Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.     

Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling

Extracellular interactions involving both secreted and membrane-tethered receptor proteins are essential to initiate signaling pathways that orchestrate cellular behaviors within biological systems. Because of the biochemical properties of these proteins and their interactions, identifying novel extracellular interactions remains experimentally challenging. To address this, we have recently developed an assay, AVEXIS (avidity-based extracellular interaction screen) to detect low affinity extracellular interactions on a large scale and have begun to construct interaction networks between zebrafish receptors belonging to the immunoglobulin and leucine-rich repeat protein families to identify novel signaling pathways important for early development. Here, we expanded our zebrafish protein library to include other domain families and many more secreted proteins and performed our largest screen to date totaling 16,544 potential unique interactions. We report 111 interactions of which 96 are novel and include the first documented extracellular ligands for 15 proteins. By including 77 interactions from previous screens, we assembled an expanded network of 188 extracellular interactions between 92 proteins and used it to show that secreted proteins have twice as many interaction partners as membrane-tethered receptors and that the connectivity of the extracellular network behaves as a power law. To try to understand the functional role of these interactions, we determined new expression patterns for 164 genes within our clone library by using whole embryo in situ hybridization at five key stages of zebrafish embryonic development. These expression data were integrated with the binding network to reveal where each interaction was likely to function within the embryo and were used to resolve the static interaction network into dynamic tissue- and stage-specific subnetworks within the developing zebrafish embryo. All these data were organized into a freely accessible on-line database called ARNIE (AVEXIS Receptor Network with Integrated Expression; www.sanger.ac.uk/arnie) and provide a valuable resource of new extracellular signaling interactions for developmental biology.The individual cells within a multicellular organism communicate with each other to provide coordinated and appropriate cellular responses that ensure the normal development and maintenance of the organism as a whole. Frequently, these intercellular dialogues are initiated by specific binding events mediated by cell surface receptor glycoproteins, the molecular bridges through which cells receive information from their immediate environment and subsequently relay it to cytoplasmic signaling networks. Despite their importance in many different biological contexts, identifying novel extracellular protein interactions remains technically challenging because membrane proteins are difficult to biochemically manipulate, and their interactions are typified by extremely low interaction strengths (1, 2). Several scalable methods to identify this class of interactions have been developed that account for some or all of these challenges: they include approaches based on protein complementation (3), phage display (4), mass spectrometry (5), surface plasmon resonance (6–9), and detection of direct binding between multivalent recombinant proteins (Refs. 10–12; for a review, see Ref. 1). Our own approach, called avidity-based extracellular interaction screen (AVEXIS)1 (see Fig. 1A), involves expression by mammalian cells of soluble recombinant ectodomain regions of cell surface receptor and secreted proteins as either a monobiotinylated bait or a pentamerized β-lactamase-tagged prey. The pentamerization is achieved through a peptide derived from the cartilage oligomeric matrix protein (COMP) (13) and is necessary to increase the local concentration of the ectodomain fragments to effect gains in the overall binding avidity so that even very transient interactions can be detected. We have determined the parameters of the assay and shown that it can reliably detect interactions that are very transient, having monomeric half-lives of ≤0.1 s when subsequently measured in their monomeric state by surface plasmon resonance using purified proteins (10). This assay now permits the systematic screening of thousands of binary interactions and can be used to identify novel receptor-ligand pairs that were previously difficult to detect.Open in a separate windowFig. 1.Summary of AVEXIS method and overall approach to construct and resolve extracellular protein interaction networks. A, the entire ectodomains of endogenous cell surface receptors (pink) are expressed as both monomeric biotinylated baits (B) and pentamerized β-lactamase-tagged (β) preys (P). Bait proteins are immobilized in individual wells of streptavidin-coated microtiter plates and probed with a normalized prey, which, if the two proteins physically interact, is captured within the well. B, a flowchart presenting an overall summary of the work described here, including how interactions from two previous screens (Refs. 10 (Bushell 2008) and 27 (Söllner 2009)) were integrated into the larger network of interactions. C, positive interactions are detected by adding a colorimetric β-lactamase substrate, nitrocefin, which is converted from a yellow to red product. Two typical screening plates are shown illustrating a heterophilic interaction between Cadm3 and Cadm4 that is detected in both bait-prey orientations and a homophilic interaction involving Cadm3. The controls for each prey included a negative bait, the rat Cd4d3+4 protein tag alone (well G6), and a biotinylated anti-rat Cd4 monoclonal antibody (OX68), which captured the Cd4-tagged preys (well G7). Each plate also contained positive control interactions: the rat Cd200R prey was probed against rat Cd200 baits immobilized at the normalized screening threshold and at 1:500 and 1:1,000 dilutions (wells G8–G10, respectively) and against the negative Cd4d3+4 bait (well G12). An additional negative bait (Fgfr1b) was included for both preys (wells G5 and G11). SLRPs, small leucine-rich proteoglycans.To identify novel extracellular receptor-ligand interactions that initiate signaling pathways important for vertebrate development, we have selected two protein families that constitute a significant proportion of the extracellular proteome: the immunoglobulin superfamily (IgSF) (14) and leucine-rich repeat (LRR) (15) families. These two families, which both contain membrane-tethered receptors and secreted proteins, are disproportionately expanded in vertebrates relative to invertebrates and are therefore likely to be involved in initiating vertebrate-specific signaling processes (16). IgSF proteins are used as cell surface recognition molecules in a diverse array of tissues, including the immune and nervous systems (17). They function by forming specific receptor-ligand pairs with other IgSF receptors but also other protein domain families, including the LRR (18). Extracellular LRR domain-containing proteins can be separated into two main families: the small leucine-rich proteoglycans, which are secreted and form a major component of the extracellular matrix (19), and cell surface receptors that are involved in the development and maintenance of the nervous system (20). To elucidate the functional role of novel interactions in vertebrate development, we have chosen to screen for novel receptor-ligand pairs from the zebrafish. This popular model organism has many experimental advantages to study early developmental processes, including the amenability to forward (21–23) and reverse (24, 25) genetics approaches. Importantly, the ready accessibility of large numbers of externally developing translucent embryos and the ability to determine large scale gene expression patterns at different stages of embryonic development make whole organism spatiotemporal gene expression profiling possible (26).Using this approach, we first reported a network of 43 interactions within the zebrafish IgSF proteins (10) and subsequently a neural network of 34 interactions between membrane-tethered receptors from both the IgSF and LRR families (27). Here, we expanded our zebrafish recombinant library by 87 proteins to include many more secreted factors and proteins from other families and discovered 111 new interactions. By combining them with interactions identified in our previous screens, we compiled an expanded static network containing 188 interactions that we analyzed to identify properties of extracellular interaction networks. To begin the functional validation of identified interactions, we determined the developmental expression patterns of all genes encoding proteins within our library and describe here 164 new gene expression patterns, many of which are highly dynamic and tissue-restricted. By integrating the spatiotemporal expression patterns with the interaction network, we were able to determine in which stages and tissues each interaction was likely to function and resolved the aggregate interaction network into stage- and tissue-specific subnetworks. The work described in this study is summarized schematically in Fig. 1B. To facilitate navigation of these data, they were organized into a freely accessible on-line database that provides a valuable and accessible resource of new extracellular signaling interactions for developmental biology.     

The impact of gene expression regulation on evolution of extracellular signaling pathways

Extracellular protein interactions are crucial to the development of multicellular organisms because they initiate signaling pathways and enable cellular recognition cues. Despite their importance, extracellular protein interactions are often under-represented in large scale protein interaction data sets because most high throughput assays are not designed to detect low affinity extracellular interactions. Due to the lack of a comprehensive data set, the evolution of extracellular signaling pathways has remained largely a mystery. We investigated this question using a combined data set of physical pairwise interactions between zebrafish extracellular proteins, mainly from the immunoglobulin superfamily and leucine-rich repeat families, and their spatiotemporal expression profiles. We took advantage of known homology between proteins to estimate the relative rates of changes of four parameters after gene duplication, namely extracellular protein interaction, expression pattern, and the divergence of extracellular and intracellular protein sequences. We showed that change in expression profile is a major contributor to the evolution of signaling pathways followed by divergence in intracellular protein sequence, whereas extracellular sequence and interaction profiles were relatively more conserved. Rapidly evolving expression profiles will eventually drive other parameters to diverge more quickly because differentially expressed proteins get exposed to different environments and potential binding partners. This allows homologous extracellular receptors to attain specialized functions and become specific to tissues and/or developmental stages.     

Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis

Dysregulated cell-cell adhesion plays a critical role in epithelial cancer development. Studies of human and mouse cancers have indicated that loss of adhesion complexes known as adherens junctions contributes to tumor progression and metastasis. In contrast, little is known regarding the role of the related cell-cell adhesion junction, the desmosome, during cancer development. Studies analyzing expression of desmosome components during human cancer progression have yielded conflicting results, and therefore genetic studies using knockout mice to examine the functional consequence of desmosome inactivation for tumorigenesis are essential for elucidating the role of desmosomes in cancer development. Here, we investigate the consequences of desmosome loss for carcinogenesis by analyzing conditional knockout mice lacking Perp, a p53/p63 regulated gene that encodes an important component of desmosomes. Analysis of Perp-deficient mice in a UVB-induced squamous cell skin carcinoma model reveals that Perp ablation promotes both tumor initiation and progression. Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion. Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status. Similarly, human squamous cell carcinomas display loss of PERP expression with retention of adherens junctions components, indicating that this is a relevant stage of human cancer development. Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis. Together, these studies suggest that Perp-deficiency promotes cancer by enhancing cell survival, desmosome loss, and inflammation, and they highlight a fundamental role for Perp and desmosomes in tumor suppression. An understanding of the factors affecting cancer progression is important for ultimately improving the diagnosis, prognostication, and treatment of cancer.     

Why allometric scaling enhances stability in food web models

It has recently been shown that the incorporation of allometric scaling into the dynamic equations of food web models enhances network stability if predators are assigned a higher body mass than their prey. We investigate the underlying mechanisms leading to this stability increase. The dynamic equations can be written such that allometric scaling influences these equations at three places: the time scales of predator and prey dynamics become separated, the energy outflow to the predators is decreased, and intraspecific competition is increased relative to metabolic rates. For five food web topologies and various network sizes (i.e., species richness), we study the effect of each of these modifications on the percentage of surviving species separately and find that the decreased interaction strengths and the increased intraspecific competition are responsible for the enhanced stability. We also investigate the range of parameter values for which an enhanced stability is observed.     

Rapid Microbiological Testing: Monitoring the Development of Bacterial Stress

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H₂O₂. The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery.     

Do Fleas Affect Energy Expenditure of Their Free-Living Hosts?

Background

Parasites can cause energetically costly behavioural and immunological responses which potentially can reduce host fitness. However, although most laboratory studies indicate that the metabolic rate of the host increases with parasite infestation, this has never been shown in free-living host populations. In fact, studies thus far have shown no effect of parasitism on field metabolic rate (FMR).

Methodology and Results

We tested the effect of parasites on the energy expenditure of a host by measuring FMR using doubly-labelled water in free-living Baluchistan gerbils (Gerbillus nanus) infested by naturally occurring fleas during winter, spring and summer. We showed for the first time that FMR of free-living G. nanus was significantly and positively correlated with parasite load in spring when parasite load was highest; this relationship approached significance in summer when parasite load was lowest but was insignificant in winter. Among seasons, winter FMRs were highest and summer FMRs were lowest in G. nanus.

Discussion

The lack of parasite effect on FMR in winter could be related to the fact that FMR rates were highest among seasons. In this season, thermoregulatory costs are high which may indicate that less energy could be allocated to defend against parasites or to compensate for other costly activities. The question about the cost of parasitism in nature is now one of the major themes in ecological physiology. Our study supports the hypothesis that parasites can elevate FMR of their hosts, at least under certain conditions. However, the effect is complex and factors such as season and parasite load are involved.     

Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae

We have devised an approach for analyzing shotgun proteomics datasets based on the normalized spectral abundance factor that can be used for quantitative proteomics analysis. Three biological replicates of samples enriched for plasma membranes were isolated from S. cerevisiae grown in 14N-rich media and 15N-minimal media and analyzed via quantitative multidimensional protein identification technology. The natural log transformation of NSAF values from S. cerevisiae cells grown in 14N YPD media and 15N-minimal media had a normal distribution. The t-test analysis demonstrated 221 of 1316 proteins were significantly overexpressed in one or the other growth conditions with a p value <0.05. Notably, amino acid transporters were among the 14 membrane proteins that were significantly upregulated in cells grown in minimal media, and we functionally validated these increases in protein expression with radioisotope uptake assays for selected proteins.