Comparative proteomic analysis of blood eosinophils reveals redox signaling modifications in patients with FIP1L1-PDGFRA-associated chronic eosinophilic leukemia

The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos). Using 2D-DIGE and mass spectrometry techniques, we identified 41 proteins significantly overexpressed between F/P-Eos and C-Eos. Among them, 17.8% belonged to the oxidoreductase family. We further observed a down-expression of peroxiredoxin-2 (PRX-2) and an overexpression of src-homology-2 domain containing tyrosine phosphatase (SHP-1), enzymes regulating PDGFR downstream pathways, and especially intracellular reactive oxygen species (ROS) production. This profile, confirmed in immunoblot analysis, appears specific to F/P-Eos compared to controls and patients with idiopathic HES. In this clonal disorder possibly involving a pluripotent hematopoietic stem cell, we postulate that the well documented relationships between PDGFRA downstream signals and intracellular ROS levels might influence the phenotype of this leukemia.     

Multiplicative versus additive selection in relation to genome evolution: a simulation study.

The evolution of molecular quantitative traits, such as codon usage bias or base frequencies, can be explained as the result of mutational biases alone, or as the result of mutation and selection. Whereas mutation models can be investigated easily, realistic modelling of selection-directed genome evolution is analytically intractable, and numerical calculations require substantial computer resources. We investigated the evolution of optimal codon frequency under additive and multiplicative effects of selected linked codons. We show that additive selective effects of many linked sites cannot be effective in genomes when the number of selected sites is greater than the effective population size, a realistic assumption according to current molecular data. We then discuss the implications of these results for isochore evolution in vertebrates.     

Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation

Background

Addictive drugs have in common that they cause surges in dopamine (DA) concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA). Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs) at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine) cause similar changes through their effects on the mesolimbic DA system.

Methodology / Principal Findings

We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT) is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine.

Conclusions / Significance

We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.     

Clues about the genetic basis of adaptation emerge from comparing the proteomes of two Ostreococcus ecotypes (Chlorophyta, Prasinophyceae)

We compared the proteomes of two picoplanktonic Ostreococcus unicellular green algal ecotypes to analyze the genetic basis of their adaptation with their ecological niches. We first investigated the function of the species-specific genes using Gene Ontology databases and similarity searches. Although most species-specific genes had no known function, we identified several species-specific functions involved in various cellular processes, which could be critical for environmental adaptations. Additionally, we investigated the rate of evolution of orthologous genes and its distribution across chromosomes. We show that faster evolving genes encode significantly more membrane or excreted proteins, consistent with the notion that selection acts on cell surface modifications that is driven by selection for resistance to viruses and grazers, keystone actors of phytoplankton evolution. The relationship between GC content and chromosome length also suggests that both strains have experienced recombination since their divergence and that lack of recombination on the two outlier chromosomes could explain part of their peculiar genomic features, including higher rates of evolution.     

Screening the Sargasso Sea metagenome for data to investigate genome evolution in Ostreococcus (Prasinophyceae, Chlorophyta)

The Sargasso Sea water shotgun sequencing unveiled an unprecedented glimpse of marine prokaryotic diversity and gene content. The sequence data was gathered from 0.8 microm filtered surface water extracts, and revealed picoeukaryotic (cell size<2 microm) sequences alongside the prokaryotic data. We used the available genome sequence of the picoeukaryote Ostreococcus tauri (Prasinophyceae, Chlorophyta) as a benchmark for the eukaryotic sequence content of the Sargasso Sea metagenome. Sequence data from at least two new Ostreococcus strains were identified and analyzed, and showed a bias towards higher coverage of the AT-rich organellar genomes. The Ostreococcus nuclear sequence data retrieved from the Sargasso metagenome is divided onto 731 scaffolds of average size 3917 bp, and covers 23% of the complete nuclear genome and 14% of the total number of protein coding genes in O. tauri. We used this environmental Ostreococcus sequence data to estimate the level of constraint on intronic and intergenic sequences in this compact genome.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Marine Prasinovirus Genomes Show Low Evolutionary Divergence and Acquisition of Protein Metabolism Genes by Horizontal Gene Transfer

Although marine picophytoplankton are at the base of the global food chain, accounting for half of the planetary primary production, they are outnumbered 10 to 1 and are largely controlled by hugely diverse populations of viruses. Eukaryotic microalgae form a ubiquitous and particularly dynamic fraction of such plankton, with environmental clone libraries from coastal regions sometimes being dominated by one or more of the three genera Bathycoccus, Micromonas, and Ostreococcus (class Prasinophyceae). The complete sequences of two double-stranded (dsDNA) Bathycoccus, one dsDNA Micromonas, and one new dsDNA Ostreococcus virus genomes are described. Genome comparison of these giant viruses revealed a high degree of conservation, both for orthologous genes and for synteny, except for one 36-kb inversion in the Ostreococcus lucimarinus virus and two very large predicted proteins in Bathycoccus prasinos viruses. These viruses encode a gene repertoire of certain amino acid biosynthesis pathways never previously observed in viruses that are likely to have been acquired from lateral gene transfer from their host or from bacteria. Pairwise comparisons of whole genomes using all coding sequences with homologous counterparts, either between viruses or between their corresponding hosts, revealed that the evolutionary divergences between viruses are lower than those between their hosts, suggesting either multiple recent host transfers or lower viral evolution rates.Phytoplankton is responsible for about half of the photosynthetic activity of the planet (13), with the other half being ensured by terrestrial plants. Phytoplankton is essentially composed of unicellular organisms which have a high turnover rate, and whereas terrestrial plants are renewed on average once every 9 years, the global phytoplankton population is replaced approximately every week (13). Although the ecological importance of viruses has previously been debated, they are now recognized as major players in regulating these highly dynamic phytoplankton populations. Indeed, viruses are the most numerous biological entities in the ocean, infecting all marine organisms from prokaryotes to uni- and multicellular eukaryotes (36). Cell death following viral infection produces particulate and dissolved organic matter that in turn fuels the growth of other phytoplankton. The importance of this viral shunt is not yet well understood although some studies suggest that it constitutes an important flux that must be taken into account in marine trophic transfer models.Among viruses affecting the eukaryotic phytoplankton, several large double-stranded DNA (dsDNA) viruses have been described, and these viruses have been named phycodnaviruses because they infect algae (12). However, the term “alga” has no evolutionary significance, and phycodnaviruses infect phylogenetically distantly related organisms. Thus, comparisons of dsDNA viruses infecting organisms as diverse as haptophytes, dinoflagellates, and green algae likely span the same order of evolutionary distances as comparisons of viruses of animals with those of plants. In order to understand the evolution of these viruses, comparisons between more closely related host-virus combinations are desirable and are even more valuable if DNA sequence information about their host species'' genomes is available. Viruses infecting Chlorophyta, which include most green algae, thus present attractive systems for such analyses. In this phylum, both prasinoviruses and chloroviruses, infecting Prasinophyceae and Trebouxiophyceae, respectively, have been described.Several dsDNA viruses have been described infecting different Chlorella sp. unicellular green algae (Trebouxiophyceae), which are symbionts of the ciliate Paramecium bursaria (14, 15, 44) or of the heliozoon Acanthocystis turfacea (16). They belong to the nucleocytoplasmic large DNA viruses (NCLDV), indicating that they either replicate exclusively in the cytoplasm of the host cell or start their life cycle in the host nucleus but complete it in the cytoplasm (20, 46). NCLDV can also infect members of the Prasinophyceae, an ecologically important class of microalgae that are found in all oceans (39). Prasinophyceae can dominate the eukaryotic picoplankton fraction in coastal areas, and a high proportion of the DNA sequences in many environmental DNA clone libraries can be attributed to one or more of the three genera Bathycoccus, Micromonas, and Ostreococcus (31, 42). Two dsDNA Ostreococcus viruses have been sequenced (9, 40), but no viruses specific to Bathycoccus have yet been reported (2, 6). Both dsDNA and RNA Micromonas viruses have been described although information about their genomes is not yet available (5, 8). Phylogenetic analyses based on their DNA polymerase or major capsid gene sequences suggest that chloroviruses and prasinoviruses form a monophyletic group (4). Since host genomes of two Chlorella species and three Prasinophyceae genera are available, the possibility of horizontal gene transfer (HGT) between hosts and their viruses can be investigated and might provide key insights into their coevolution. Both chloroviruses and prasinoviruses have a DNA polymerase gene but no DNA-dependent RNA polymerase, in contrast to the Emiliania huxleyi virus EhV-86 (41), which is consistent with a large evolutionary divergence between these viruses.Here, we describe the complete sequences of two dsDNA Bathycoccus virus genomes, one dsDNA Micromonas virus genome, and one new dsDNA Ostreococcus virus genome. Comparison between them revealed a high degree of conservation, both for orthologous genes and for synteny. Several specific pathways, such as amino acid biosynthesis, are encoded differentially by genes never previously identified before in viruses, and we compared these genomes with those of the six available Chlorella viruses. We propose a new phylogeny to reconcile the wide evolutionary distances between phycodnavirus genomes with those of their hosts.