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.     

Iron depletion affects nitrogenase activity and expression of nifH and nifA genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.     

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.     

Extracellular proteoglycans modify TGF-beta bio-availability attenuating its signaling during skeletal muscle differentiation.

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Satellite cells constitute the main source of muscle precursor cells for growth and repair. After skeletal muscle injury, cell-derived signals induce their re-entry into the cell cycle and their migration into the damaged zone, where they proliferate and differentiate into mature myofibers. The surrounding extracellular matrix (ECM) together with inhibitory growth factors, such as transforming growth factor-beta (TGF-beta), also likely play an important role in growth control and muscle differentiation. Decorin, biglycan and betaglycan are proteoglycans that bind TGF-beta during skeletal muscle differentiation. In this paper, we show that the binding of TGF-beta to the receptors TGF-betaRI and-betaRII diminished in a satellite cell-derived cell line during differentiation, in spite of an increase expression of both receptors. In contrast, during the differentiation of decorin-null myoblasts (Dcn null), which lack decorin expression, the binding of TGF-beta to TGF-betaRI and -betaRII increased concomitantly with receptors levels. Both the addition and re-expression of decorin, in these myoblasts, diminished the binding of TGF-beta to its transducing receptors. Similar results were obtained when biglycan was added or over-expressed in Dcn null myoblasts. The binding of TGF-beta to TGF-betaRIII, alternatively known as betaglycan, was also augmented in Dcn null myoblasts and diminished by decorin, biglycan and betaglycan. These results suggest that decorin, biglycan and betaglycan compete for the binding of TGF-beta to its transducing receptors. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1, coupled with luciferase, revealed that the addition of each proteoglycan diminished TGF-beta-dependent activity, for both TGF-beta1 and -beta2. The modulation of TGF-beta signaling by ECM proteoglycans diminishing the bio-availability of TGF-beta for its transducing receptors appears to be a feasible mechanism for the attenuation of this inhibitory growth factor during skeletal muscle formation.     

The effect of Trichoderma harzianum and T. koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina

The effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot (Pyrenophora tritici-repentis) and leaf blotch of wheat (Mycosphaerella graminicola) was evaluated under field conditions. Significant differences between wheat cultivars, inoculum types and growth stages were found. Three of the isolates tested (T2 for M. graminicola, T7 for P. tritici-repentis and T5 for both of them) showed the best performance in controlling leaf blotch and tan spot when coated onto seed or sprayed onto wheat leaves at different growth stages, with significant severity reduction up to 56%. At tillering, six of the isolates reduced the severity of P. tritici-repentis and M. graminicola compared to the control by up to 39% and 12-53%, respectively. In some experiments, the biocontrol preparation (T2 and T5) gave a level of disease control similar to that obtained with Tebuconazole (70 and 48%, respectively). The effect of Trichoderma against P. tritici-repentis was also observed at the heading stage, when six of the treatments reduced disease severity by 16-35%. This is the first report on the efficacy of Trichoderma spp. against wheat necrotrophic pathogens under field conditions in Argentina.     

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.