Adaptive covariation between the coat and movement proteins of prunus necrotic ringspot virus

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.     

Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue

Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.     

Rate asymmetry after genome duplication causes substantial long-branch attraction artifacts in the phylogeny of Saccharomyces species

Whole-genome duplication (WGD) produces sets of gene pairs that are all of the same age. We therefore expect that phylogenetic trees that relate these pairs to their orthologs in other species should show a single consistent topology. However, a previous study of gene pairs formed by WGD in the yeast Saccharomyces cerevisiae found conflicting topologies among neighbor-joining (NJ) trees drawn from different loci and suggested that this conflict was the result of "asynchronous functional divergence" of duplicated genes (Langkjaer, R. B., P. F. Cliften, M. Johnston, and J. Piskur. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848-852). Here, we test whether the conflicting topologies might instead be due to asymmetrical rates of evolution leading to long-branch attraction (LBA) artifacts in phylogenetic trees. We constructed trees for 433 pairs of WGD paralogs in S. cerevisiae with their single orthologs in Saccharomyces kluyveri and Candida albicans. We find a strong correlation between the asymmetry of evolutionary rates of a pair of S. cerevisiae paralogs and the topology of the tree inferred for that pair. Saccharomyces cerevisiae gene pairs with approximately equal rates of evolution tend to give phylogenies in which the WGD postdates the speciation between S. cerevisiae and S. kluyveri (B-trees), whereas trees drawn from gene pairs with asymmetrical rates tend to show WGD pre-dating this speciation (A-trees). Gene order data from throughout the genome indicate that the "A-trees" are artifacts, even though more than 50% of gene pairs are inferred to have this topology when the NJ method as implemented in ClustalW (i.e., with Poisson correction of distances) is used to construct the trees. This LBA artifact can be ameliorated, but not eliminated, by using gamma-corrected distances or by using maximum likelihood trees with robustness estimated by the Shimodaira-Hasegawa test. Tests for adaptive evolution indicated that positive selection might be the cause of rate asymmetry in a substantial fraction (19%) of the paralog pairs.     

CAPS: coevolution analysis using protein sequences

Coevolution Analysis using Protein Sequences (CAPS) is a PERL based software that identifies co-evolution between amino acid sites. Blosum-corrected amino acid distances are used to identify amino acid co-variation. The phylogenetic sequence relationships are used to remove the phylogenetic and stochastic dependencies between sites. The 3D protein structure is used to identify the nature of the dependencies between co-evolving amino acid sites. Friendly interpretable output files are generated. AVAILABILITY: CAPS version 1 is available at http://bioinf.gen.tcd.ie/~faresm/software/caps/. Distribution versions for Linux/Unix, Mac OS X and Windows operating systems are available, including manual and example files.     

The Roles of Whole-Genome and Small-Scale Duplications in the Functional Specialization of Saccharomyces cerevisiae Genes

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD–duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.     

Tropospheric ozone reduces carbon assimilation in trees: estimates from analysis of continuous flux measurements

High ground‐level ozone concentrations are typical of Mediterranean climates. Plant exposure to this oxidant is known to reduce carbon assimilation. Ozone damage has been traditionally measured through manipulative experiments that do not consider long‐term exposure and propagate large uncertainty by up‐scaling leaf‐level observations to ecosystem‐level interpretations. We analyzed long‐term continuous measurements (>9 site‐years at 30 min resolution) of environmental and eco‐physiological parameters at three Mediterranean ecosystems: (i) forest site dominated by Pinus ponderosa in the Sierra Mountains in California, USA; (ii) forest site composed of a mixture of Quercus spp. and P. pinea in the Tyrrhenian sea coast near Rome, Italy; and (iii) orchard site of Citrus sinensis cultivated in the California Central Valley, USA. We hypothesized that higher levels of ozone concentration in the atmosphere result in a decrease in carbon assimilation by trees under field conditions. This hypothesis was tested using time series analysis such as wavelet coherence and spectral Granger causality, and complemented with multivariate linear and nonlinear statistical analyses. We found that reduction in carbon assimilation was more related to stomatal ozone deposition than to ozone concentration. The negative effects of ozone occurred within a day of exposure/uptake. Decoupling between carbon assimilation and stomatal aperture increased with the amount of ozone pollution. Up to 12–19% of the carbon assimilation reduction in P. ponderosa and in the Citrus plantation was explained by higher stomatal ozone deposition. In contrast, the Italian site did not show reductions in gross primary productivity either by ozone concentration or stomatal ozone deposition, mainly due to the lower ozone concentrations in the periurban site over the shorter period of investigation. These results highlight the importance of plant adaptation/sensitivity under field conditions, and the importance of continuous long‐term measurements to explain ozone damage to real‐world forests and calculate metrics for ozone‐risk assessment.     

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.     

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand,in human osteoblast-like cells

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [³H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [¹⁸F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10⁻⁵ M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.