Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

11 pagesInternational audienceThe formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods)

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information

International audienceAffinity photo-cross-linking coupled to mass spec-trometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ 9), 18:1 (trans Δ 9) PG, and DLoPG (18:2 cis Δ 9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: CC double bond formation on saturated fatty acids, covalent adducts formation via classical C−C bond, and Paterno-Buchi oxetane formation followed or not by fragmentation (retro-Paterno-Buchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction

Purification and multi-omics characterization of human pre-beta-HDL reveals specific proteomic and lipidomic signature relevant to biological activities

International audienceAIM: Multiple biological activities of high-density lipoprotein (HDL) particles may reflect their wide diversity. While alpha-migrating spherical HDLs are well characterised in terms of composition and function, small, dense, discoid pre-beta-migrating HDLs which are thought to display potent biological activities, remain difficult to purify and thus poorly characterised. Methods: We developed an original purification protocol yielding up to 2 mg of pre-beta-HDL (d 1.21-1.25 g/ml) from 70ml plasma of healthy volunteers (n=4). Alpha-HDL (d 1.08-1.11 g/ml) were purified concomitantly. Proteomic and lipidomic analyses of pre-beta- and alpha-HDL were performed and major biological activities, including cholesterol efflux and anti-inflammatory capacities, were evaluated in human THP-1 macrophages. Results: Pre-beta-HDL proteomic analysis identified 36 scaffold proteins gathered in three GO-biological processes. Proteins enriched in pre-beta-HDL were associated with inflammatory response, (e.g. PON1, serpin A1, haptoglobin) and immune response (e.g. IGHA1, C4A/B, haptoglobin-related-protein). Most of proteins related to lipid metabolism and transport were common to alpha- and pre-beta-HDL. Importantly, several functional proteins, including LCAT, PLTP, CETP and PON3, were exclusively detected in pre-beta-HDL. Lipidomic analysis revealed that pre-beta- and alpha-HDL contained similar lipid species. Yet, pre-beta-HDL was enriched in negatively-charged biologically-active phosphatidic acid (+35%; p<0.05) and phosphatidylserine (+20%; p<0.05), as compared to alpha-HDL. Cholesterol efflux from human macrophages to pre-beta-HDL was 3.5-fold elevated relative to alpha-HDL, being similar to that to lipid-free apolipoprotein A-I. Incubation with pre-beta-HDL prior to LPS stimulation reduced mRNA levels of IL-6 (-45%, p<0.05), IL-1-beta (- 44%, p<0.05) and TNF-alpha (- 60%, p<0.01) in THP-1 macrophages, while alpha-HDL exerted no effect.Conclusion: The present study, provides the first-ever structure-function characterization of human pre-beta-HDL using multi-omics analysis allowing identification of proteins and lipids potentially accounting for the enhanced biological activities of pre-beta-HDL. Our findings may provide novel clues to understand physiological and physiopathological roles of pre-beta-HDL

Supplementary figures from Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida <i>Elliptio complanata</i> and <i>Villosa lienosa</i>

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Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver

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Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach

International audienceSexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues