INTRODUCTION: NLRP3-dependent inflammasome signalling is a key pathway during inflammatory processes and its deregulation is implicated in several diseases. NLRP3-inflammasome pathway activation leads to the rapid, phosphorylation-driven NF$\kappa$κB-pathway signalling, subsequently proceeds via slower transcription/translation process for producing pro-enzymes, and finally leads to the medium-speed enzymatic activation of the central inflammatory mediator IL-$1\beta$1β[1] . We here were interested in how the timing of the rate-limiting step of transcription/translation and the presence of a positive and negative auto-regulation would pose conditions for meaningful and stable IL-$1\beta$1β-activation. METHODS: We extracted the essential topology of the inflammasome pathway network using a linear chain of first-order reaction and a second-order reaction for inhibitory feedback. We then performed an analytical treatment of the resulting ODE set to obtain closed-form formulae. We therefore looked for the steady states and characterized their stability by using a Jacobian-based, local analysis. We employed the Small Gain Theorem from Control Theory as recently applied by us [2] and the Gershgorin Circle Theorem to obtain mathematically exact conditions for a positive ON state and stabilities for ON and OFF steady states. RESULTS: We identified an ON- and one OFF- steady state whose properties we characterized in terms of the kinetic parameters by closed-form formulae. We found that under the assumption of a first-order information flow through the network, the existence of a biologically reasonable ON steady state required the simultaneous presence of the positive and the negative feedback. Assuming non-competitivity between IL-$1\beta$1β entities binding to different receptors, we found that a minimum kinetics for protein production is required to sustain a steady state with IL-$1\beta$1β activation. Assuming competitivity between IL-$1\beta$1β entities introduced additional restrictions on the maximum protein production speed to guarantee a biologically reasonable ON steady state. Finally, for both models, we ruled out bistability, suggesting that IL-$1\beta$1β activation would undergo a smooth change upon alterations of its parameters. CONCLUSION: Exemplified by the core pathway of NLRP3-inflammasome signalling, we here demonstrate that a mostly linear activation cascade containing an intermediate rate limiting step poses kinetic restrictions on this step and requires positive and negative autoregulation for obtaining a meaningful ON steady state. Due to the generality of our framework, our results are important for a wide class of receptor mediated-pathways, where a fast initial phosphorylation cascade is followed by a (slower) transcriptional response and subsequent autoregulation. Our results may further provide important design principles for synthetic biological networks involving biochemical activation and transcription/translation, by relating timing considerations and autoregulation to stable pathway activation.

The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure.

Fluorescence microscopy imaging is a constant trade off between signal to noise ratio, total observation time and spatio-temporal resolution due to photo toxicity. In this paper, we propose a method to estimate the quality of a fluorescent image acquisition, from a single image, taking into account both signal dependent and signal independent noise. We propose a method for the calculation of the signal to noise ratio globally and locally. We validated our algorithm on real experimental data and data with known simulated noise. Results allow us to conclude that this fully automatic method provides a good quantification of the image quality.

UNLABELLED: We have developed a web service that provides a comprehensive analysis of the susceptibility of cells to undergo apoptosis in response to an activation of the mitochondrial apoptotic pathway. Based on ordinary differential equations, (pre-determined) protein concentrations and release kinetics of mitochondrial pro-apoptotic factors, a network of 52 reactions and 19 reaction partners can be employed as a tool to display temporal protein profiles, to identify key regulatory proteins and to determine critical threshold concentrations required for the execution of apoptosis in HeLa cancer cells or other cell types. The web service also provides an interactive database function for the deposition of cell-type-specific quantitative data. In addition, the web service provides an output that can be compared directly to experimental results obtained from real-time single-cell experiments, making this a widely applicable systems biology tool for apoptosis and cancer researchers. AVAILABILITY: http://systemsbiology.rcsi.ie/apopto-cell.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.