Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

A systems model of phosphorylation for inflammatory signaling events

published_or_final_versio

Characterization of the Subcellular Localization of DCBLD2 and its Regulation by Tyrosine Phosphorylation

DCBLD2 is a scaffolding receptor possibly involved in neuronal migration or differentiation. It contains seven intracellular tyrosine sites that can be phosphorylated to allow Crk/CrkL binding. Crk/CrkL are involved in the Reelin signaling pathway that regulates neuronal migration in the developing cortex. My research was aimed at understanding the subcellular localization of DCBLD2 and to determine if its ability to become tyrosine phosphorylated altered its localization. I hypothesized that DCBLD2 would localize similarly to NP1 and PlxnA2 based on their similar functions relayed to axon guidance and their similar ectodomains. DCBLD2 is termed a neuropilin-like protein as they have similar ectodomains. NP1 is a co-receptor to PlxnA2 and together they respond to semaphorins to regulate axonal guidance. Therefore, DCBLD2 might localize similarly to NP-1 and PlxnA2 within a cell. It is possible that the mutant version of DCBLD2, lacking the seven tyrosine sites, will localize differently than the wild type, perhaps driving it more or less towards the plasma membrane. I created three constructs to study DCBLD2 in human embryonic kidney cells. These constructs were visualized via immunofluorescence. From the images and statistical results, both DCBLD2 wild type and the phosphorylation site mutant localize similarly to PlxnA2

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

Photo shows a man taking in the view from the Harts Point scenic overlook, Canyonlands National Park, Uta

Tracking Cell Fate with Synthetic Memory and Pulse Detecting Transcriptional Circuits

Synthetic biology aims to engineer biological systems to meet new challenges and teach us more about natural biological systems. These pursuits range from the building of relatively simple transcriptional circuits, to engineering the metabolism of an organism, to reconstructing entire genomes. While we are still emerging from the foundational stages of this new field, we are already using engineered cells to discover underlying biological mechanisms, develop new therapeutics, and produce natural products. In this dissertation, we discuss the application of synthetic biology principles to the development of memory and pulse-detecting genetic circuits. In Chapter 2, we use novel transcriptional positive-feedback based memory devices integrated in human cells to study heterogeneous responses to cellular stresses. We built doxycycline, hypoxia, and DNA damage sensing versions of the device, demonstrating its modularity. In Chapter 3, we discuss further applications of the memory device in the study of long-term responses to hypoxia, gamma radiation, and inflammation. Finally, in Chapter 4 we describe work leading to the future construction of a pulse-detecting genetic circuit integrated in the E. coli genome. The work presented here illustrates the general applicability of synthetic biology in the study of biological phenomena and brings us one step closer to achieving a more exquisite understanding and control of natural systems

Quantitative analysis of microbial sensing and motility

Microbes need to extract relevant information from their environment and use this information to produce adequate behavioral responses that ensure their survival. Quantitative, mathematical analysis of microbial sensory systems (such as various signaling pathways) and their effectors (such as bacterial motors) forms the basis of the field of systems biology. Because of their relative simplicity, in comparison with analogous systems in multicellular organisms, these structures are more amenable to quantitative modelling. In this dissertation I present the quantitative analysis of three microbial sensory-effector systems, two in bacteria and one in the unicellular eukaryote Saccharomyces cerevisiae. In all three cases I look at a behavior that is an evolutionarily selected response to a given problem that the microorganism is confronted with. I then explain the mechanistic basis of this response in the sensory or effector system by a mathematical model. In the first case, mating in yeast cells, the problem the cells need to solve is to establish the likelihood of mating and invest cellular resources accordingly to prepare for the mating event. The solution that wild-type yeast MATa cells have evolved to tackle this problem is fractional sensing, the ability to sense robustly the fraction of partner cells in a mixed population. The mechanism that enables this behavior is the degradation of the partner cells’ pheromone signal by a secreted enzyme. I show mathematically that the necessary consequence of this mechanism is the rescaling of the signal strength proportionally to the fraction of partner cells, as opposed to their absolute quantity. Additionally, I also explain the experimentally observed difference between the fractionally sensing wild-type cells and the mutants performing absolute sensing, due to the latter’s lack of a signal attenuation mechanism. Moreover, by a cost-benefit model of mating, I show that the strategy of fractional sensing and resource investment is optimal, as compared to sensing the absolute amount of partners. In the second case, I look at the most prevalent bacterial signaling systems, the so-called two-component systems and their capacity to generate bistability, or, in behavioral terms, memory. In the case of two-component systems that control developmental processes, an irreversible shift is required at the level of individual cells: once the system is turned ‘on’, it should not revert to its ‘off’ state, within some range of the input. At the population level, because of the stochasticity of chemical reactions and variation in expression levels, a bistable control system can result in a bimodal distribution with some cells in ‘on’ and others in ‘off’ state. In fluctuating and unpredictable environments this strategy of ‘bet hedging’ is another advantageous feature of bistability. I first describe post-translational mechanisms that can generate bistable behavior and analyze the parametric properties of bistable systems. Second, I show that the transcriptional auto-induction of pathway components can lead to bistability in the ‘canonical’ two-component system with a bifunctional sensor kinase as well, a question not resolved in the previous literature. In the third case, I analyze the motility of the marine bacteria Shewanella putrefaciens. Higher efficiency of spreading and chemotaxis is expected to lead to higher fitness as it enables a bacterial population to better explore and exploit the resources of its environment. Wild-type Shewanella cells achieve this higher efficiency by inducing a lateral flagellar system, leading to a lower mean turning angle. By lowering the mean of the turning angle distribution, the presence of the lateral flagella leads to higher directional persistence and hence increased spreading efficiency. By both analytical calculations and stochastic simulations I reproduce the experimentally observed trends of spreading. Furthermore, I show that in shallow gradients the higher directional persistence also leads to higher chemotactic efficiency. By mathematical analysis I was able to identify the mechanisms underlying these evolutionarily selected behaviors. Moreover, in the case of yeast mating, I also showed that the observed behavior of fractional sensing is optimal in cost-benefit terms. In the case of transcriptionally induced bistability in bacterial two-component systems, the analysis identified parametric properties of bistable systems that can be potentially used to engineer monostable signaling systems into bistable ones experimentally

Mathematical Modelling of Inter- and Intracellular Signal Transduction: The Regulatory Role of Multisite Interactions

Signalling processes regulate various aspects of living cells via modulation of protein activity. The interactions between the signalling proteins can occur at single or multiple sites. Although single site protein interactions are relatively easy to understand, these rarely occur in living systems. It is therefore important to investigate multisite interactions. Despite the recent progress in experimental studies, the underlying molecular mechanisms and molecular functions of the multisite interactions are still not clear and therefore require systems approaches for deeper understanding, for example to understand how the system will react to perturbation of one of its components. The examples of the molecular functions that are studied in this thesis are: kinetics of multisite calcium binding to proteins such as calmodulin (CaM), multisite phosphorylation of interferon regulatory factor 5 (IRF-5) and signal transducers and activators of transcription (STATs). We also study the role of STATs in the overall immune response and in T cell phenotype switching as well as multisite phosphorylation of high osmolarity glycerol factor 1 (Hog1) in mitogen activated protein kinase (MAPK) cascade during the adaptation of Candida glabrata to osmotic stress. In this thesis, these examples are studied using the systems approach in the context of human diseases: cancer, candidiasis, immunity-related pathologies such as rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus. We discuss potential therapeutic implications of the proposed models in these diseases. The predictions of the models developed in this thesis are supported by the experimental data and propose possible mechanisms of the multisite interactions involved in the cellular regulation