Homo- and hetero-oligomerization of beta-arrestins in living cells

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, beta-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of beta-arrestin2 and beta-arrestin1 prevented beta-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that beta-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.     

Roscovitine targets, protein kinases and pyridoxal kinase

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Characterization of the DNA-binding site in the ferric uptake regulator protein from Escherichia coli by UV crosslinking and mass spectrometry

Ferric uptake regulator protein (Fur) is activated by its cofactor iron to a state that binds to a specific DNA sequence called 'Fur box'. Using mass spectrometry-based methods, we showed that Tyr 55 of Escherichia coli Fur, as well as the two thymines in positions 18 and 19 of the consensus Fur Box, are involved with binding. A conformational model of the Fur-DNA complex is proposed, in which DNA is in contact with each H4 [A52-A64] Fur helix. We propose that this interaction is a common feature for the Fur-like proteins, such as Zur and PerR, and their respective DNA boxes.     

Genetics of species differences in the wild annual sunflowers, Helianthus annuus and H. petiolaris

Much of our knowledge of speciation genetics stems from quantitative trait locus (QTL) studies. However, interpretations of the size and distribution of QTL underlying species differences are complicated by differences in the way QTL magnitudes are estimated. Also, many studies fail to exploit information about QTL directions or to compare inter- and intraspecific QTL variation. Here, we comprehensively analyze an extensive QTL data set for an interspecific backcross between two wild annual sunflowers, Helianthus annuus and H. petiolaris, interpret different estimates of QTL magnitudes, identify trait groups that have diverged through selection, and compare inter- and intraspecific QTL magnitudes. Our results indicate that even minor QTL (in terms of backcross variance) may be surprisingly large compared to levels of standing variation in the parental species or phenotypic differences between them. Morphological traits, particularly flower morphology, were more strongly or consistently selected than life history or physiological traits. Also, intraspecific QTL were generally smaller than interspecific ones, consistent with the prediction that larger QTL are more likely to spread to fixation across a subdivided population. Our results inform the genetics of species differences in Helianthus and suggest an approach for the simultaneous mapping of inter- and intraspecific QTL.     

Exploring the evolution of Wolbachia compatibility types: a simulation approach

Wolbachia-induced cytoplasmic incompatibility (CI) is observed when males bearing the bacterium mate with uninfected females or with females bearing a different Wolbachia variant; in such crosses, paternal chromosomes are lost at the first embryonic mitosis, most often resulting in developmental arrest. The molecular basis of CI is currently unknown, but it is useful to distinguish conceptually the male and female sides of this phenomenon: in males, Wolbachia must do something, before it is shed from maturing sperm, that will disrupt paternal chromosomes functionality [this is usually termed "the modification (mod) function"]; in females, Wolbachia must somehow restore embryonic viability, through what is usually called "the rescue (resc) function." The occurrence of CI in crosses between males and females bearing different Wolbachia variants demonstrates that the mod and resc functions interact in a specific manner: different mod resc pairs make different compatibility types. We are interested in the evolutionary process allowing the diversification of compatibility types. In an earlier model, based on the main assumption that the mod and resc functions can mutate independently, we have shown that compatibility types can evolve through a two-step process, the first involving drift on mod variations and the second involving selection on resc variations. This previous study has highlighted the need for simulation-based models that would include the effects of nondeterministic evolutionary forces. This study is based on a simulation program fulfilling this condition, allowing us to follow the evolution of compatibility types under mutation, drift, and selection. Most importantly, simulations suggest that in the frame of our model, the evolution of compatibility types is likely to be a gradual process, with new compatibility types remaining partially compatible with ancestral ones.     

Intermediate mutation frequencies favor evolution of multidrug resistance in Escherichia coli

In studying the interplay between mutation frequencies and antibiotic resistance among Escherichia coli natural isolates, we observed that modest modifications of mutation frequency may significantly influence the evolution of antibiotic resistance. The strains having intermediate mutation frequencies have significantly more antibiotic resistances than strains having low and high mutation frequencies.