Papers by Ernesto Ambroggio
PLoS ONE, 2013
Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the... more Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the Arf/Arl family in their active GTP-bound state. It has been proposed that BAR domains are able to sense membrane curvature and to induce membrane tubulation. We report here that active Arf1 is required for the recruitment of Arfaptin2 to artificial liposomes mimicking the Golgi apparatus lipid composition. The Arf1-dependent recruitment of Arfaptin2 increases with membrane curvature, while the recruitment of Arf1 itself is not sensitive to curvature. At high protein concentrations, the binding of Arfaptin2 induces membrane tubulation. Finally, membrane-bound Arfaptin2 is released from the liposome when ArfGAP1 catalyzes the hydrolysis of GTP to GDP in Arf1. These results show that both Arf1 activation and high membrane curvature are required for efficient recruitment of Arfaptin2 to membranes. Citation: Ambroggio EE, Sillibourne J, Antonny B, Manneville J-B, Goud B (2013) Arf1 and Membrane Curvature Cooperate to Recruit Arfaptin2 to Liposomes. PLoS ONE 8(4): e62963.
Journal of Peptide Science, 2007
Journal of Molecular Biology, 2006
b-Amyloid peptide (Ab), which is cleaved from the larger trans-membrane amyloid precursor protein... more b-Amyloid peptide (Ab), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Ab(1-42) and the effect of metal ions (Cu 2C and Zn 2C ) on model membranes using 31 P and 2 H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Ab(1-42) was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Ab(1-42) adopted more b-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Ab in disease states.
Biophysical Journal, 2005
Membrane lysis caused by antibiotic peptides is often rationalized by means of two different mode... more Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the socalled carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, Maculatin 1.1, Citropin 1.1, and Aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for Maculatin compared with Aurein or Citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that Maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, Citropin and Aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of Maculatin, the same experimental approach was used for Maculatin-Ala and Maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of Maculatin-Ala and Maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring Maculatin sequence. As observed for Maculatin, the lytic action of Maculatin-Ala and Maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.
Biophysical Journal, 2005
Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibri... more Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a b-sheet conformation. One of the most abundant components in amyloid aggregates is the b-amyloid peptide 1-42 (Ab 1-42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Ab 1-42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Ab 1-42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Ab 1-42 triggers the induction of a sequential leakage of low-and highmolecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Ab 1-42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.
Biophysical Chemistry, 2006
We have studied the thermodynamic, surface, and structural properties of αM1 transmembrane sequen... more We have studied the thermodynamic, surface, and structural properties of αM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of − 7.2 kcal/mol. A cross-sectional molecular area of 210 Å 2 /molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl 4 , respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoylphosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyloleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Surface behaviour of Maculatin 1.1 and Citropin 1.1 antibiotic peptides have been studied using t... more Surface behaviour of Maculatin 1.1 and Citropin 1.1 antibiotic peptides have been studied using the Langmuir monolayer technique in order to understand the peptide -membrane interaction proposed as critical for cellular lysis. Both peptides have a spontaneous adsorption at the air -water interface, reaching surface potentials similar to those obtained by direct spreading. Collapse pressures (P c , stability to lateral compression), molecular areas at maximal packing and surface potentials (DV ) obtained from compression isotherms of both pure peptide monolayers are characteristic of peptides adopting mainly a-helical structure at the interface. The stability of Maculatin monolayers depended on the subphase and increased when pH was raised. In an alkaline environment, Maculatin exhibits a molecular reorganization showing a reproducible discontinuity in the P -A compression isotherm. Both peptides in lipid films with the zwitterionic palmitoyl-oleoylphosphatidylcholine (POPC) showed an immiscible behaviour at all lipid -peptide proportions studied. By contrast, in films with the anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG), the peptides showed miscible behaviour when the peptides represented less than 50% of total surface area. Additional penetration experiments also demonstrated that both peptides better interact with POPG compared with POPC monolayers. This lipid preference is discussed as a possible explanation of their antibiotic properties. D
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013
Several protein transport processes in the cell are mediated by signal sequence peptides located ... more Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide-lipid interaction of three signal sequence peptides at the air-NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide-lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.
Biophysical Journal, 2009
Cytoplasmic coat proteins are required for cargo selection and budding of tubulovesicular transpo... more Cytoplasmic coat proteins are required for cargo selection and budding of tubulovesicular transport intermediates that shuttle between intracellular compartments. To better understand the physical parameters governing coat assembly and coat-induced membrane deformation, we have reconstituted the Arf1-dependent assembly of the COPI coat on giant unilamellar vesicles by using fluorescently labeled Arf1 and coatomer. Membrane recruitment of Arf1-GTP occurs exclusively on disordered lipid domains and does not induce optically visible membrane deformation. In the presence of Arf1-GTP, coatomer self-assembles into weakly curved coats on membranes under high tension, while it induces extensive membrane deformation at low membrane tension. These deformations appear to have a composition different from the parental membrane because they are protected from phase transition. These findings suggest that the COPI coat is adapted to liquid disordered membrane domains where it could promote lipid sorting and that its mechanical effects can be tuned by membrane tension.
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Papers by Ernesto Ambroggio