Toggle Switch Research Articles

Potential of orthogonal and cross-talk quorum sensing for dynamic regulation in cocultivation

Quorum sensing (QS) can be used to regulate bacterial population in a cell-density-dependent manner through devices such as synchronized lysis circuit (SLC) and metabolic toggle switch (MTS). However, there is still a lack of studies on cocultivation with different QS-based devices, and the widespread QS crosstalk is usually ignored in metabolic engineering. Taking microbial production of isopropanol from cellobiose with two cross-feeding strains as an example, we have mathematically modeled a comprehensive set of cocultivation configurations equipped with different QS combinations from four QS systems (lux, rpa, tra, las), with a focus on the consequence of designs with various QS crosstalk. The simulation results predict the superior performance of cocultivation with both strains adopted QS-based regulation. Furthermore, several combined QS devices with QS crosstalk could outperform the natural QS devices, and most of QS combinations with crosstalk could deliver a similar or better performance than those simulated with crosstalk ignored, thanks to the positive contribution of cross regulation in coordinating competitive growth and concomitant activation of regulated cellular processes. This work reveals theoretical potential of cocultivation with sophisticated QS-based regulation and potential value of QS devices with crosstalk in the future design of such systems.

A Novel Curcumin Isomer, cis‐transCurcumin, Activates Adenosine Receptor Subtypes A1 And A3: Potential for Pain Therapy

A wide variety of plants produce compounds that contain the 4‐hydroxy‐3‐methoxybenzyl or “vanillyl” group. These vanilloid compounds include such well‐known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory Gαsubunit (subtypes A1and A3). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis‐transcurcumin (CTCUR), at AR subtypes A1and A3. Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A1 and A3. Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A1 with a Ki of 306 nM and to subtype A3 with a Ki of 400 nM. Microscopy results confirm sub‐micromolar affinity for A1, but not for A3. Docking results confirm sub‐micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the “toggle switch” domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.

Structural basis for the tethered peptide activation of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-β-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif(φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.

MD Simulations Revealing Special Activation Mechanism of Cannabinoid Receptor 1.

Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) that is gaining much interest for its regulating role in the central nervous system and its value as a drug target. Structures of CB1 in inactive and active states have revealed conformational change details that are not common in other GPCRs. Here, we performed molecular dynamics simulations of CB1 in different ligand binding states and with mutations to reveal its activation mechanism. The conformational change of the “twin toggle switch” residues F2003.36 and W3566.48 that correlates with ligand efficacy is identified as a key barrier step in CB1 activation. Similar conformational change of residues 3.36/6.48 is also observed in melanocortin receptor 4, showing this “twin toggle switch” residue pair is crucial for the activation of multiple GPCR members.

Designing Biological Circuits: From Principles to Applications.

Genetic circuit design is a well-studied problem in synthetic biology. Ever since the first genetic circuits─the repressilator and the toggle switch─were designed and implemented, many advances have been made in this area of research. The current review systematically organizes a number of key works in this domain by employing the versatile framework of generalized morphological analysis. Literature in the area has been mapped on the basis of (a) the design methodologies used, ranging from brute-force searches to control-theoretic approaches, (b) the modeling techniques employed, (c) various circuit functionalities implemented, (d) key design characteristics, and (e) the strategies used for the robust design of genetic circuits. We conclude our review with an outlook on multiple exciting areas for future research, based on the systematic assessment of key research gaps that have been readily unravelled by our analysis framework.

Mechanistic origin of partial agonism of tetrahydrocannabinol for cannabinoid receptors

Cannabinoid receptor 1 (CB1) is a therapeutically relevant drug target for controlling pain, obesity, and other central nervous system disorders. However, full agonists and antagonists of CB1 have been reported to cause serious side effects in patients. Therefore, partial agonists have emerged as a viable alternative as they can mitigate overstimulation and side effects. One of the key bottlenecks in the design of partial agonists, however, is the lack of understanding of the molecular mechanism of partial agonism itself. In this study, we examine two mechanistic hypotheses for the origin of partial agonism in cannabinoid receptors and predict the mechanistic basis of partial agonism exhibited by Δ9-Tetrahydrocannabinol (THC) against CB1. In particular, we inspect whether partial agonism emerges from the ability of THC to bind in both agonist and antagonist-binding poses or from its ability to only partially activate the receptor. We used extensive molecular dynamics simulations and Markov state modeling to capture the THC binding in both antagonist and agonist-binding poses in the CB1 receptor. Furthermore, we predict that binding of THC in the agonist-binding pose leads to rotation of toggle switch residues and causes partial outward movement of intracellular transmembrane helix 6 (TM6). Our simulations also suggest that the alkyl side chain of THC plays a crucial role in determining partial agonism by stabilizing the ligand in the agonist and antagonist-like poses within the pocket. Taken together, this study provides important insights into the mechanistic origin of the partial agonism of THC.

The Toggle Switch Model for Gene Expression Change during the Prenatal-to-Postnatal Transition in Mammals.

The prenatal-to-postnatal transition is a pivotal process in the life cycle whereby an organism shifts from responding to intrauterine cues to undergoing extrauterine stresses with many physiological adaptations. However, the molecular basis underlying the evolutionarily conserved physiological adaptations remains elusive. Here, we analyze the transcriptomes of seven organs across developmental time points from five mammalian species by constructing computational coexpression networks and report a developmental shift of gene expression at the perinatal stage. The low-to-high and high-to-low expressed genes tightly coalesce in the functional categories and gene regulatory pathways that implicate the physiological adaptions during the prenatal-to-postnatal transition, including lipid metabolism, circadian rhythm, immune response, cell cycle, and cell division. The low-to-high and high-to-low expressed genes around the perinatal stage tend to form the mutually inhibitory toggle switch gene pairs linking the gene regulatory networks in response to the environmental changes. We thus propose the toggle switch model for the developmental shift of gene expression as a mechanic framework to investigate how the physiological adaptations occur during the prenatal-to-postnatal transition.

Building an RNA-Based Toggle Switch Using Inhibitory RNA Aptamers.

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in degrading conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.

Cryo-EM structures of human bradykinin receptor-Gq proteins complexes

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.

Time‐optimal control of piecewise affine bistable gene‐regulatory networks

AbstractWe study the minimal‐time problem for a piecewise affine bistable switch. Motivated by applications in synthetic biology and biotechnology, the aim is to minimize the time needed for this system to achieve transitions between its two stable steady states. The latter represents the two possible states of a genetic toggle switch, a synthetic flip‐flop device playing a fundamental role in biocomputing and gene therapy. Results show that a time‐optimal transition between states should pass by an undifferentiated state, which is well known in cell biology for its importance in fate differentiation of cells. In order to characterize the capacity of the system to achieve transitions, we provide a lower bound on the minimal time, whose knowledge becomes relevant when considering realistic systems involving subsystems evolving on different time scales. Then, we show numerical simulations of optimal trajectories illustrating the structure of the bang‐bang optimal control for different scenarios.

A model of fluid expulsion from compacting tight sedimentary rocks based on the Toggle-Switch algorithm

Expulsion of fluids from tight rocks is an important process in sedimentary basins for phenomena such as distribution of overpressure and primary migration of hydrocarbons. This paper shows that the “Toggle Switch” (TS) algorithm of Miller and Nur is well-suited to model expulsion of brine by hydraulic fracturing from tight rocks undergoing compaction. Local random overpressure is generated in the TS algorithm using a void ratio function with random compaction length. The TS algorithm equilibrates critical fluid pressure by local exchange of excess fluid. We show that the algorithm gives net migration of fluid upwards, because the least compressive stress is decreasing towards the surface. The random void ratio makes the total expulsion rate from the layer intermittent. In the limit of zero cell size the expulsion rate approaches a mean value. When the rock has a random strength, it is shown that critically pressured clusters remain after the TS algorithm has been applied. The implementation of the algorithm is mass conservative, which ensures that the expelled mass is exactly the mass of pore fluid lost by compaction. Expressions are derived for the rate of fluid expulsion and for an effective fracture permeability.

Stability Analysis of Parameter Varying Genetic Toggle Switches Using Koopman Operators

The genetic toggle switch is a well known model in synthetic biology that represents the dynamic interactions between two genes that repress each other. The mathematical models for the genetic toggle switch that currently exist have been useful in describing circuit dynamics in rapidly dividing cells, assuming fixed or time-invariant kinetic rates. There is a growing interest in being able to model and extend synthetic biological function for growth conditions such as stationary phase or during nutrient starvation. As cells transition from one growth phase to another, kinetic rates become time-varying parameters. In this paper, we propose a novel class of parameter varying nonlinear models that can be used to describe the dynamics of genetic circuits, including the toggle switch, as they transition from different phases of growth. We show that there exists unique solutions for this class of systems, as well as for a class of systems that incorporates the microbial phenomena of quorum sensing. Further, we show that the domain of these systems, which is the positive orthant, is positively invariant. We also showcase a theoretical control strategy for these systems that would grant asymptotic monostability of a desired fixed point. We then take the general form of these systems and analyze their stability properties through the framework of time-varying Koopman operator theory. A necessary condition for asymptotic stability is also provided as well as a sufficient condition for instability. A Koopman control strategy for the system is also proposed, as well as an analogous discrete time-varying Koopman framework for applications with regularly sampled measurements.

