed in the immune system. Experimental evidence suggests that proteins phosphorylated at different phosphorylation sites may have selective activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-ChangForeman1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Hochrainer1" target="_blank">[61]</a> and give rise to distinct T cell populations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Macian1" target="_blank">[62]</a>. (B) Computational model predictions for distribution of IRF-5 phosphorylated species. Extracellular environment is hypothesized ratio causing the distribution of IRF-5 phosphorylated species: one site phosphorylated (black), two (magenta), three (yellow), four (cyan), five (grey) and six (blue). According to the proposed model extracellular environment can actively change the ratio of IRF-5 phosphorylated species and thereby contribute to the mechanism of T cell plasticity by modulating the numbers of T cell phenotypes.</p

A schematic diagram for the dependence of T cell differentiation on intracellular phosphorylation signaling.

<p>A vast amount of experimental evidence suggests that T cell phenotypes strongly depend on the intracellular phosphorylation signaling mechanisms <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Krausgruber1" target="_blank">[44]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Shi1" target="_blank">[50]</a>. Environmental factors, genetic mutations, cellular and intracellular factors influence the underlying phosphorylation mechanics. The cartoon summarizes possible differential responses of TLR downstream phosphorylation signaling events to pathogens leading to the distinct polarization of naive T cells into three distinct phenotypes Th17, Th1 and Treg. According to this model activation or interplay of phosphorylation pathways is responsible for selective differentiation as well as for T cell phenotype switching. The model suggests that the cell plasticity observed under pathological conditions can be due to altered intracellular phosphorylation patterns, which are, in turn, dependent on the extracellular cytokine environment.</p

Model predictions for the concentration of STAT3_p phosphorylated by JAK and dephosphorylated by SHP-1.

<p>Investigation of the dependence of STAT3 phosphorylation on the relative activities of JAK and SHP. (A) Cartoon diagram of STAT3 phosphorylation and dephosphorylation by JAK and SHP-1, respectively. (B) A comparative analysis of the proposed and applications of the previously published models for STAT3 phosphorylation. Ratios of JAK and SHP-1 are found to be critical for STAT3 phosphorylation response and the differences between the model predictions. The red line shows the predictions by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Goldbeter1" target="_blank">[6]</a> whereas the black line offers predictions from the presented model. The STAT3 phosphorylation predictions coincide when STAT3 significantly exceeds SHP-1 concentration. (C) The effects of phosphorylation and dephosphorylation rates are studied on the proposed (black line) and previously reported (red line) models <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Goldbeter1" target="_blank">[6]</a>. We found that our model predicts the modulation of phosphorylated STAT as opposed to the prediction of STAT3 phosphorylation rate offered by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Goldbeter1" target="_blank">[6]</a>. (D) The comparison between the proposed and the previously model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110913#pone.0110913-Goldbeter1" target="_blank">[6]</a> is shown as a function of phosphorylation/dephosphorylation rates for various ratios of JAK and SHP-1. This analysis clearly demonstrates the differences in STAT3 phosphorylation predictions due to the underlying assumptions employed in the models.</p

Theoretical investigation of the regulation of IRF-5 multisite phosphorylation.

<p>The distribution of IRF-5 species was investigated as a function of kinase to phosphatase (TBK to AP) ratio for comparable IRF-5 and AP concentrations (A), IRF-5 significantly exceeds AP (B). Similar analysis was also performed when the phosphorylation rate was significantly lower than dephosphorylation rate and comparable IRF-5 and AP concentrations (C), IRF-5 significantly exceeds AP (D). The effects of changes in the phosphorylation to dephosphorylation ratio on the IRF-5 species were also investigated with comparable IRF-5 and AP concentrations (E), IRF-5 significantly exceeds AP (F). The presented analyses clearly show that the phosphorylation/dephosphorylation parameters, modulated via extracellular cytokines have prominent impact on the distribution of phosphorylated species. Therefore, physiological or pathological alterations of these parameters represent the multisite phosphorylation-mediated mechanism of T cell plasticity.</p