Signalling pathways in the pathogenesis of Cryptococcus

Efficient communication with the environment is critical for all living organisms. Fungi utilize complex signalling systems to sense their environments and control proliferation, development and in some cases virulence. Well-studied signalling pathways include the protein kinase A/cyclic AMP (cAMP), protein kinase C (PKC)/mitogen-activated protein kinase (MAPK), lipid signalling cascades, and the calcium–calcineurin signalling pathway. The human pathogenic basidiomycetous fungus Cryptococcus neoformans deploys sensitive signalling systems to survive in the human host, leading to life-threatening meningoencephalitis. Known virulence traits of this fungus, including the antioxidant melanin production, the antiphagocytic polysaccharide capsule and the ability to grow at 37°C, are orchestrated by complex signalling networks, whose understanding is crucial to better treat, diagnose and prevent cryptococcosis.     

Towards targeting signalling networks by functional phosphoproteomics

We present the development of Proteo-Mode, an instrument for automated, high-throughput preparation of phosphoproteins for proteomics analysis of complex cellular signalling networks involving multiple, time-dependent protein phosphorylation events. Proteo-Mode automates all steps in the network analysis of phosphoproteins by proteomics method. This enables the integrated response of complex cellular signalling networks to be analyzed in normal and abnormal (disease) states and provides new perspectives in targeting and evaluation of the effects of therapeutic compounds on such networks.     

The proteomics of feather development in pied flycatchers (Ficedula hypoleuca) with different plumage coloration

The genetic theory of morphological evolution postulates that form evolves largely by changing the expression proteins that are functionally conserved. It follows that understanding the function of proteins during different phases of development as well as the mechanisms by which the functions are modified is a prerequisite for understanding evolutionary change. Male pied flycatchers exhibit marked phenotypic variation in their breeding plumage. This variation has repeatedly been shown to have adaptive significance, but the molecular basis of this variation is not known. Here, we characterize the proteome of developing pied flycatcher feathers from differently pigmented males and also introduce a new method for examining the effect sizes of expression differences in protein interaction networks. Approximately 300 proteins were identified in the developing feathers of males. Gene products associated with cellular transport, cell metabolism and protein synthesis formed a large part of the developing feather proteome. Sixty‐five proteins associated with the development of the epidermis and/or pigmentation were detected in the data. The examination of expression level differences of protein–protein interaction networks revealed an immunological signalling–related network to exhibit significantly higher expression in black compared to brown males. Additionally, indications of differences in energy balance and oxidative stress related characteristics were detected. Together, these results provide new insight into the molecular mechanisms and evolutionary significance of plumage colour variation.     

Discrete logic modelling as a means to link protein signalling networks with functional analysis of mammalian signal transduction

Large‐scale protein signalling networks are useful for exploring complex biochemical pathways but do not reveal how pathways respond to specific stimuli. Such specificity is critical for understanding disease and designing drugs. Here we describe a computational approach—implemented in the free CNO software—for turning signalling networks into logical models and calibrating the models against experimental data. When a literature‐derived network of 82 proteins covering the immediate‐early responses of human cells to seven cytokines was modelled, we found that training against experimental data dramatically increased predictive power, despite the crudeness of Boolean approximations, while significantly reducing the number of interactions. Thus, many interactions in literature‐derived networks do not appear to be functional in the liver cells from which we collected our data. At the same time, CNO identified several new interactions that improved the match of model to data. Although missing from the starting network, these interactions have literature support. Our approach, therefore, represents a means to generate predictive, cell‐type‐specific models of mammalian signalling from generic protein signalling networks.     

Kolmogorov complexity of epithelial pattern formation: The role of regulatory network configuration

The tissues of multicellular organisms are made of differentiated cells arranged in organized patterns. This organization emerges during development from the coupling of dynamic intra- and intercellular regulatory networks. This work applies the methods of information theory to understand how regulatory network structure both within and between cells relates to the complexity of spatial patterns that emerge as a consequence of network operation. A computational study was performed in which undifferentiated cells were arranged in a two dimensional lattice, with gene expression in each cell regulated by identical intracellular randomly generated Boolean networks. Cell–cell contact signalling between embryonic cells is modeled as coupling among intracellular networks so that gene expression in one cell can influence the expression of genes in adjacent cells. In this system, the initially identical cells differentiate and form patterns of different cell types. The complexity of network structure, temporal dynamics and spatial organization is quantified through the Kolmogorov-based measures of normalized compression distance and set complexity. Results over sets of random networks that operate in the ordered, critical and chaotic domains demonstrate that: (1) ordered and critical networks tend to create the most information-rich patterns; (2) signalling configurations in which cell-to-cell communication is non-directional mostly produce simple patterns irrespective of the internal network domain; and (3) directional signalling configurations, similar to those that function in planar cell polarity, produce the most complex patterns, but only when the intracellular networks function in non-chaotic domains.     

Syntrophins entangled in cytoskeletal meshwork: Helping to hold it all together

Syntrophins are a family of 59 kDa peripheral membrane‐associated adapter proteins, containing multiple protein‐protein and protein‐lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub‐cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin‐glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer’s disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.     

Involvement of protein kinases and calcium in the * NO-signalling cascade for defence-gene induction in ozonated tobacco plants

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.     

GPCR signalling to the translation machinery

G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and β-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a “GPCR signature” impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.     

Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.