Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous nephropathy (MN), a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1, and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for Regulated Intramembrane Proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C inducer PMA, cytokines and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer and MN.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1.

Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study.

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2.

BACKGROUND: Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity. METHODS: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids. RESULTS: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs. CONCLUSION: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity. GENERAL SIGNIFICANCE: New informations are given to further establish the structure-function relationships of the Sm-PLGV.

A Low Molecular Weight Protein from the Sea Anemone Anemonia viridis with an Anti-Angiogenic Activity.

Sea anemones are a remarkable source of active principles due to a decentralized venom system. New blood vessel growth or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have limited efficacy. In this study, a protein fraction, purified from tentacles of Anemonia viridis, was able to limit endothelial cells proliferation and angiogenesis at low concentration (14 nM). Protein sequences were determined with Edman degradation and mass spectrometry in source decay and revealed homologies with Blood Depressing Substance (BDS) sea anemones. The presence of a two-turn alpha helix observed with circular dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to an Arginine Glycin Aspartate (RGD) motif. Molecular modeling showed that this RGD motif was well exposed to solvent. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the Vascular Endothelial Growth Factor (VEGF).

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.

Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa.

Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

The green cupredoxin CopI is a multicopper protein able to oxidize Cu(I).

Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.

Characterization of the Achromobacter xylosoxidans Type VI Secretion System and Its Implication in Cystic Fibrosis.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.

Preliminary crystallographic analysis of a possible transcription factor encoded by the mimivirus L544 gene.

Mimivirus is the prototype of a new family (the Mimiviridae) of nucleocytoplasmic large DNA viruses (NCLDVs), which already include the Poxviridae, Iridoviridae, Phycodnaviridae and Asfarviridae. Mimivirus specifically replicates in cells from the genus Acanthamoeba. Proteomic analysis of purified mimivirus particles revealed the presence of many subunits of the DNA-directed RNA polymerase II complex. A fully functional pre-transcriptional complex appears to be loaded in the virions, allowing mimivirus to initiate transcription within the host cytoplasm immediately upon infection independently of the host nuclear apparatus. To fully understand this process, a systematic study of mimivirus proteins that are predicted (by bioinformatics) or suspected (by proteomic analysis) to be involved in transcription was initiated by cloning and expressing them in Escherichia coli in order to determine their three-dimensional structures. Here, preliminary crystallographic analysis of the recombinant L544 protein is reported. The crystals belonged to the orthorhombic space group C222(1) with one monomer per asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal present in a selenomethionine-substituted protein crystal.

The mimivirus R355 gene product: preliminary crystallographic analysis of a putative ubiquitin-like protein-specific protease.

The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal.

Nanobacteria are mineralo fetuin complexes.

"Nanobacteria" are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of "nanobacteria" susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of "nanobacteria" as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call "nanons." The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices.

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Isolation of an Anti-Tumour Disintegrin: Dabmaurin-1, a Peptide Lebein-1-Like, from Daboia mauritanica Venom.

In the soft treatment of cancer tumours, consequent downregulation of the malignant tissue angiogenesis constitutes an efficient way to stifle tumour development and metastasis spreading. As angiogenesis requires integrin-promoting endothelial cell adhesion, migration, and vessel tube formation, integrins represent potential targets of new therapeutic anti-angiogenic agents. Our work is a contribution to the research of such therapeutic disintegrins in animal venoms. We report isolation of one peptide, named Dabmaurin-1, from the hemotoxic venom of snake Daboia mauritanica, and we evaluate its potential anti-tumour activity through in vitro inhibition of the human vascular endothelial cell HMECs functions involved in tumour angiogenesis. Dabmaurin-1 altered, in a dose-dependent manner, without any significant cytotoxicity, HMEC proliferation, adhesion, and their mesenchymal migration onto various extracellular matrix proteins, as well as formation of capillary-tube mimics on MatrigelTM. Via experiments involving HMEC or specific cancers cells integrins, we demonstrated that the above Dabmaurin-1 effects are possibly due to some anti-integrin properties. Dabmaurin-1 was demonstrated to recognize a broad panel of prooncogenic integrins (αvß6, αvß3 or αvß5) and/or particularly involved in control of angiogenesis α5ß1, α6ß4, αvß3 or αvß5). Furthermore, mass spectrometry and partial N-terminal sequencing of this peptide revealed, it is close to Lebein-1, a known anti-ß1 disintegrin from Macrovipera lebetina venom. Therefore, our results show that if Dabmaurin-1 exhibits in vitro apparent anti-angiogenic effects at concentrations lower than 30 nM, it is likely because it acts as an anti-tumour disintegrin.

Identification of a new natural gastric lipase inhibitor from star anise.

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

Chemical synthesis and characterization of J46 peptide, an atypical class IIa bacteriocin from Lactococcus lactis subsp. cremoris J46 Strain.

Bacteriocin J46 is a 27-residue polypeptide produced by Lactococcus lactis subsp. cremoris J46 in fermented milk. The natural form of J46 (nJ46) exhibits a broad antimicrobial spectrum. Herein, we produced the synthetic form of J46 (sJ46) by solid-phase chemical synthesis. The biochemical and physico-chemical properties of sJ46, as well as its antimicrobial activity, were found to be identical to those of its natural counterpart nJ46. It showed a potent antimicrobial activity against both lactic acid bacteria and other Gram-positive microorganisms. (1)H-NMR conformational analysis of sJ46 indicates that it adopts a flexible random coil structure.

Characterization of pepsin from rabbit gastric extract, its action on ß-casein and the effects of lipids on proteolysis.

Rabbit gastric extract (RGE) is a source of gastric enzymes for in vitro digestion studies. While its gastric lipase activity has been characterized and compared to other lipases, its pepsin activity has not been studied. We measured pepsin activity in RGE using both hemoglobin and azocoll as substrates, and identified the protein separated by SDS-PAGE as a type II-4 mature pepsin of 328 amino acid residues using Edman sequencing, LC-MS/MS analysis and intact mass measurement. As a proof-of-concept that RGE was suitable for in vitro digestion of both proteins and lipids, it was used for studying the proteolysis of ß-casein under conditions mimicking the early stages of intragastric digestion. ß-Casein was displayed either in solution or at the surface of a ß-casein-stabilized rapeseed oil emulsion to investigate the impact of lipids and lipolysis on proteolysis. Proteolysis of ß-casein was quantified based on the kinetics of ß-casein disappearance, the identification of various peptides generated upon digestion and their variation with time. The results obtained with RGE were highly similar to those obtained with equivalent amounts of porcine pepsin used as a reference standard. Digestion of ß-casein was slower when it was displayed at the oil-water interface and some degradation peptides were transiently observed at higher levels and for a longer time than with ß-casein in solution, or accumulated upon digestion. N-terminal sequencing of the main isolated peptides revealed a sequential action of pepsin starting from the hydrophobic C-terminal end of ß-casein, which was impaired by the interaction of ß-casein with lipids.