Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.     

Evidence for the Concerted Evolution between Short Linear Protein Motifs and Their Flanking Regions

Background

Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein–protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids.

Results

The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co–evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif–mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions.

Conclusion

The results suggest that flanking regions are relevant for linear motif–mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise.     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

Characterisation of structural genes involved in phytic acid biosynthesis in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Phytate (myo-inositol hexakisphosphate), the major form of phosphorous storage in plant seeds, is an inositol phosphate compound poorly digested by humans and monogastric animals. A major goal for grain crop improvement is the reduction of its content in the seed to improve micronutrient bioavailability and phosphorus utilisation by humans and non-ruminant animals, respectively. We are interested in lowering phytic acid in common bean seed and to this goal we have undertaken a two-strategy approach: the isolation of mutants from an EMS mutagenised population (Campion et al. 2009) and the identification of genes coding for candidate enzymes involved in inositol phosphate metabolism for future targeted mutant isolation and/or study. In this paper we report data referred to the second approach and concerning the isolation and genomic organisation of Phaseolus vulgaris genes coding for myo-inositol 1-phosphate synthase (PvMIPSs and PvMIPSv), inositol monophosphatase (PvIMP), myo-inositol kinase (PvMIK), inositol 1,4,5-tris-phosphate kinase (PvIPK2), inositol 1,3,4-triphosphate 5/6-kinase (PvITPKα and PvITPKβ) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1). All these genes have been mapped on the common bean reference genetic map of McClean (NDSU) 2007 using a virtual mapping strategy. Bean markers, presumably associated to each gene of the phytic acid pathway, have also been identified. In addition, we provide a picture of the expression, during seed development, of the genes involved in phytic acid synthesis, including those such as MIK, IMP and IPK2, for which this information was lacking.     

Nitric oxide production inhibited by xenobiotic compounds in the protozoan Paramecium primaurelia

The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds.Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia. In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l-NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl₂ and CdCl₂), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca²⁺ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.     

mRNPs, polysomes or granules: FMRP in neuronal protein synthesis

mRNA localization and regulated translation play central roles in neurite outgrowth and synaptic plasticity. A key molecule in these processes is the Fragile X mental retardation protein, FMRP, which is involved in the metabolism of neuronal mRNAs. Absence or mutation of FMRP leads to spine dysmorphogenesis and impairs synaptic plasticity. Studies that have mainly been performed on the mouse and Drosophila models for Fragile X Syndrome showed that FMRP is involved in translational regulation at synapses, but even 15 years after discovery of the FMR1 gene, the precise working mechanisms remain elusive.