Genetic noise control via protein oligomerization

Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamic role of protein-protein interactions. Here we have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch), integrating quantitative results from previous in vivo and in vitro studies. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its random switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced) state from randomly being induced (uninduced). The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise

From Endogenous to Synthetic microRNA-Mediated Regulatory Circuits: An Overview

MicroRNAs are short non-coding RNAs that are evolutionarily conserved and are pivotal post-transcriptional mediators of gene regulation. Together with transcription factors and epigenetic regulators, they form a highly interconnected network whose building blocks can be classified depending on the number of molecular species involved and the type of interactions amongst them. Depending on their topology, these molecular circuits may carry out specific functions that years of studies have related to the processing of gene expression noise. In this review, we first present the different over-represented network motifs involving microRNAs and their specific role in implementing relevant biological functions, reviewing both theoretical and experimental studies. We then illustrate the recent advances in synthetic biology, such as the construction of artificially synthesised circuits, which provide a controlled tool to test experimentally the possible microRNA regulatory tasks and constitute a starting point for clinical applications

Receptomics, design of a microfluidic receptor screening technology

This thesis describes the development of a G Protein-Coupled Receptor (GPCR) screening technology that combines a receptor cell array (~300 spots) with microfluidics. This technology was developed for the purpose of sensing the taste of, or active components in complex samples. GPCR activation was monitored using a genetically encoded calcium indicator (GECI) which was based on a change in Förster Resonance Energy Transfer (FRET) between two fluorescent proteins linked by a calcium binding domain which, upon binding of calcium, induces a conformational change between the fluorophores. The receptor cell arrays were created by reverse transfection of printed plasmid DNA. The arrays were assembled in a flowcell, connected to a microfluidic system, and mounted on a stereo fluorescence microscope. This setup allowed for controlled and importantly, repeated sample exposure while monitoring the changes in intracellular calcium in real-time. GPCRs play an important role in many physiological or disease-related processes. These membrane proteins have evolved to sense a wide range of molecules that can be of either exogenous or endogenous origin. Their sensing mechanisms are complex and potentially involve many cellular signalling events depending the cell type. The introductory chapter of this thesis presents a brief overview of the GPCR types and their signalling pathways with a focus on taste signalling. This chapter also places the microfluidic receptomics technology within the framework of existing receptor screening technologies. The second chapter explores the general principles, setup and characterization of the microfluidic biosensor to measure GPCR activation via imaging of [Ca2+] changes in recombinant human HEK293 cells. These cells expressed a combination of the Neurokinin 1-receptor and Cameleon YC3.6 protein as calcium indicator. Here, a stable cell line was employed for robust expression with little variation Next to GPCRs, the system was also used for the detection of transient receptor potential channel Vanilloid 1 (TRPV1) ion channel activation by means of the Cameleon YC3.6 calcium sensor as is reported in Chapter 3. This assay was performed with LCMS fractions and whole extracts of chilli pepper fruits which led to the identification of new ion channel agonists. This chapter also discusses the possibility of coupling the receptomics assay directly to an LCMS as an additional on-line bioactivity detector. The general discussion of this thesis (Chapter 7) elaborates on this topic with additional perspectives on the feasibility of coupling the two systems. Chapter 4 provides an extensive technical characterization of the preparation and measurement of reverse transfected cell arrays using fluorescent proteins. The response of the Neurokinin 1-receptor in relation to its gene dose in reverse transfection was studied, as well as response reproducibility during repeated activations. These results led to a study of bitter taste receptors in relation to sensitivity-determining parameters such as sensor type and calcium buffering (Chapter 5). This chapter aimed to enhance the sensitivity and robustness of the receptor assay and showed proof of concept with bitter receptor arrays that performed in the same range as existing state-of-the-art platforms. Such bitter taste receptor arrays may be employed for future screenings of new bitter taste agonists or modulators and the identification of bitter principles in foods. Development of software and statistical models -the linear mixed model, as presented in Chapter 6- to analyse this new type of data showed that a spot-based comparison of sequentially-tested samples yielded the most reliable data and largely eliminated inter-spot differences in signal strength. The method could also visualize receptor specific differences between samples in the presence of a simulated host cell response. A host cell response, induced by ATP, was used to show that specific bitter receptor responses from compound spikes were cumulative to the host cell response and can be retrieved from a host cell response signal by means of comparative analysis. The general discussion (Chapter 7) critically discusses the advantages and limitations of this new micro-fluidics approach and details which additional developments are needed to advance the technology further. The receptomics technology as described in this thesis is argued to be complementary to microplate screening technologies and represents a new analytical paradigm. The microfluidics aspect and overall assay size reduction are more cost efficient and allow both a high content dynamics analysis as well as the development of novel applications such as direct identification of bioactive compounds by coupling of LCMS to receptomics. All in all, this thesis presents an enabling receptor screening technology that is based on new design principles. This receptomics technology offers novel applications and has potential in the bioactivity screening of crude extracts.</p

Partial differential equations for self-organization in cellular and developmental biology

Understanding the mechanisms governing and regulating the emergence of structure and heterogeneity within cellular systems, such as the developing embryo, represents a multiscale challenge typifying current integrative biology research, namely, explaining the macroscale behaviour of a system from microscale dynamics. This review will focus upon modelling how cell-based dynamics orchestrate the emergence of higher level structure. After surveying representative biological examples and the models used to describe them, we will assess how developments at the scale of molecular biology have impacted on current theoretical frameworks, and the new modelling opportunities that are emerging as a result. We shall restrict our survey of mathematical approaches to partial differential equations and the tools required for their analysis. We will discuss the gap between the modelling abstraction and biological reality, the challenges this presents and highlight some open problems in the field

Evidence that fold-change, and not absolute level, of β-catenin dictates Wnt signaling

In response to Wnt stimulation, β-catenin accumulates and activates target genes. Using modeling and experimental analysis, we found that the level of β-catenin is sensitive to perturbations in the pathway, such that cellular variation would be expected to alter the signaling outcome. One unusual parameter was robust: the fold-change in β-catenin level (post-Wnt/pre-Wnt). In Xenopus, dorsal-anterior development and target gene expression are robust to perturbations that alter the final level but leave the fold-change intact. These suggest, first, that despite cellular noise, the cell responds reliably to Wnt stimulation by maintaining a robust fold-change in β-catenin. Second, the transcriptional machinery downstream of the Wnt pathway does not simply read the β-catenin level after Wnt stimulation but computes fold-changes in β-catenin. Analogous to Weber's Law in sensory physiology, some gene transcription networks must respond to fold-changes in signals, rather than absolute levels, which may buffer stochastic, genetic, and environmental variation

Model-guided Design of RNA-based Synthetic Circuits for the Dynamic Regulation of Gene Expression

A longstanding goal in synthetic biology has been to build synthetic gene circuits with the ability to harness nature’s capability of precise gene expression regulation. Advancements in RNA technology have established RNA-based regulators with distinct advantages over traditional protein-based regulators such as faster signal propagation, versatile programmability, and low cellular burden, which has created an interest in the field to construct innovative synthetic gene circuits using de novo RNA- based regulators. However, our understanding of the behavior and kinetics of RNA-RNA interactions for the construction of gene circuits is incomplete. This thesis proposes a model-guided design framework that integrates mechanistic modeling and statistical analysis with experimental efforts to overcome this challenge. The proposed framework features: first, define the application of gene circuit; second, select the circuit\u27s architecture and relevant gene regulatory components based on desired dynamics; third, develop a mathematical model to describe the involved biomolecular reaction in the system and identify relevant kinetic information from literature; fourth, perform in vivo or in vitro experimental construction and validation of the circuit. The feasibility of the framework is first demonstrated by assessing the viability of an RNA-only I1-FFL gene circuit. The proposed design is evaluated using a combined experimental and mathematical approach to elucidate the kinetics of RNA-RNA interactions for timescale critical circuit architectures. The framework is then extended to evaluate the relationship between regulation level (transcription or translation) and circuit dynamics using four design variations of the I1-FFL circuit. The performance of each circuit is compared using mechanistic modeling, statistical analysis, and standard control theory concepts, which provide a quantitative way to reveal the effect of regulation level and circuit behavior. The major contributions of this thesis include: (1) it demonstrates the utility of modeling to troubleshoot and debug circuit design (2) it reveals the importance of regulation level in designing synthetic circuits. Together, the findings presented in this thesis aim to facilitate the design and implementation of gene circuits with increased complexity and functionality

The biological and pharmacological allosteric modulation of inositol monophosphatase

The purpose of this project was to develop novel pharmacological tools against Inositol Monophosphatase (IMPase) and gain new insights into the Calbindin-IMPase interaction. IMPase is an enzyme that has been of significant therapeutic interest since the development of the inositol depletion hypothesis of lithium’s efficacy in the treatment of bipolar disorder. More recent studies have implicated IMPase inhibition in the induction of autophagy and the clearance of peptides involved in the pathogenesis of neurodegenerative diseases. Despite this there is a lack of specific inhibitors and those that have been developed lack favorable physiochemical properties to explore specific IMPase inhibition in vivo. My aim was to find novel inhibiters and binding chemical fragments that could later be developed into potent specific inhibitors of IMPase that would allow for the specific evaluation of IMPase inhibition in vivo. To achieve this, two approaches were tried: first a crystallographic fragment soaking screen was used to identify novel binding sites and chemical matter. This resulted in the discovery of a novel fragment binding site at the homodimer interface and mutagenesis at this site inhibited IMPase activity, indicating that the site could be therapeutically relevant. The second approach was to screen a specially curated isothiazolone library based on the previously identified covalent inhibitor Ebselen. Here I identified a new class of isothiazolone inhibitors of IMPase. The calcium binding protein Calbindin-D28k can allosterically modulate IMPase, this results in the increase in the catalytic activity of IMPase. I aimed to explore this interaction by fusing the proteins using a flexible amino acid linker. The resulting complexes had activity in excess of that of the wild type IMPase, confirming that Calbindin-D28k activated IMPase. Low resolution structural analysis using small angle X-ray scattering (SAXS) revealed a V-shaped IMPase-calbindin complex which was in contrast to the more linear shape predicted by computational docking models. Here we also obtained the first crystal structure of Calbindin-D28k. This crystal structure gave the first direct visualization of the calcium binding sites of the protein and is a better model of Calbindin-D28k in solution than the previous NMR structure

Linearity in Cell Signaling Pathways

Accurate cellular communication is of paramount importance for the development, growth, and maintenance of multi-cellular organisms. Communication between cells is carried out by a highly conserved set of signaling pathways, whose dysregulation can lead to many diseases. The molecular details of these signaling pathways are now well-characterized, allowing researchers to investigate the emergent properties that arise from the complex signaling networks. These properties often arise from counter-intuitive or paradoxical mechanisms, meaning that systems-level analysis is necessary. Importantly, mathematical models have been constructed for many pathways that capture measured reaction rates and protein levels. These mathematical models successfully recapitulate dynamic responses of each pathway. Here, I investigated the input-output response of the Wnt, MAPK/ERK, and Tgfβ pathways using analytical and numerical treatment of mathematical models. Using this approach, I found that the distinct architectures of the three signaling pathways lead to a convergent behavior, linear input-output response. Specifically, mathematical analysis reveals that a futile cycle in the Wnt pathway, a kinase cascade coupled to feedback in the ERK pathway, and nucleocytoplasmic shuttling in the Tgfβ pathways all yield linear signal transmission. I then verified this finding experimentally in the Wnt and ERK pathways. For the Wnt pathway, direct measurements of the input-output response reveal that β-catenin is linear with respect to Wnt co-receptor LRP5/6 activity up until receptor saturation. For the ERK pathway, direct measurements indicate a linear relationship between phosphorylated ERK1/2 and the concentration of EGF ligand, up until saturation of ERK1/2. Finally, mathematical modeling reveals that linear response in the Wnt pathway, in conjunction with a recently identified cis-regulatory motif, is sufficient to explain gene expression buffering to perturbations. Therefore, this thesis demonstrates how linearity emerges across three dissimilar architectures, and introduces a novel benefit for linear signal transmission in biology.</p

Protein-responsive ribozyme switches in eukaryotic cells

Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers