Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.     

Asymmetric usage of antagonist charged residues drives interleukin-5 receptor recruitment but is insufficient for receptor activation

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.     

Metazoan OXPHOS gene families: evolutionary forces at the level of mitochondrial and nuclear genomes

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.     

Cell growth-dependent subcellular localization of p8

p8 is a stress-induced protein, biochemically related to the architectural factor HMG-I/Y, overexpressed in many cancers and required for tumor expansion. The molecular mechanisms by which p8 may exert its effect in aspects of growth is unknown. Using immunocytochemistry, we found that p8 presents nuclear localization in sub-confluent cells, but it localizes throughout the whole cell in high density grown cells. Cells arrested in Go/G1, either by serum deprivation or by hydroxyurea treatment, show a nucleo-cytoplasmic localization of p8, whether in the rest of the cell cycle stages of actively dividing cells the localization is nuclear. A comparison of p8 sequences from human to fly predicts a conserved bipartite nuclear localization sequence (NLS). The putative NLS has been demonstrated to be functional, since nuclear import is energy dependent (inhibited by sodium azide plus 2-deoxyglucose), and fusion proteins GFP-p8 and GFP-NLSp8 localize to the nucleus, whereas GFP-p8NLSmut in which with Lys 65, 69, 76, and 77 mutated to Ala localized to the whole cell. p8 localization does not involve the CRM1 transporter, since it is insensitive to leptomycin B. Inhibitors of MAPK pathways did not affect p8 subcellular localization. The inhibition of deacetylation with Trichostatin A promotes cytoplasmic accumulation of p8. The results suggest that p8 growth stage-dependent localization is regulated by acetylation, that p8 is not free within the cell but forming part of a complex and that it may exert a role in both subcellular localizations.     

Synthesis and alpha4beta2 nicotinic affinity of unichiral 5-(2-pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes

The RS and SR enantiomers of 2-oxazolidinone and 1,4-benzodioxane bearing a 2-pyrrolidinyl substituent at the 5- and 2-position, respectively, were synthesized as candidate nicotinoids. One of the two benzodioxane stereoisomers reasonably fits the pharmacophore elements of (S)-nicotine and binds at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity. Interestingly, both the synthesized pyrrolidinylbenzodioxanes exhibit analogous affinity at alpha(2) adrenergic receptor resembling the behaviour of some known alpha(2)-AR ligands recently proved to possess neuronal nicotinic affinity.     

Discovery of N-[(1-aryl-1H-indazol-5-yl)methyl]amides derivatives as smoothened antagonists for inhibition of the hedgehog pathway

We report the synthesis and biological evaluation of N-[(1-aryl-1H-indazol-5-yl)methyl]amide derivatives as Smoothened antagonists and inhibitors of the Hedgehog pathway. Identification of the lead structure 1 by HTS, followed by SAR study on the amide and aryl portions led to the discovery of antagonists with nanomolar activity.     

Systematic conservation assessment for the Mesoamerica, Chocó, and Tropical Andes biodiversity hotspots: a preliminary analysis

Using IUCN Red List species as biodiversity surrogates, supplemented with additional analyses based on ecoregional diversity, priority areas for conservation in Mesoamerica, Chocó, and the Tropical Andes were identified using the methods of systematic conservation planning. Species’ ecological niches were modeled from occurrence records using a maximum entropy algorithm. Niche models for 78 species were refined to produce geographical distributions. Areas were prioritized for conservation attention using a complementarity-based algorithm implemented in the ResNet software package. Targets of representation for Red List species were explored from 10 to 90% of the modeled distributions at 10% increments; for the 53 ecoregions, the target was 10% for each ecoregion. Selected areas were widely dispersed across the region, reflecting the widespread distribution of Red List species in Mesoamerica, Chocó, and the Tropical Andes, which underscores the region’s importance for biodiversity. In general, existing protected areas were no more representative of biodiversity than areas outside them. Among the countries in the region, the protected areas of Belize performed best and those of Colombia and Ecuador worst. A high representation target led to the selection of a very large proportion of each country except Colombia and Ecuador (for a 90% target, 83–95% of each country was selected). Since such large proportions of land cannot realistically be set aside as parks or reserves, biodiversity conservation in Mesoamerica, Chocó, and the Tropical Andes will require integrative landscape management which combines human use of the land with securing the persistence of biota. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.