Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte‐derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte‐derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3‐L1 adipocytes cultured for 24 h in serum‐free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte‐derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low‐grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3‐L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte‐derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

A comparative study of proteome and transcriptome changes during Medicago truncatula (cultivar Jemalong) seed development has been carried out. Transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. These data, combined with the analysis of transcript and protein distribution in the isolated seed coat, endosperm, and embryo, demonstrated the major contribution made to the embryo by the surrounding tissues. First, a remarkable compartmentalization of enzymes involved in methionine biosynthesis between the seed tissues was revealed that may regulate the availability of sulfur-containing amino acids for embryo protein synthesis during seed filling. This intertissue compartmentalization, which was also apparent for enzymes of sulfur assimilation, is relevant to strategies for modifying the nutritional value of legume seeds. Second, decreasing levels during seed filling of seed coat and endosperm metabolic enzymes, including essential steps in Met metabolism, are indicative of a metabolic shift from a highly active to a quiescent state as the embryo assimilates nutrients. Third, a concomitant persistence of several proteases in seed coat and endosperm highlighted the importance of proteolysis in these tissues as a supplementary source of amino acids for protein synthesis in the embryo. Finally, the data revealed the sites of expression within the seed of a large number of transporters implied in nutrient import and intraseed translocations. Several of these, including a sulfate transporter, were preferentially expressed in seeds compared with other plant organs. These findings provide new directions for genetic improvement of grain legumes.

Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens. In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.

The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase–transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P–P and N–P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120–150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P–P and N–P complexes, and P controls the RNA binding activity of N.

The transcriptional regulator PlcR and its cognate cell–cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR–PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli .