A unified approach to dissecting biphasic responses in cell signaling.

Biphasic responses are encountered at all levels in biological systems. At the cellular level, biphasic dose-responses are widely encountered in cell signaling and post-translational modification systems and represent safeguards against overactivation or overexpression of species. In this paper, we provide a unified theoretical synthesis of biphasic responses in cell signaling systems, by assessing signaling systems ranging from basic biochemical building blocks to canonical network structures to well-characterized exemplars on one hand, and examining different types of doses on the other. By using analytical and computational approaches applied to a range of systems across levels (described by broadly employed models), we reveal (i) design principles enabling the presence of biphasic responses, including in almost all instances, an explicit characterization of the parameter space (ii) structural factors which preclude the possibility of biphasic responses (iii) different combinations of the presence or absence of enzyme-biphasic and substrate-biphasic responses, representing safeguards against overactivation and overexpression, respectively (iv) the possibility of broadly robust biphasic responses (v) the complete alteration of signaling behavior in a network due to biphasic interactions between species (biphasic regulation) (vi) the propensity of different co-existing biphasic responses in the Erk signaling network. These results both individually and in totality have a number of important consequences for systems and synthetic biology.

Network regulation meets substrate modification chemistry.

Biochemical networks are at the heart of cellular information processing. These networks contain distinct facets: (i) processing of information from the environment via cascades/pathways along with network regulation and (ii) modification of substrates in different ways, to confer protein functionality, stability and processing. While many studies focus on these factors individually, how they interact and the consequences for cellular systems behaviour are poorly understood. We develop a systems framework for this purpose by examining the interplay of network regulation (canonical feedback and feed-forward circuits) and multisite modification, as an exemplar of substrate modification. Using computational, analytical and semi-analytical approaches, we reveal distinct and unexpected ways in which the substrate modification and network levels combine and the emergent behaviour arising therefrom. This has important consequences for dissecting the behaviour of specific signalling networks, tracing the origins of systems behaviour, inference of networks from data, robustness/evolvability and multi-level engineering of biomolecular networks. Overall, we repeatedly demonstrate how focusing on only one level (say network regulation) can lead to profoundly misleading conclusions about all these aspects, and reveal a number of important consequences for experimental/theoretical/data-driven interrogations of cellular signalling systems.

Symmetry breaking meets multisite modification.

Multisite modification is a basic way of conferring functionality to proteins and a key component of post-translational modification networks. Additional interest in multisite modification stems from its capability of acting as complex information processors. In this paper, we connect two seemingly disparate themes: symmetry and multisite modification. We examine different classes of random modification networks of substrates involving separate or common enzymes. We demonstrate that under different instances of symmetry of the modification network (invoked explicitly or implicitly and discussed in the literature), the biochemistry of multisite modification can lead to the symmetry being broken. This is shown computationally and consolidated analytically, revealing parameter regions where this can (and in fact does) happen, and characteristics of the symmetry-broken state. We discuss the relevance of these results in situations where exact symmetry is not present. Overall, through our study we show how symmetry breaking (i) can confer new capabilities to protein networks, including concentration robustness of different combinations of species (in conjunction with multiple steady states); (ii) could have been the basis for ordering of multisite modification, which is widely observed in cells; (iii) can significantly impact information processing in multisite modification and in cell signalling networks/pathways where multisite modification is present; and (iv) can be a fruitful new angle for engineering in synthetic biology and chemistry. All in all, the emerging conceptual synthesis provides a new vantage point for the elucidation and the engineering of molecular systems at the junction of chemical and biological systems.

Proteins help our cells perform the chemical reactions necessary for life. Once proteins are made, they can also be modified in different ways. This can simply change their activity, or otherwise make them better suited for their specific jobs within the cell. Biological 'catalysts' called enzymes carry out protein modifications by reversibly adding (or removing) chemical groups, such as phosphate groups. 'Multisite modifications' occur when a protein has two or more modifications in different areas, which can be added randomly or in a specific sequence. The combination of all the modifications attached to a protein acts like a chemical barcode and confers a specific function to the protein. Modification networks add levels of complexity above individual proteins. These encompass not only the proteins in a cell or tissue, but also the different enzymes that can modify them, and how they all interact with each other. Although our knowledge of these networks is substantial, basic aspects, such as how the ordering of multisite modification systems emerges, is still not well understood. Using a simple set of multisite modifications, Ramesh and Krishnan set out to study the potential mechanisms allowing the creation of order in this context. Symmetry is a pervasive theme across the sciences. In biology, symmetry and how it may be broken, is important to understand, for example, how organism develop. Ramesh and Krishnan used the perspective of symmetry in protein networks to uncover the origins of ordering. First, mathematical models of simple modification networks were created based on their basic descriptions. This system centred on proteins that could have phosphate modifications at two possible sites. The network was 'symmetric', meaning that the rate of different sets of chemical reactions was identical, as were the amounts of all the enzymes involved. Dissecting the simulated network using a variety of mathematical approaches showed that its initial symmetry could break, giving rise to sets of ordered multisite modifications. Breaking symmetry did not require any additional features or factors; the basic chemical 'ingredients' of protein modification were all that was needed. The prism of symmetry also revealed other aspects of these multisite modification networks, such as robustness and oscillations. This study sheds new light on the mechanism behind ordering of protein modifications. In the future, Ramesh and Krishnan hope that this approach can be applied to the study of not just proteins but also a wider range of biochemical networks.

Exploring cyclic networks of multisite modification reveals origins of information processing characteristics.

Multisite phosphorylation (and generally multisite modification) is a basic way of encoding substrate function and circuits/networks of post-translational modifications (PTM) are ubiquitous in cell signalling. The information processing characteristics of PTM systems are a focal point of broad interest. The ordering of modifications is a key aspect of multisite modification, and a broad synthesis of the impact of ordering of modifications is still missing. We focus on a basic class of multisite modification circuits: the cyclic mechanism, which corresponds to the same ordering of phosphorylation and dephosphorylation, and examine multiple variants involving common/separate kinases and common/separate phosphatases. This is of interest both because it is encountered in concrete cellular contexts, and because it serves as a bridge between ordered (sequential) mechanisms (representing one type of ordering) and random mechanisms (which have no ordering). We show that bistability and biphasic dose response curves of the maximally modified phosphoform are ruled out for basic structural reasons independent of parameters, while oscillations can result with even just one shared enzyme. We then examine the effect of relaxing some basic assumptions about the ordering of modification. We show computationally and analytically how bistability, biphasic responses and oscillations can be generated by minimal augmentations to the cyclic mechanism even when these augmentations involved reactions operating in the unsaturated limit. All in all, using this approach we demonstrate (1) how the cyclic mechanism (with single augmentations) represents a modification circuit using minimal ingredients (in terms of shared enzymes and sequestration of enzymes) to generate bistability and oscillations, when compared to other mechanisms, (2) new design principles for rationally designing PTM systems for a variety of behaviour, (3) a basis and a necessary step for understanding the origins and robustness of behaviour observed in basic multisite modification systems.