A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila

The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway—including a new protein complex modulating RAF activation—we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.     

A Targeted Library Screen Reveals a New Inhibitor Scaffold for Protein Kinase D

Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.     

A Proteomic Screen of Neuronal Cell-Surface Molecules Reveals IgLONs as Structurally Conserved Interaction Modules at the Synapse

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A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4

Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.     

Spatial Fingerprints of Community Structure in Human Interaction Network for an Extensive Set of Large-Scale Regions

Human interaction networks inferred from country-wide telephone activity recordings were recently used to redraw political maps by projecting their topological partitions into geographical space. The results showed remarkable spatial cohesiveness of the network communities and a significant overlap between the redrawn and the administrative borders. Here we present a similar analysis based on one of the most popular online social networks represented by the ties between more than 5.8 million of its geo-located users. The worldwide coverage of their measured activity allowed us to analyze the large-scale regional subgraphs of entire continents and an extensive set of examples for single countries. We present results for North and South America, Europe and Asia. In our analysis we used the well-established method of modularity clustering after an aggregation of the individual links into a weighted graph connecting equal-area geographical pixels. Our results show fingerprints of both of the opposing forces of dividing local conflicts and of uniting cross-cultural trends of globalization.     

Analysis of the Protein Phosphotome of Entamoeba histolytica Reveals an Intricate Phosphorylation Network

Phosphorylation is the most common mechanism for the propagation of intracellular signals. Protein phosphatases and protein kinases play a dynamic antagonistic role in protein phosphorylation. Protein phosphatases make up a significant fraction of eukaryotic proteome. In this article, we report the identification and analysis of protein phosphatases in the intracellular parasite Entamoeba histolytica. Based on an in silico analysis, we classified 250 non-redundant protein phosphatases in E. histolytica. The phosphotome of E. histolytica is 3.1% of its proteome and 1.3 times of the human phosphotome. In this extensive study, we identified 42 new putative phosphatases (39 hypothetical proteins and 3 pseudophosphatases). The presence of pseudophosphatases may have an important role in virulence of E. histolytica. A comprehensive phosphotome analysis of E. histolytica shows spectacular low similarity to human phosphatases, making them potent candidates for drug target.     

A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.     

Meta-Analytically Informed Network Analysis of Resting State fMRI Reveals Hyperconnectivity in an Introspective Socio-Affective Network in Depression

Alterations of social cognition and dysfunctional interpersonal expectations are thought to play an important role in the etiology of depression and have, thus, become a key target of psychotherapeutic interventions. The underlying neurobiology, however, remains elusive. Based upon the idea of a close link between affective and introspective processes relevant for social interactions and alterations thereof in states of depression, we used a meta-analytically informed network analysis to investigate resting-state functional connectivity in an introspective socio-affective (ISA) network in individuals with and without depression. Results of our analysis demonstrate significant differences between the groups with depressed individuals showing hyperconnectivity of the ISA network. These findings demonstrate that neurofunctional alterations exist in individuals with depression in a neural network relevant for introspection and socio-affective processing, which may contribute to the interpersonal difficulties that are linked to depressive symptomatology.     

System-Wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.     

Lambda CIN-1, a New Mutation Which Enhances Lysogenization by Bacteriophage Lambda, and the Genetic Structure of the Lambda CY Region

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm⁴³⁴ region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3–5 nucleotide pairs. Lying approximately 11–21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that λimm⁴³⁴ cin-1 cy2001 lysogenizes efficiently, but not λimm⁴³⁴ cin-1 cy2001 cII68 nor any other λimm⁴³⁴ cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.     

Kinetics of Ligand-Receptor Interaction Reveals an Induced-Fit Mode of Binding in a Cyclic Nucleotide-Activated Protein

Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼10⁷ M⁻¹ s⁻¹ are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Analysis of Nonlinear Gene Expression Progression Reveals Extensive Pathway and Age-Specific Transitions in Aging Human Brains

Several recent gene expression studies identified hundreds of genes that are correlated with age in brain and other tissues in human. However, these studies used linear models of age correlation, which are not well equipped to model abrupt changes associated with particular ages. We developed a computational algorithm for age estimation in which the expression of each gene is treated as a dichotomized biomarker for whether the subject is older or younger than a particular age. In addition, for each age-informative gene our algorithm identifies the age threshold with the most drastic change in expression level, which allows us to associate genes with particular age periods. Analysis of human aging brain expression datasets from three frontal cortex regions showed that different pathways undergo transitions at different ages, and the distribution of pathways and age thresholds varies across brain regions. Our study reveals age-correlated expression changes at particular age points and allows one to estimate the age of an individual with better accuracy than previously published methods.     

Large-Scale Sequence Analysis of Hemagglutinin of Influenza A Virus Identifies Conserved Regions Suitable for Targeting an Anti-Viral Response

Background

Influenza A viral surface protein, hemagglutinin, is the major target of neutralizing antibody response and hence a main constituent of all vaccine formulations. But due to its marked evolutionary variability, vaccines have to be reformulated so as to include the hemagglutinin protein from the emerging new viral strain. With the constant fear of a pandemic, there is critical need for the development of anti-viral strategies that can provide wider protection against any Influenza A pathogen. An anti-viral approach that is directed against the conserved regions of the hemaggutinin protein has a potential to protect against any current and new Influenza A virus and provide a solution to this ever-present threat to public health.

Methodology/Principal Findings

Influenza A human hemagglutinin protein sequences available in the NCBI database, corresponding to H1, H2, H3 and H5 subtypes, were used to identify highly invariable regions of the protein. Nine such regions were identified and analyzed for structural properties like surface exposure, hydrophilicity and residue type to evaluate their suitability for targeting an anti-peptide antibody/anti-viral response.

Conclusion/Significance

This study has identified nine conserved regions in the hemagglutinin protein, five of which have the structural characteristics suitable for an anti-viral/anti-peptide response. This is a critical step in the design of efficient anti-peptide antibodies as novel anti-viral agents against any Influenza A pathogen. In addition, these anti-peptide antibodies will provide broadly cross-reactive immunological reagents and aid the rapid development of vaccines against new and emerging Influenza A strains.     

Comparative Genome Analysis of Listeria Bacteriophages Reveals Extensive Mosaicism,Programmed Translational Frameshifting,and a Novel Prophage Insertion Site

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3′ overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3′ end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and −1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3′ ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.The opportunistic pathogen Listeria monocytogenes is ubiquitous in nature and can become endemic in food processing environments, causing contamination of dairy products, meats, vegetables, and processed ready-to-eat food (14). L. monocytogenes is the causative agent of epidemic and sporadic listeriosis. The risk of infection is markedly increased among immunocompromised patients, newborns, pregnant women, and the elderly and is associated with a mortality rate of about 20 to 30% (37).Although all strains of L. monocytogenes are considered potentially pathogenic, epidemiological evidence has shown that certain serovars are more frequently associated with both sporadic cases and larger food-borne outbreaks. However, genetic variation within the virulence genes of wild-type strains appears to be limited and could not be directly linked to differences in pathogenicity (30) or environmental distribution.It is becoming increasingly clear that bacteriophages have an important role in bacterial biology, diversity, and evolution, as indicated by the advances in genome sequencing which revealed a high incidence of phage-related sequences in bacterial genomes. Many phages have been described for the genus Listeria, and lysogeny appears to be widespread (28). Availability of the genome sequences of different L. monocytogenes and L. innocua strains also revealed the presence of several different putative prophages in the genomes of these bacteria, constituting up to 7% of the coding capacities of the genomes (15, 32). Although the genomes of L. monocytogenes were found to be essentially syntenic, a significant portion of sequence variability is apparently based upon phage insertions and subsequent rearrangements. Investigations of prophage contributions to population dynamics in Salmonella suggest that prophages can improve the competitive fitness of the lysogenized host strains (5). This type of selective pressure also results in diversification and generation of new strains by lysogenic conversion. In the case of Listeria, however, the potential influence of prophages on their host strains, such as phenotypic variation or provision of selective benefits, has not been investigated. To gain more insight in bacteriophage-host interactions and the molecular biology and characteristics of Listeria phages, more information on the structure, information content, and variability of different Listeria phage genomes is required.Although a number of Listeria phages have been isolated and described (25, 27, 42), only limited information was available at the sequence level for phages PSA, A118, A511, and P100 (9, 19, 26, 41). As the result of a comprehensive study to determine the diversity of this group of bacterial viruses, we here report the complete nucleotide sequences of a representative set of six different Listeria phages from the Siphoviridae (A006, A500, B025, P35, and P40) and Myoviridae (B054) families in the order Caudovirales. In addition to molecular and in silico analyses, we also determined the physical genome structures and attachment loci of the temperate phages, and we describe integration of the B054 prophage into a highly conserved elongation factor gene. Another interesting finding is the unusual decoding used by some of the phages, which use programmed frameshifting to generate C-terminally modified structural proteins required for assembly of the capsid and tail.