Transfer Functions for Protein Signal Transduction: Application to a Model of Striatal Neural Plasticity

We present a novel formulation for biochemical reaction networks in the context of signal transduction. The model consists of input-output transfer functions, which are derived from differential equations, using stable equilibria. We select a set of 'source' species, which receive input signals. Signals are transmitted to all other species in the system (the 'target' species) with a specific delay and transmission strength. The delay is computed as the maximal reaction time until a stable equilibrium for the target species is reached, in the context of all other reactions in the system. The transmission strength is the concentration change of the target species. The computed input-output transfer functions can be stored in a matrix, fitted with parameters, and recalled to build discrete dynamical models. By separating reaction time and concentration we can greatly simplify the model, circumventing typical problems of complex dynamical systems. The transfer function transformation can be applied to mass-action kinetic models of signal transduction. The paper shows that this approach yields significant insight, while remaining an executable dynamical model for signal transduction. In particular we can deconstruct the complex system into local transfer functions between individual species. As an example, we examine modularity and signal integration using a published model of striatal neural plasticity. The modules that emerge correspond to a known biological distinction between calcium-dependent and cAMP-dependent pathways. We also found that overall interconnectedness depends on the magnitude of input, with high connectivity at low input and less connectivity at moderate to high input. This general result, which directly follows from the properties of individual transfer functions, contradicts notions of ubiquitous complexity by showing input-dependent signal transmission inactivation.Comment: 13 pages, 5 tables, 15 figure

Signaling pathways for transduction of the initial message of the glycocode into cellular responses

The sugar units of glycan structures store information and establish an alphabet of life. The language of the oligosaccharide coding units is deciphered by receptors such as lectins and the decoded message can be transduced by multiple signaling pathways. Similar to glycoconjugates, these receptors can exhibit pronounced changes in quantitative and qualitative aspects of expression, as attested by a wealth of lectin and immunohistochemical studies. Since histochemistry provides a static picture, it is essential to shed light on the mechanisms of how a recognitive protein-carbohydrate interplay can be transduced into cellular responses. Their consequences for example for cell morphology will then be visible to the histochemist. Therefore, basic signaling routes will be graphically outlined and their trigger potential will be explained by selected examples from the realm of glycosciences

Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers

The chemokine prokineticin 2 (PK2) activates its cognate G protein-coupled receptor (GPCR) PKR2 to elicit various downstream signaling pathways involved in diverse biological processes. Many GPCRs undergo dimerization that can modulate a number of functions including membrane delivery and signal transduction. The aim of this study was to elucidate the interface of PKR2 protomers within dimers by analyzing the ability of PKR2 transmembrane (TM) deletion mutants to associate with wild type (WT) PKR2 in yeast using co-immunoprecipitation and mammalian cells using bioluminescence resonance energy transfer. Deletion of TMs 5-7 resulted in a lack of detectable association with WT PKR2, but could associate with a truncated mutant lacking TMs 6-7 (TM1-5). Interestingly, TM1-5 modulated the distance, or organization, between protomers and positively regulated Gαs signaling and surface expression of WT PKR2. We propose that PKR2 protomers form type II dimers involving TMs 4 and 5, with a role for TM5 in modulation of PKR2 function

P2 purinoceptors signaling in fibroblasts of rat subcutaneous tissue

Mestrado em Biologia Molecular e CelularO tecido conjuntivo parece estar envolvido na génese de diversas condições patológicas. O aumento da rigidez do tecido conjuntivo, resultante da fibrose, pode constituir um factor importante no mecanismo patogénico da dor crónica resistente a fármacos (Langevin & Sherman, 2007). Por outro lado, os nucleótidos extracelulares parecem estar envolvidos na fisiopatologia da dor crónica (Burnstock, 2001). Assim, este estudo teve como objectivo averiguar o efeito dos nucleótidos de adenina e uridina na proliferação e síntese de colagénio tipo I de fibroblastos do tecido subcutâneo de rato em cultura. Os resultados obtidos mostram que a incubação com UTP (0.3-100 M, n=5) induz um aumento da proliferação e da produção de colagénio tipo I, o qual é dependente da concentração. Contrariamente, o agonista selectivo dos receptores P2Y2, o MRS 2768 (10 μM, n=3), não teve qualquer efeito no que se refere à proliferação, mas diminuiu significativamente (P<0.05) a síntese de colagénio tipo I. Uma vez que o aumento da produção de colagénio induzida pelo UTP (100 μM) foi proporcional ao aumento do número de células (proliferação celular),podemos especular que este aumento se deve ao aumento do número de células per si do que a uma maior actividade sintética de cada célula. Assim, ao normalizar os valores do colagénio tipo I em relação aos valores obtidos do MTT para os mesmos momentos/dias, deixamos de observar diferenças estatisticamente significativas entre o controlo e as células expostas ao UTP. Uma vez que os receptores P2Y2 não parecem estar envolvidos nesta resposta do UTP (100 μM), esta poderá estar a ser mediada pela activação dos receptores P2Y4 e/ou P2Y6. Considerando que o RB-2 (10 μM, n=5), um antagonista não selectivo que actua preferencialmente no subtipo de receptores P2Y4, não foi capaz de modificar a resposta induzida pelo UTP (100 μM), os receptores P2Y4 parecem também não estar envolvidos. Por outro lado, o MRS 2578 (100 nM), um antagonista selectivo dos receptores P2Y6, atenuou de forma significativa o aumento induzido pelo UTP (100 μM). A corroborar os nossos resultados, uma análise imunocitoquímica mostrou uma imunorreactividade positiva contra os receptores P2Y2 e P2Y6, mostrando um padrão de marcação citoplasmático/membranar, o qual é típico para este tipo de receptores, ao contrário do padrão nuclear exibido pelo anticorpo contra os receptores P2Y4. Relativamente ao envolvimento dos receptores sensíveis ao ADP, os resultados obtidos mostraram que o ADPβS (10-100 μM, n=3-6), um análogo estável do ADP, não parece induzir efeitos significativamente diferentes (P>0.05) na proliferação celular. Contudo, a sua incubação continuada aumentou a produção de colagénio tipo I de forma dependente da concentração (P<0.05). De modo a identificar os receptores purinérgicos envolvidos neste efeito, testamos o ADPβS (100 μM) na presença do MRS 2179 (0.3 μM), do AR-C 66096 (0.1 μM), e do MRS 2211 (10 μM), os quais antagonizam selectivamente os receptores P2Y1, P2Y12 e P2Y13, respectivamente. O efeito facilitatório induzido pelo ADPβS (100 μM) foi atenuado de forma significativa na presença do antagonista dos receptores P2Y1, o MRS 2179 (0.3 μM, n=3), sem ser afectado pelo antagonista dos receptores P2Y12, o AR- C 66096 (0.1 μM, n=3). Pelo contrário, o MRS 2211 (10 μM, n=2) potenciou o aumento da produção de colagénio induzida pelo ADPβS (100 μM), indicando assim que a síntese de colagénio tipo I induzida pelo receptor P2Y1 pode estar a ser parcialmente influenciada por uma activação síncrona do receptor inibitório P2Y13. Por último, uma análise por imunocitoquímica mostrou que estas células apresentam imunorreactividade positiva para os receptores P2Y1 e P2Y13, exibindo um padrão citoplasmático/membranar, contrariamente ao padrão nuclear dos receptores ostentado pelo anticorpo contra os receptores P2Y12. Concluindo, a remodelação da fáscia superficial induzida pelos fibroblastos parece ser regulada por um balanço entre a activação dos receptores P2Y2 e P2Y6, assim como dos receptores P2Y13 e P2Y1. Clarificar as vias que conduzem ao processo de fibrose pode representar uma oportunidade para esclarecer o seu envolvimento na patogénese da dor crónica musculo-esquelética, bem como ser útil no desenvolvimento de novas estratégias terapêuticas.Connective tissue may be involved in the pathogenesis of a wide variety of disease conditions. Increased connective tissue stiffness due to fibrosis may be an important link to the pathogenic mechanism leading to drug-resistant chronic pain (Langevin & Sherman, 2007). In addition, extracellular nucleotides seem to be involved in the pathophysiology of chronic pain (Burnstock, 2001). Therefore, we aimed at investigating the effect of adenine and uridine nucleotides on the proliferation and synthesis of type I collagen by rat fibroblasts from subcutaneous connective tissue. The results showed that continuous incubation of UTP (0.3-100 M, n=5) concentration-dependently increased fibroblasts proliferation, as also increased the synthesis of type I collagen above the control levels. Conversely, the selective P2Y2 agonist, MRS 2768 (10 μM, n=3), was devoid of effect in what concerns proliferation, but significantly (P<0.05) decreased type I collagen synthesis. Since the increase in type I collagen synthesis induced by UTP (100 μM) was proportional to the increase in the amount of cells in the culture (fibroblasts proliferation), we speculated that such an increase could be related to the increase in the cell number rather than a higher synthetic activity. Thus, we performed a more detailed data analysis, in which we normalized type I collagen production taking into consideration the MTT values obtained at the same time points, and we observed no longer significant differences between control and UTP-exposed cells. Discounting the contribution of MRS 2768-sensitive P2Y2 receptors, UTP (100 μM)-induced increase in cells proliferation could be due to P2Y4 and/or P2Y6 receptor activation. Since RB-2 (10 μM, n=5), a non-selective antagonist that acts preferentially on the P2Y4 subtype, did not modify the effect of UTP (100 μM), P2Y4 does not seem to be involved. In turn, MRS 2578 (100 nM), which is a selective P2Y6 antagonist, significantly attenuated UTP (100 μM)-induced increase. To corroborate our results, an immunocytochemistry analysis showed a positive immunoreactivity against the P2Y2 and P2Y6 receptors exhibiting a cytoplasmic/membrane labeling pattern, which is typical for those receptors in many different cells, conversely to the nuclear labeling pattern exhibited by the antibody against the P2Y4. To investigate the involvement of ADP-sensitive P2 receptors on cell proliferation and extracellular matrix production, fibroblast cultures were continuously incubated with the stable ADP analogue, ADPβS (10-100 μM). Results obtained with ADPβS (10-100 μM, n=3-6) showed no significant (P>0.05) differences in fibroblast cells proliferation. However, a continuous incubation with ADPβS (10-100 μM, n=2-5) concentration-dependently increased type I collagen production by fibroblasts (P<0.05). In order to identify which purinoceptor(s) that could be mediating this effect, we tested ADPβS (100 μM) in the presence of MRS 2179 (0.3 μM), AR-C 66096 (0.1 μM), and MRS 2211 (10 μM), which antagonize selectively ADP-sensitive P2Y1, P2Y12 and P2Y13 receptors, respectively. The facilitatory effect of ADPβS (100 μM) was significantly attenuated in the presence of the P2Y1 antagonist, MRS 2179 (0.3 μM, n=3), without being affected by the P2Y12 antagonist, AR- C 66096 (0.1 μM, n=3). In contrast, MRS 2211 (10 μM, n=2) potentiated the effect of ADPβS (100 μM) on type I collagen synthesis, thus indicating that the P2Y1-receptor-induction of type I collagen synthesis may be partially counteracted by synchronous activation of the inhibitory P2Y13 receptor. Finally, an immunocytochemistry analysis showed that these cells exhibit immunoreactivity to P2Y1 and P2Y13 receptors with a cytoplasmic/membrane staining pattern, conversely to the nuclear pattern of P2Y12. Concluding, a delicate balance between the activation of P2Y2 and P2Y6, as well as P2Y13 and P2Y1 purinoceptors, might regulate fibroblast’s induced superficial fascia remodeling. Targeting the pathways leading to fibrosis may represent an opportunity to clarify its involvement in the pathogenesis of musculoskeletal chronic pain and it may be useful for designing novel therapeutic strategies to overcome this disease

