Necessary conditions for a minimal model of receptor to show adaptive response over a wide range of levels of stimulus

Sensory systems respond to temporal changes in the stimulus and adapt to the new level when it persists, this pattern of response being maintained in a wide range of levels of stimulus. Here we use a simple model of adaptation developed by Segel et al. (J. Theor. Biol. 120 (1986) 151-179) and extended by Hauri and Ross (Biophys. J. 68 (1995) 708-722) to study the conditions in which it shows wide range of response. The model consists of a receptor that switches between a variable number of states, either by mass action law or by covalent modification. Using a global optimization procedure, we have optimized the adaptive response of the alternatives of the model with different number of states. We find that it is impossible to obtain a wide range of response if the receptor switches between states following mass-action laws, irrespective of the number of states. Instead, a wide range (of five orders of magnitude of ligand concentration) can be obtained if the receptor switches between several states by irreversible covalent modification, in agreement with previous models. Therefore, in this model, expenditure of energy to maintain a large number of covalent modification cycles operating outside equilibrium is necessary to achieve a wide range of response. The optimal values of the parameters present similar patterns to those reported for specific receptors, but there is no quantitative agreement. For instance, ligand affinity varies several orders of magnitude between the different states of the receptor, what is unlikely to be fulfilled by real systems. To see if the minimal model can show adaptive response and range with quantitatively plausible parameter values a sub-optimal receptor was studied, finding that adaptive response of high intensity can still be obtained in at least three orders of magnitude.     

Intrinsic Feedbacks in MAPK Signaling Cascades Lead to Bistability and Oscillations

Previous studies have demonstrated that double phosphorylation of a protein can lead to bistability if some conditions are fulfilled. It was also shown that the signaling behavior of a covalent modification cycle can be quantitatively and, more importantly, qualitatively modified when this cycle is coupled to a signaling pathway as opposed to being isolated. This property was named retroactivity. These two results are studied together in this paper showing the existence of interesting phenomena—oscillations and bistability—in signaling cascades possessing at least one stage with a double-phosphorylation cycle as in MAPK cascades.     

Template-directed assembly of receptor signaling complexes

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.     

The CBL–CIPK network mediates different signaling pathways in plants

The calcineurin B-like protein–CBL-interacting protein kinase (CBL–CIPK) signaling pathway in plants is a Ca²⁺-related pathway that responds strongly to both abiotic and biotic environmental stimuli. The CBL–CIPK system shows variety, specificity, and complexity in response to different stresses, and the CBL–CIPK signaling pathway is regulated by complex mechanisms in plant cells. As a plant-specific Ca²⁺ sensor relaying pathway, the CBL–CIPK pathway has some crosstalk with other signaling pathways. In addition, research has shown that there is crosstalk between the CBL–CIPK pathway and the low-K⁺ response pathway, the ABA signaling pathway, the nitrate sensing and signaling pathway, and others. In this paper, we summarize and review research discoveries on the CBL–CIPK network. We focus on the different modification and regulation mechanisms (phosphorylation and dephosphorylation, dual lipid modification) of the CBL–CIPK network, the expression patterns and functions of CBL–CIPK network genes, the responses of this network to abiotic stresses, and its crosstalk with other signaling pathways. We also discuss the technical research methods used to analyze the CBL–CIPK network and some of its newly discovered functions in plants.     

Adaptation Dynamics in Densely Clustered Chemoreceptors

In many sensory systems, transmembrane receptors are spatially organized in large clusters. Such arrangement may facilitate signal amplification and the integration of multiple stimuli. However, this organization likely also affects the kinetics of signaling since the cytoplasmic enzymes that modulate the activity of the receptors must localize to the cluster prior to receptor modification. Here we examine how these spatial considerations shape signaling dynamics at rest and in response to stimuli. As a model system, we use the chemotaxis pathway of Escherichia coli, a canonical system for the study of how organisms sense, respond, and adapt to environmental stimuli. In bacterial chemotaxis, adaptation is mediated by two enzymes that localize to the clustered receptors and modulate their activity through methylation-demethylation. Using a novel stochastic simulation, we show that distributive receptor methylation is necessary for successful adaptation to stimulus and also leads to large fluctuations in receptor activity in the steady state. These fluctuations arise from noise in the number of localized enzymes combined with saturated modification kinetics between the localized enzymes and the receptor substrate. An analytical model explains how saturated enzyme kinetics and large fluctuations can coexist with an adapted state robust to variation in the expression levels of the pathway constituents, a key requirement to ensure the functionality of individual cells within a population. This contrasts with the well-mixed covalent modification system studied by Goldbeter and Koshland in which mean activity becomes ultrasensitive to protein abundances when the enzymes operate at saturation. Large fluctuations in receptor activity have been quantified experimentally and may benefit the cell by enhancing its ability to explore empty environments and track shallow nutrient gradients. Here we clarify the mechanistic relationship of these large fluctuations to well-studied aspects of the chemotaxis system, precise adaptation and functional robustness.     

A Discrete Dynamical System Approach to Pathway Activation Profiles of Signaling Cascades

In living organisms, cascades of covalent modification cycles are one of the major intracellular signaling mechanisms, allowing to transduce physical or chemical stimuli of the external world into variations of activated biochemical species within the cell. In this paper, we develop a novel method to study the stimulus–response of signaling cascades and overall the concept of pathway activation profile which is, for a given stimulus, the sequence of activated proteins at each tier of the cascade. Our approach is based on a correspondence that we establish between the stationary states of a cascade and pieces of orbits of a 2D discrete dynamical system. The study of its possible phase portraits in function of the biochemical parameters, and in particular of the contraction/expansion properties around the fixed points of this discrete map, as well as their bifurcations, yields a classification of the cascade tiers into three main types, whose biological impact within a signaling network is examined. In particular, our approach enables to discuss quantitatively the notion of cascade amplification/attenuation from this new perspective. The method allows also to study the interplay between forward and “retroactive” signaling, i.e., the upstream influence of an inhibiting drug bound to the last tier of the cascade.     

