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.     

The heritage of pathogen pressures and ancient demography in the human innate-immunity CD209/CD209L region

The innate immunity system constitutes the first line of host defense against pathogens. Two closely related innate immunity genes, CD209 and CD209L, are particularly interesting because they directly recognize a plethora of pathogens, including bacteria, viruses, and parasites. Both genes, which result from an ancient duplication, possess a neck region, made up of seven repeats of 23 amino acids each, known to play a major role in the pathogen-binding properties of these proteins. To explore the extent to which pathogens have exerted selective pressures on these innate immunity genes, we resequenced them in a group of samples from sub-Saharan Africa, Europe, and East Asia. Moreover, variation in the number of repeats of the neck region was defined in the entire Human Genome Diversity Panel for both genes. Our results, which are based on diversity levels, neutrality tests, population genetic distances, and neck-region length variation, provide genetic evidence that CD209 has been under a strong selective constraint that prevents accumulation of any amino acid changes, whereas CD209L variability has most likely been shaped by the action of balancing selection in non-African populations. In addition, our data point to the neck region as the functional target of such selective pressures: CD209 presents a constant size in the neck region populationwide, whereas CD209L presents an excess of length variation, particularly in non-African populations. An additional interesting observation came from the coalescent-based CD209 gene tree, whose binary topology and time depth (approximately 2.8 million years ago) are compatible with an ancestral population structure in Africa. Altogether, our study has revealed that even a short segment of the human genome can uncover an extraordinarily complex evolutionary history, including different pathogen pressures on host genes as well as traces of admixture among archaic hominid populations.     

HIV type 1 glycoprotein 120 inhibits human B cell chemotaxis to CXC chemokine ligand (CXCL) 12, CC chemokine ligand (CCL)20, and CCL21

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40-50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V(H)3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cbeta3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Galpha(i), and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials.     

Fine-scale genetic structure and gene dispersal in Centaurea corymbosa (Asteraceae). II. Correlated paternity within and among sibships

The fine-scale pattern of correlated paternity was characterized within a population of the narrow-endemic model plant species, Centaurea corymbosa, using microsatellites and natural progeny arrays. We used classical approaches to assess correlated mating within sibships and developed a new method based on pairwise kinship coefficients to assess correlated paternity within and among sibships in a spatio-temporal perspective. We also performed numerical simulations to assess the relative significance of different mechanisms promoting correlated paternity and to compare the statistical properties of different estimators of correlated paternity. Our new approach proved very informative to assess which factors contributed most to correlated paternity and presented good statistical properties. Within progeny arrays, we found that about one-fifth of offspring pairs were full-sibs. This level of correlated mating did not result from correlated pollen dispersal events (i.e., pollen codispersion) but rather from limited mate availability, the latter being due to limited pollen dispersal distances, the heterogeneity of pollen production among plants, phenological heterogeneity and, according to simulations, the self-incompatibility system. We point out the close connection between correlated paternity and the "TwoGener" approach recently developed to infer pollen dispersal and discuss the conditions to be met when applying the latter.     

The putative RNA helicase Dbp6p functionally interacts with Rpl3p, Nop8p and the novel trans-acting Factor Rsa3p during biogenesis of 60S ribosomal subunits in Saccharomyces cerevisiae

Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.     

Quantitative 2H NMR at natural abundance can distinguish the pathway used for glucose fermentation by lactic acid bacteria

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.     

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.     

Dendritic cell maturation controls adhesion, synapse formation, and the duration of the interactions with naive T lymphocytes

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.     

The uncoupling protein 2 modulates the cytokine balance in innate immunity

The uncoupling protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.