Allo-network drugs: Extension of the allosteric drug concept to protein-protein interaction and signaling networks

Allosteric drugs are usually more specific and have fewer side effects than orthosteric drugs targeting the same protein. Here, we overview the current knowledge on allosteric signal transmission from the network point of view, and show that most intra-protein conformational changes may be dynamically transmitted across protein-protein interaction and signaling networks of the cell. Allo-network drugs influence the pharmacological target protein indirectly using specific inter-protein network pathways. We show that allo-network drugs may have a higher efficiency to change the networks of human cells than those of other organisms, and can be designed to have specific effects on cells in a diseased state. Finally, we summarize possible methods to identify allo-network drug targets and sites, which may develop to a promising new area of systems-based drug design

Development and application of a bioassay for follicle-stimulating hormone : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of vital reproductive processes, such as gametogenesis, follicular development and ovulation. Produced in the anterior pituitary, FSH is a glycoprotein hormone that exists as a family of isohormones. Follicle-stimulating hormone concentrations have traditionally been measured by radioimmunoassay (RIA). However, results generated using RIA are a determination of the immunological activity of FSH. The potential of FSH to generate a biological response cannot be measured by RIA. Therefore, the identification of physiologically significant differences in the activity of these isoforms requires the use of assay systems that can differentiate between the biological activity of the FSH isoforms. Commonly used assays for measuring the biological activity of FSH are based on the measurement of aromatase activity in cultured rat Sertoli cells following stimulation with FSH. However, these assays have an inherently high ethical cost involved due to the use of primary tissue culture. In addition, the variation in these assays associated with differences between animals is difficult to eliminate. Recently a bioassay for human FSH has been described based on FSH stimulation of cyclic AMP production by a Chinese hamster ovary (CHO) cell line stably expressing the human FSH receptor (FSH-R). The purpose of this study was to evaluate the potential usefulness of this CHO FSH-R cell line expressing the human receptor for FSH to be used as a bioassay to measure the biological activity of ovine FSH. The receptor cell line bioassay described in this study is based on the ability of FSH to stimulate cAMP production by cultured CHO FSH-R cells. Optimisation of the culture system to enable the bioactivity of ovine FSH to be measured by bioassay was undertaken. This involved optimising the density of cultured cells, the time in culture and time exposed to FSH and the most suitable dose range for FSH. The influence of matrix effects, such as those exerted by serum was also investigated. The specificity of the assay towards FSH was also determined as was the sensitivity, accuracy and precision of the assay. No stimulation of cAMP production was seen in CHO FSH-R cells following treatment with α-FSH, β-FSH, LH, TSH, GH, prolactin or vasopressin at concentrations up to 10 μg/ml. Although the methodology used differed slightly depending on the presence or absence of serum, all assayed were performed using the following methods and materials. Freshly thawed FSH-R cells were bulked up in culture, and aliquots of 1 x 105 to 5 x 105 cells/well dispensed into 48 well culture dishes and incubated overnight at 37°C. The assay culture media was then replaced with 0.25 ml fresh media (α-MEM + 0.1% BSA + 0.25 mM 3-isobutyl-1-methyl-xanthine) containing varying doses of NIH-FSH-RP2 (RP2) FSH preparations or FSH containing samples, and the cells incubated for 4 hours at 37°C. The assay culture media was then removed and stored frozen at -20°C until assayed for cyclic adenosine monophosphate (cAMP) by RIA. Once optimal assay conditions were determined, the CHO FSH-R cell bioassay was used to measure FSH concentrations in ovine serum, pituitary extracts and medium from cultures of ovine pituitary cells. It was found that the concentrations of FSH in serum from intact sheep was close to the detection limit of the assay. Thus, while FSH concentrations could be measured in serum from some sheep, other animals had concentrations that were too low to be accurately measured by the bioassay in its present form. The assay was, however, well suited to measuring FSH concentrations in serum from sheep that had elevated concentrations of FSH. In one study, FSH concentrations measured by the bioassay were compared to those measured by RIA in sheep that had been ovariectomised and then hypophysectomised. It was found that the profile of FSH concentrations following hypophysectomy was similar whether measured by RIA or by bioassay (R2=0.7513), though absolute concentrations sometimes differed. This suggested that the immunoassay and bioassay were not always measuring the same characteristics of FSH. The assay was also used to measure FSH concentrations in samples of ovine hypophyseal venous blood. However, the results obtained for these samples indicated a poor correlation between FSH concentrations obtained by bioassay and RIA. Levels of bioactive FSH in hypophyseal venous blood fluctuated markedly and were up to 10-fold higher than the associated RIA concentrations. The CHO-cell bioassay was also found to be very suitable for measuring pituitary concentrations of FSH. In one study, pituitary extracts underwent chromatography and the separated isoforms of FSH were analysed by bioassay and RIA. Again, there was excellent correlation (R2=0.9328) between the concentrations of FSH measured both assay types. However, some differences were apparent suggesting a discrepancy in the biological and immunological characteristics of different FSH isoforms. The bioassay was also used to measure FSH concentrations in media from pituitary cells in tissue culture where serially diluted samples displayed good parallelism with the RP2 FSH standard curve. Results of this study demonstrate that the CHO FSH-R cell bioassay is suitable for measuring the biological activity of ovine FSH in a variety of biological fluids. The use of a permanent cell line eliminates the high ethical cost associated with primary tissue culture that other bioassay systems have. The inherent variation associated with culture systems utilising tissue from different sources is also avoided. The sensitivity of the bioassay is suitable for measuring FSH in surgically altered sheep or hypophyseal blood concentrations where FSH levels are generally higher than those in the peripheral circulation. In addition to blood samples, the bioassay is also excellent for monitoring FSH activity in pituitary extracts and in media from tissue culture. However, the sensitivity of the bioassay currently does not always allow measurement of bioactive FSH concentrations in serum samples with low FSH levels. In summary, the CHO FSH-R cell bioassay described in this study offers a useful alternative to RIA and other bioassays for monitoring the biological activity of ovine FSH and its isoforms in various biological fluids. It is concluded that this convenient and robust bioassay may have considerable application in future investigations of ovine FSH bioactivity