Selective cysteine modification of metal‐free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI‐MS

Human metallothionein 1a, a protein with two cysteine‐rich metal‐binding domains (α with 11 Cys and β with 9), was analyzed in its metal‐free form by selective, covalent Cys modification coupled with ESI‐MS. The modification profiles of the isolated β‐ and α‐fragments reacted with p‐benzoquinone (Bq), N‐ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI‐mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.     

Ca++/CaMKII switches nociceptor‐sensitizing stimuli into desensitizing stimuli

Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3‐induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second ‘sensitizing’ stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII‐dependent manner. This mechanism was also observed by in vivo C‐fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.     

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.     

Signaling output: it's all about timing and feedbacks

The central questions in understanding signaling pathway specificity are how these pathways encode which stimulus is present and how this stimulus is decoded to yield the correct cell fate decision. In their recent work, Ryu et al (2015) show by stimulation experiments with different ligands how the differential engagement of feedback and feed‐forward regulation leads to different dynamics of pathway activity, which in turn alters cell fate. Moreover, they show that by considering the timescales of the feedback regulations, the different cellular responses can be triggered with pulsed stimulations by a single ligand.     

Simple molecular model for sensing and adaptation based on receptor modification with application to bacterial chemotaxis

A series of simple models to explain adaptation in a sensory system based on reversible covalent modification is developed. The models are applied to the reversible methylation of chemoreceptors in bacteria and by analogy to other sensory transduction systems. The receptor modification system exhibits sensing and adaptation, i.e. raising the stimulus to a new level generates a transient response followed by a return to prestimulus behavior. By means of an analytical solution of the kinetic equation that governs the evolution of the receptor system. an exact expression is obtained for the time required for adaptation. The results account for the most conspicuous properties of the bacterial sensory system; namely, the response times in relation to stimulus changes, the proportionality of receptor modification to receptor occupancy, and the additivity of response times. The analysis indicates how these properties depend upon the parameters of the system, e.g. the rates of covalent modification and demodification, the accuracy of the detector, and the molecular nature of the response regulator. The theory developed for analysis of the bacterial system revealed properties that will be applicable to any system processing sensory information.     

Wanted and wanting: Antibody against methionine sulfoxide

Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35-40; 2009). However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.     

Covalent modification regulates ligand binding to receptor complexes in the chemosensory system of Escherichia coli

In the Escherichia coli chemosensory pathway, receptor modification mediates adaptation to ligand. Evidence is presented that covalent modification influences ligand binding to receptors in complexes with CheW and the kinase CheA. Kinase inhibition was measured with serine receptor complexes in different modification levels; Ki for serine-mediated inhibition increased 10,000-fold from the lowest to the highest level. Without CheA and CheW, ligand binding is unaffected by covalent modification; thus, the influence of covalent modification is mediated only in the receptor complex, a conclusion supported by an analogy to allosteric enzymes and the observation of cooperative kinase inhibition. Also, the finding that a subsaturating serine concentration accelerates active receptor-kinase complex assembly implies that the assembly/disassembly process may also contribute to kinase regulation.     

A Minimal Model of Signaling Network Elucidates Cell-to-Cell Stochastic Variability in Apoptosis

Background

Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli.

Methodology

We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. Gillespie''s stochastic simulation algorithm (SSA) is used in this study.

Conclusions/Significance

We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways.     

Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.

A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.     

Emerging Roles and Research Tools of Atypical Ubiquitination

Ubiquitination is a posttranslational modification characterized by the covalent attachment of ubiquitin molecules to protein substrates. The ubiquitination modification process is reversible, dynamic, and involved in the regulation of various biological processes, such as autophagy, inflammatory responses, and DNA damage responses. The forms of ubiquitin modification are very diverse, incorporating either a single ubiquitin molecule or a complicated ubiquitin polymer, and different types of ubiquitination usually elicit corresponding cellular responses. The development of research tools and strategies has afforded more detailed insight into atypical ubiquitin signaling pathways that were previously poorly understood. Here, an update on the understanding of atypical ubiquitin chain signaling pathways is provided and the recent development of representative research tools for ubiquitin systems is discussed. In addition, the future challenges in ubiquitin research are reflected on and summarized.     

Elucidating the Sources of β-Catenin Dynamics in Human Neural Progenitor Cells

Human neural progenitor cells (hNPCs) form a new prospect for replacement therapies in the context of neurodegenerative diseases. The Wnt/[Formula: see text]-catenin signaling pathway is known to be involved in the differentiation process of hNPCs. RVM cells form a common cell model of hNPCs for in vitro investigation. Previous observations in RVM cells raise the question of whether observed kinetics of the Wnt/[Formula: see text]-catenin pathway in later differentiation phases are subject to self-induced signaling. However, a concern when investigating RVM cells is that experimental results are possibly biased by the asynchrony of cells w.r.t. the cell cycle. In this paper, we present, based on experimental data, a computational modeling study on the Wnt/[Formula: see text]-catenin signaling pathway in RVM cell populations asynchronously distributed w.r.t. to their cell cycle phases. Therefore, we derive a stochastic model of the pathway in single cells from the reference model in literature and extend it by means of cell populations and cell cycle asynchrony. Based on this, we show that the impact of the cell cycle asynchrony on wet-lab results that average over cell populations is negligible. We then further extend our model and the thus-obtained simulation results provide additional evidence that self-induced Wnt signaling occurs in RVM cells. We further report on significant stochastic effects that directly result from model parameters provided in literature and contradict experimental observations.