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International audience; Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depends greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. It is responsible for the effective hydrolysis of the main lipids composing plant photosynthetic membranes, which represent an excellent source of omega-3 for human nutrition. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and physical state of its substrate at the nanoscopic and microscopic scales. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipid, and/or phospholipid, and/or phytosterol, presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their digestion kinetics by gPLRP2 were investigated in the presence or absence of bile salts (NaTDC) during static in vitro, so called “bulk”, digestion. The enzymatic activity of gPLRP2 onto mixed monolayer of galactolipids and/or phospholipids, and/or phytosterols was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. Additionally, gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids, but the compositional complexity in these systems induced a lag phase before the initiation of lipolysis, and a slowdown in enzymatic activity. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes. The understanding of PLRP2 enzymatic activity at the level of natural plant-based assemblies open the way to new strategies to vectorize bioaccessible omega-3 and rebalance human and animal diets.; La lipase pancréatique apparentée à la protéine 2 (PLRP2) présente des activités galactolipase et phospholipase A1 remarquables, qui dépendent fortement de l'organisation supramoléculaire des substrats et de la présence de molécules tensioactives telles que les sels biliaires. Elle est responsable de l'hydrolyse efficace des principaux lipides composant les membranes photosynthétiques des plantes, qui représentent une excellente source d'oméga-3 pour la nutrition humaine. L'objectif de l'étude était de comprendre la modulation des mécanismes d'adsorption et de l'activité enzymatique de la PLRP2 de cobaye (gPLRP2), par l'environnement physique de l'enzyme et l'état physique de son substrat à l'échelle nanoscopique et microscopique. Des monocouches de Langmuir ont été utilisées pour reproduire des membranes modèles photosynthétiques homogènes et hétérogènes contenant des galactolipides, et/ou des phospholipides, et/ou des phytostérols, présentant des interfaces chargées ou non. Les mêmes mélanges lipidiques ont également été utilisés pour former des liposomes micrométriques, et leur cinétique de digestion par la gPLRP2 a été étudiée en présence ou en absence de sels biliaires (NaTDC) au cours d'une digestion statique in vitro, dite "bulk". L'activité enzymatique de la gPLRP2 sur une monocouche mixte de galactolipides et/ou phospholipides, et/ou phytostérols a été caractérisée avec une activité optimale à 15 mN/m, en l'absence de sels biliaires. De plus, la gPLRP2 a montré une meilleure adsorption sur des monocouches modèles biomimétiques contenant des lipides chargés négativement, mais la complexité de la composition de ces systèmes a induit une phase de latence avant l'initiation de la lipolyse, et un ralentissement de l'activité enzymatique. En bulk, aucune activité enzymatique n'a pu être démontrée sur les liposomes à base de GL en l'absence de sels biliaires, probablement en raison de la pression latérale élevée des bicouches lipidiques. En présence de NaTDC (4 mM), cependant, la gPLRP2 a montré des activités galactolipase élevées et phospholipase A1 modérées sur les liposomes, probablement en raison d'une diminution du packing et de la pression latérale lors de l'adsorption du NaTDC, et de la rupture ultérieure des liposomes. La compréhension de l'activité enzymatique de la PLRP2 au niveau des assemblages naturels à base de plantes ouvre la voie à de nouvelles stratégies pour vectoriser des oméga-3 bioaccessibles et rééquilibrer les régimes alimentaires humains et animaux.

International audience; The microalgae Chlorella vulgaris represent a good model for the study of photosynthetic membranes and are rich in photosynthetic proteins, galactolipids, and pigments (Demarco et al., 2022). These photosynthetic organisms have been consumed in some parts of the world for centuries and their nutritional potential has been highlighted in many reports, explaining a growing interest in their use in new food applications (Torres-Tiji et al., 2020). However, their digestibility by gastrointestinal enzymes has not been extensively studied. So we used the Pancreatic lipase related-protein 2 (PLRP2) to get more information on the digestive mechanisms. Indeed, it has been shown to be responsible for the effective hydrolysis of galactolipids, the main lipids composing plant photosynthetic membranes. Guinea pig pancreatic lipase related-protein 2 (gPLRP2) is a model enzyme close to its human analogue, and that has been extensively studied for its ability to hydrolyze a wide range of acylglycerol substrates, exhibiting remarkable galactolipase and phospholipase A1 activity. In this study, Langmuir model monolayers were used to fellow the interfacial behavior of gPLRP2 at the level of natural plant lipid monolayers from Chlorella microalgae extracts, and to understand the impact of the chemical and physical heterogeneity of natural photosynthetic plant membranes on the enzymatic activity of gPLRP2. We compared also with data obtained on lipid extracts from nuts and almonds for which the composition is mainly composed of phospholipids. The results have shown that the enzymatic activity of gPLRP2 is with an optimum activity obtained at 15 mN/m. Interfacial studies showed enhanced adsorption of gPLRP2 onto natural monolayers containing negatively charged lipids, and that the compositional complexity in these systems induced a lag phase before the initiation of lipolysis, and a slowdown in enzymatic activity.; Les microalgues Chlorella vulgaris représentent un bon modèle pour l'étude des membranes photosynthétiques et sont riches en protéines photosynthétiques, galactolipides et pigments (Demarco et al., 2022). Ces organismes photosynthétiques sont consommés dans certaines régions du monde depuis des siècles et leur potentiel nutritionnel a été mis en évidence dans de nombreux rapports, expliquant un intérêt croissant pour leur utilisation dans de nouvelles applications alimentaires (Torres-Tiji et al., 2020). Cependant, leur digestibilité par les enzymes gastro-intestinales n'a pas été étudiée de manière approfondie. Nous avons donc utilisé la lipase pancréatique apparentée à la protéine de type 2 (PLRP2) pour obtenir plus d'informations sur les mécanismes digestifs. En effet, il a été démontré qu'elle est responsable de l'hydrolyse efficace des galactolipides, les principaux lipides composant les membranes photosynthétiques des plantes. La lipase pancréatique de cochon d'Inde (gPLRP2) est une enzyme modèle proche de son analogue humain, et qui a été largement étudiée pour sa capacité à hydrolyser une large gamme de substrats d'acylglycérol, présentant une remarquable activité galactolipase et phospholipase A1. Dans cette étude, des monocouches modèles de Langmuir ont été utilisées pour étudier le comportement interfacial de la gPLRP2 au niveau de monocouches lipidiques végétales naturelles provenant d'extraits de microalgues Chlorella, et pour comprendre l'impact de l'hétérogénéité chimique et physique des membranes végétales photosynthétiques naturelles sur l'activité enzymatique de la gPLRP2. Nous avons également comparé avec les données obtenues sur des extraits lipidiques de noix et d'amandes dont la composition est principalement composée de phospholipides. Les résultats ont montré que la gPLRP2 présente une activité optimale à 15 mN/m. Les études interfaciales ont montré une adsorption accrue de la gPLRP2 sur des monocouches naturelles contenant des lipides chargés négativement, mais également que la complexité de la composition de ces systèmes induisait une phase de latence avant l'initiation de la lipolyse, et un ralentissement de l'activité enzymatique.

International audience; The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

International audience; The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin–Benson–Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde−3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

Langmuir films and their analysis by tensiometry, ellipsometry and Langmuir–Blodgett transfer coupled to atomic force microscopy are suitable tools to study the interactions of various lipases with model membranes. The objective of our study was to characterize the adsorption and lipolysis behavior of different gastrointestinal enzymes onto biomimetic vegetal membranes. An heterogenous monolayer based on a mixture of unsaturated galactolipids (MGDG, DGDG), saturated phospholipids (DPPC), and phytosterols was used. Four enzymes were studied, (i) gastric lipase (GL), which has no lipolytic activity on polar lipids but whose adsorption may modulate the subsequent adsorption and activity of other lipases, and three pancreatic enzymes: (ii) pancreatic lipase 2 (PLRP2) presenting both galactolipase and phospholipase A1 activities, (iii) pancreatic triacylglycerol lipase and its cofactor, colipase (PTL/coPTL), and (iv) pancreatic secreted phospholipase A2 (sPLA2-IB). A strong surfactant property of GL and its preferential adsorption at both the expanded lipid phase and at the boundary phase were observed, in line with previous results obtained with biomimetic milk fat globule mem- brane. With PLRP2, changes in surface pressure indicated a lipolytic activity in addition to lipase adsorption, with a higher rate at surface pressure below 15 mN/m. A drastic decrease in film thickness was also observed by ellipsometry, suggesting the removal of some lipolysis products. Such variation was not observed upon PTL/coPTL adsorption and may be related to the absence of activity of PTL on polar lipids. Finally, the evolution of adsorption kinetic after injection of sPLA2-IB did not indicate a clear trend of lipolysis on the lipid film. However, a change in the morphology of condensed domains was observed by AFM, suggesting a phase separation of lipids induced by binding of sPLA2-IB. This study is a step forward in the understanding of interaction of gastrointestinal lipases with plant lipid membranes, an overlooked aspect of lipid digestion. (Texte intégral)