Systems biology perspectives on calcium signaling and DNA repair

This work deals with two paradigms of cellular signaling: a change in the intracellular Ca2+ concentration represents often one of the first steps in the transduction of incoming stimuli; the ability to properly respond to external injuries and repair damaged DNA is an essential component for life. The approaches used to investigate Ca2+ signaling and DNA repair bear common points. Mathematical models are used to asses system properties, such as efficiency in the signal processing, specificity of the response, and design principles behind the different pathways. A close collaboration with experimentalist made it possible to directly compare model predictions and experimental data, and to test different mechanistic hypothesis. The first part of this thesis focuses on the phosphoinositide-dependent Ca2+ signaling. Hormones that act through the Ca2+ releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular Ca2+ oscillations, which have been ascribed to Ca2+ feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic Ca2+ concentration. To investigate the functional significance of this phenomenon, mathematical models of the interaction of both second messengers have been developed. The models account for both positive and negative feedbacks of Ca2+ on the phosphoinositide metabolism, mediated by Ca2+ activation of phospholipase C and IP3 3-kinase, respectively. The theoretical analysis shows that each of these Ca2+ feedbacks substantially expands the range of oscillation frequencies of a core oscillator based on Ca2+ and IP3R dynamics, compared to Ca2+ fluctuations obtained with clamped IP3 (Chapter 2). The action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. This suggests to perturb the IP3 turnover in order to study the feedbacks present (Chapter 3): Whereas increasing the IP3 turnover by overexpressing IP3 metabolizing enzymes gives no information on the underlying feedbacks, slowing the IP3 dynamics with an IP3 binding protein can reveal positive feedback. This theory has been tested in chinese hamster ovary cells by transiently expressing an IP3 binding protein. The overexpression of this fusion protein exerted a dose-dependent suppression of agonist-induced Ca2+ oscillations that is consistent with an oscillator model including positive feedback of Ca2+ on IP3 generation. This prominent role of the IP3 dynamics in modulating Ca2+ oscillations demanded a more deeper analysis of the fate of the IP3 precursor, phosphatidylinositol-4,5-bisphosphate (PIP2). To this end, a detailed model for the phosphoinositide pathway based on measured metabolite concentrations has been developed (Chapter 4). The model illustrates the importance of futile (de)phosphorylation cycles for regenerating PIP2 during stimulation, an essential property to support long-lasting Ca2+ signals. Alternatively, when futile cycling is weak, the presence of positive feedback of Ca2+ on the IP3 production also allows to regenerate the PIP2 pool. Taken together, the experimental data and theoretical analysis presented in this thesis indicate that IP3 oscillations are an essential component of the Ca2+ oscillator, they are physiologically important for supporting the efficient frequency encoding of hormone dose and the long-lasting Ca2+ signals observed in many cell types. The second part of the thesis is devoted to nucleotide excision repair (NER). It is a versatile DNA repair mechanism that can remove different type of lesions, such as UV light induced pyrimidine dimers and bulky adducts caused by chemical agents. It requires the concerted action of many different proteins that assemble at sites of damaged DNA. Despite our detailed biochemical knowledge about NER, many questions concerning its dynamic behavior remain unanswered, in particular, it is controversial whether repair factors are assembled sequentially or in a random way at the site of damage, or whether they exist as a holocomplex. Therefore, to understand the mechanisms underlying the protein assembly and the performance of repair, a mathematical model, delineating hallmarks and general characteristics of NER, has been developed. First, the binding and dissociation kinetics of repair factors are related to the structural properties of the system, such as the sequential order in which the factors enter repair (Chapter 7). Second, using in vivo kinetic data for the recruitment of three different protein factors at local damaged nuclei, the model parameters are determined and the dynamic behavior of the repair process is scrutinized in detail. The observed saturation of NER is predicted to rely on the high engagement of the recognition factor in repair. Furthermore, the model predicts a quiescent phase, where the amounts of factors involved in repair remains high and constant for a long time period, a forecast that has been validated experimentally. The theoretical analysis of repair performance indicates that a sequential assembly process is remarkably advantageous in terms of repair efficiency and can show a marked selectivity for the damaged substrate (Chapter 8). Alternative mechanisms for repairosome formation, including random assembly and preassembly, can readily become kinetically unfavorable. Based on the model, new experiments are proposed to gain further insight into the early and late steps of this complex process and to critically test model predictions (Chapter 10). Taken together, this thesis provides a kinetic framework for NER and rationalizes why many processes within the nucleus show a sequential assembly strategy.