The multifaceted roles of PI3Kγ in hypertension, vascular biology, and inflammation

PI3Kγ is a multifaceted protein, crucially involved in cardiovascular and immune systems. Several studies described the biological and physiological functions of this enzyme in the regulation of cardiovascular system, while others stressed its role in the modulation of immunity. Although PI3Kγ has been historically investigated for its role in leukocytes, the last decade of research also dedicated efforts to explore its functions in the cardiovascular system. In this review, we report an overview recapitulating how PI3Kγ signaling participates in the regulation of vascular functions involved in blood pressure regulation. Moreover, we also summarize the main functions of PI3Kγ in immune responses that could be potentially important in the interaction with the cardiovascular system. Considering that vascular and immune mechanisms are increasingly emerging as intertwining players in hypertension, PI3Kγ could be an intriguing pathway acting on both sides. The availability of specific inhibitors introduces a perspective of further translational research and clinical approaches that could be exploited in hypertension

Both Ligand- and Cell-Specific Parameters Control Ligand Agonism in a Kinetic Model of G Protein–Coupled Receptor Signaling

G protein–coupled receptors (GPCRs) exist in multiple dynamic states (e.g., ligand-bound, inactive, G protein–coupled) that influence G protein activation and ultimately response generation. In quantitative models of GPCR signaling that incorporate these varied states, parameter values are often uncharacterized or varied over large ranges, making identification of important parameters and signaling outcomes difficult to intuit. Here we identify the ligand- and cell-specific parameters that are important determinants of cell-response behavior in a dynamic model of GPCR signaling using parameter variation and sensitivity analysis. The character of response (i.e., positive/neutral/inverse agonism) is, not surprisingly, significantly influenced by a ligand's ability to bias the receptor into an active conformation. We also find that several cell-specific parameters, including the ratio of active to inactive receptor species, the rate constant for G protein activation, and expression levels of receptors and G proteins also dramatically influence agonism. Expressing either receptor or G protein in numbers several fold above or below endogenous levels may result in system behavior inconsistent with that measured in endogenous systems. Finally, small variations in cell-specific parameters identified by sensitivity analysis as significant determinants of response behavior are found to change ligand-induced responses from positive to negative, a phenomenon termed protean agonism. Our findings offer an explanation for protean agonism reported in β2-adrenergic and α2A-adrenergic receptor systems