Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap_s). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap_β) predicted formation of a β-barrel with an N-terminal transmembrane α-helix followed by 14 transmembrane β-strands. Additional analysis revealed that the final β-strand contains an amino acid motif common to other β-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap_s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap_s. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β-barrel proteins to the bacterial outer membrane.     

The role of FGF signaling in guiding coordinate movement of cell groups: Guidance cue and cell adhesion regulator?

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.     

BAFF,APRIL, TWEAK,BCMA, TACI and Fn14 Proteins Are Related to Human Glioma Tumor Grade: Immunohistochemistry and Public Microarray Data Meta-Analysis

Gliomas are common and lethal tumors of the central nervous system (CNS). Genetic alterations, inflammatory and angiogenic processes have been identified throughout tumor progression; however, treatment still remains palliative for most cases. Biological research on parameters influencing cell survival, invasion and tumor heterogeneity identified several cytokines interfering in CNS inflammation, oxidative stress and malignant transformation, including TNF-superfamily (TNFSF) members. In this report we performed a meta-analysis of public gene-array data on the expression of a group of TNFSF ligands (BAFF, APRIL, TWEAK) and their receptors (BAFF-R, TACI, BCMA, Fn14) in gliomas. In addition, we investigated by immunohistochemistry (IHC) the tumor cells'' expression of these ligands and receptors in a series of 56 gliomas of different grade. We show that in IHC, BAFF and APRIL as well as their cognate receptors (BCMA, TACI) and Fn14 expression correlate with tumor grade. This result was not evidenced in micro-arrays meta-analysis. Finally, we detected for the first time Fn14, BAFF, BCMA and TACI in glioma-related vascular endothelium. Our data, combined with our previous report in glioma cell lines, suggest a role for these receptors and ligands in glioma biology and advance these molecules as potential markers for the classification of these tumors to the proliferative, angiogenic or stem-like molecular subtype.     

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.     

A rapid and efficient method for the synthesis of selectively S-Trt or S-Mmt protected Cys-containing peptides

Selective removal of protecting groups under different cleavage mechanisms could be an asset in peptide synthesis, since it provides the feasibility to incorporate different functional groups in similar reactive centres. However, selective protection/deprotection of orthogonal protecting groups in peptides is still challenging, especially for Cys-containing peptides, where protection of the cysteine side-chain is mandatory since the nucleophilic thiol can be otherwise alkylated, acylated or oxidized. Herein, we established a protocol for the synthesis of Cys-selective S-Trt or S-Mmt protected Cys-containing peptides, in a rapid way. This was achieved by, simply fine-tuning the carbocation scavenger in the final acidolytic release of the peptide from the solid support in the classic SPPS.     

Genetic Dissection of Anopheles gambiae Gut Epithelial Responses to Serratia marcescens

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.     

Genetic Signatures for Enhanced Olfaction in the African Mole-Rats

The Olfactory Receptor (OR) superfamily, the largest in the vertebrate genome, is responsible for vertebrate olfaction and is traditionally subdivided into 17 OR families. Recent studies characterising whole-OR subgenomes revealed a ‘birth and death’ model of evolution for a range of species, however little is known about fine-scale evolutionary dynamics within single-OR families. This study reports the first assessment of fine-scale OR evolution and variation in African mole-rats (Bathyergidae), a family of subterranean rodents endemic to sub-Saharan Africa. Because of the selective pressures of life underground, enhanced olfaction is proposed to be fundamental to the evolutionary success of the Bathyergidae, resulting in a highly diversified OR gene-repertoire. Using a PCR-sequencing approach, we analysed variation in the OR7 family across 14 extant bathyergid species, which revealed enhanced levels of functional polymorphisms concentrated across the receptors’ ligand-binding region. We propose that mole-rats are able to recognise a broad range of odorants and that this diversity is reflected throughout their OR7 gene repertoire. Using both classic tests and tree-based methods to test for signals of selection, we investigate evolutionary forces across the mole-rat OR7 gene tree. Four well-supported clades emerged in the OR phylogeny, with varying signals of selection; from neutrality to positive and purifying selection. Bathyergid life-history traits and environmental niche-specialisation are explored as possible drivers of adaptive OR evolution, emerging as non-exclusive contributors to the positive selection observed at OR7 genes. Our results reveal unexpected complexity of evolutionary mechanisms acting within a single OR family, providing insightful perspectives into OR evolutionary dynamics.     

Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg(2 + ) which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.