Identifying toggle genes from transcriptome-wide scatter: A new perspective for biological regulation

The study of gene expression variability, especially for cancer and cell differentiation studies, has become important. Here, we investigate transcriptome-wide scatter of 23 cell types and conditions across different levels of biological complexity. We focused on genes that act like toggle switches between pairwise replicates of the same cell type, i.e. genes expressed in one replicate and not expressed in the other, sometimes also referred as ON/OFF genes. The proportion of these toggle genes dramatically increases from unicellular to multicellular organization, especially for development and cancer cells. A relevant portion of toggle switches are non-coding genes: in unicellular systems the most represented classes are tRNA and rRNA, while multicellular systems more frequently show lncRNA, sncRNA and pseudogenes. Notably, disease associated microRNAs (miRNAs), pseudogenes and numerous uncharacterized transcripts are present in both development and cancer cells. On top of the known intrinsic and extrinsic factors, our work indicates toggle genes as a novel collective component creating transcriptome-wide variability. This requires further investigation for elucidating both evolutionary and disease processes.

Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of ‘tyrosine toggle switch’ required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist – cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

Structure, function and pharmacology of human itch receptor complexes.

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues(agents that promotesecretion) and is involved in itch signals and eliciting pseudoallergic reactions3-6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2-Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2-Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros-Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.

Understanding the role of the CB1 toggle switch in interaction networks using molecular dynamics simulation

The cannabinoid receptor 1 (CB1) is a class A G-protein coupled receptor (GPCR) that can exert various effects on the human body through the endocannabinoid system. Understanding CB1 activation has many benefits for the medical use of cannabinoids. A previous study reported that CB1 has two notable residues referred to as the toggle switch, F3.36 and W6.48, which are important for its activation mechanism. We performed a molecular dynamics simulation with a mutation in the toggle switch to examine its role in active and inactive states. We also examined structural changes, the residue–residue interaction network, and the interaction network among helices and loops of wildtype and mutant CB1 for both activation states. As a result, we found that the energetic changes in the hydrogen-bond network of the Na+ pocket, extracellular N-terminus–TM2–ECL1–TM3 interface including D2.63–K3.28 salt-bridge, and extracellular ECL2–TM5–ECL3–TM6 interface directly linked to the toggle switch contribute to the stability of CB1 by the broken aromatic interaction of the toggle switch. It makes the conformation of inactive CB1 receptor to be unstable. Our study explained the role of the toggle switch regarding the energetic interactions related to the Na+ pocket and extracellular loop interfaces, which could contribute to a better understanding of the activation mechanism of CB1.

Organizing Interactions Between a Robotic Manipulator and a Technological Equipment Control Panel

Algorithms for the organization of standalone operation of a robotic manipulator have been considered in this Article. The controls on the control panel of a man-made equipment piece have been the manipulated object. A robotic manipulator fitted with a control system, and a machine vision system, have been the subsystems under study. The robotic manipulator interacts with the objects to switch on and off a toggle switch, turn a rotary switch, press on a pushbutton, etc. Manipulating the objects in question involves assessing of their positions and orientations, which is achieved through the machine vision system. Coordinated operation of the subsystems is required to plan and to implement control of the manipulator, while taking into account positions of the objects relative to each other. A configuration of interactions between the manipulator and the control panel, a method for organizing coordinated functioning of the machine vision system when manipulating controls, which includes assessing positions of the object of interest relative to the robotic manipulator, and an algorithm for planning control based on artificial neural network technology have been presented in this Article.

Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.

Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.

Dynamic metabolic engineering of Escherichia coli improves fermentation for the production of pyruvate and its derivatives

Pyruvate is a key intermediate that is involved in various synthetic metabolic pathways for microbial chemical and fuel production. It is widely used in the food, chemical, and pharmaceutical industries. However, the microbial production of pyruvate and its derivatives compete with microbial cell growth, as pyruvate is an important metabolic intermediate that serves as a hub for various endogenous metabolic pathways, including gluconeogenesis, amino acid synthesis, TCA cycle, and fatty acid biosynthesis. To achieve a more efficient bioprocess for the production of pyruvate and its derivatives, it is necessary to reduce the metabolic imbalance between cell growth and target chemical production. For this purpose, we devised a dynamic metabolic engineering strategy within an Escherichia coli model, in which a metabolic toggle switch (MTS) was employed to redirect metabolic flux from the endogenous pathway toward the target synthetic pathway. Through a combination of TCA cycle interruption through MTS and reduction of pyruvate consumption in endogenous pathways, we achieved a drastic improvement (163mM, 26-fold) in pyruvate production. In addition, we demonstrated the redirection of metabolic flux from excess pyruvate toward isobutanol production. The final isobutanol production titer of the strain harboring MTS was 26% improved compared with that of the control strain.