Study of peptidelipid interactions by ITC and DSC

Study of peptide/lipid interactions by ITC and DSC Isabel ALVES Institut of Chemistry and Biology of Membranes and Nano-objects University of Bordeaux 2 nd Annual European Micro. Cal Meeting

Cell penetrating peptides (CPPs) • Small peptides (10 -20 aas), mostly cationic that are able to cross the cell membrane by a receptor-independent mechanism and to transport cargos (proteins, nanoparticles, nucleic acids, etc) inside the cell. Penetratin Origin: Homeoprotein - Drosophila Antennapedia Homeodomain Sequence: RQIKIYFQNRRMKWKK Open questions: - Are they cell selective? - What is their internalisation mechanism? - Where do they go after entering the cell? - What are the forces that govern peptide/lipid and peptide/carbohydrate interactions? D. Derossi, A. H. Joliot, G. Chassaing, A. Prochiantz, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 269 (1994) 10444 -10450. 2 nd Annual European Micro. Cal Meeting

Cells and model systems The lipid model systems: Carbohydrates: - SUV (Small Unilamellar vesicle); - LUV (Large Unilamellar vesicle); - GUV (Giant Unilamellar vesicle); - MLV (Multilamellar vesicle); 2 nd Annual European Micro. Cal Meeting

What can DSC tell us about lipids and P/L interactions? 2 nd Annual European Micro. Cal Meeting

Lipid behavior as a function of temperature - Pre-transition –transition from the phase L ’ (gel phase: fatty acid chains are ordered) to the P ’ (rippled phase– coexistence of ordered and disordered fatty acid chains); - Main phase transition – transition from P ’ to L - lamellar phase – fatty acid chains are more flexible (cis-trans isomerization of unsaturated fatty acids and trans-gauche of saturated lipids; the bilayer thickness decreases); - Lipid phase transitions depend on the lipid intrinsic properties; - Molecules that interact with lipids affect lipid phase transitions – from their effects one can obtain information about their mode of interaction ; 2 nd Annual European Micro. Cal Meeting

Information obtained by DSC about the peptide/lipid interaction Some parameters to follow : • Phase transition temperature: -decrease - favors the transition = increase in fluidity; - increase - disfavors the transition = decrease in fluidity; • Peak area: enthalpy of the reaction ( H) –decrease means weaker lipid van der Walls interactions Transition peak shape: - half-width : cooperativity of the transition – wider = less cooperative - appearence - changes in system homogeneity • 2 nd Annual European Micro. Cal Meeting

Penetratin effect on the gel/fluid phase transition of zwitterionic (DMPC) and anionic (DMPG) lipids DMPC DMPG Penetratin does not perturb DMPC phase transition but perturbs DMPG pre- and main phase transitions; 2 nd Annual European Micro. Cal Meeting

Penetratin effect on the gel/fluid phase transition of zwitterionic (DMPC) and anionic (DMPG) lipids Penetratin interacts with DMPG by inserting between the fatty acid chains; Joanne P, Galanth C, Goasdoué N, Nicolas P, Sagan S, Lavielle S, Chassaing G, El Amri C, Alves ID. (2009) Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry. Biochim Biophys Acta. 1788(9): 1772 -81. 2 nd Annual European Micro. Cal Meeting

Penetratin effect on the phase transition of lipid with tendency to form inverted micelles TH Di. Po. PE Lα - Lamellar HII - Hexagonal - Penetratin addition leads to decrease in TH – favors a negative membrane curvature; TH = 40. 4°C TH = 38. 1°C I. D. Alves, Goasdoué, N. , Correia, I. , Aubry, S. , Galanth, C. , Sagan, S. , Lavielle, S. , Chassaing, G. (2008) Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution. Biochim Biophys Acta. 1780, 948 -59. 2 nd Annual European Micro. Cal Meeting

Inverted micelle model for penetratin translocation After interacting with positively charged lipids, the Trp residues in penetratin destabilize the membrane creating a negative curvature. The peptide is encapsulated in an inverted micelle used to transport the peptide across the bilayer and deliver inside the cell. Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G, Prochiantz A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271(30): 18188 -93. 2 nd Annual European Micro. Cal Meeting

Peptide effect on the lateral lipid organization DPPC/cardiolipin (7/3 mol/mol) CL - no observable phase transition 0 -100°C; DPPC Tm = 41° C -The two lipids are miscible (even if not ideally) - Penetratin addition leads to the appearance of an additional peak at Tm ~ 40° C = DPPC Tm– the peptide interacts selectively with CL and leads to DPPC segregation – electrostatic recognition; Joanne P, Galanth C, Goasdoué N, Nicolas P, Sagan S, Lavielle S, Chassaing G, El Amri C, Alves ID. (2009) Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry. Biochim Biophys Acta. 1788(9): 1772 -81. 2 nd Annual European Micro. Cal Meeting

Peptide effect on the lateral lipid organization Lipid recruitment Electrostatics DPPC/CL Fatty acid chain lenght DMPG/DSPG Saturation DMPG/POPG yes no - Penetratin is able to recruit anionic lipids and lipids with shorter fatty acid chain 2 nd Annual European Micro. Cal Meeting

What can ITC tell us about P/L and P/carbohydrate interactions? 2 nd Annual European Micro. Cal Meeting

Isothermal Titration Calorimetry - ITC LIPIDE LUVs PEPTIDE Information obtained: - Directely: H, K, n; - Indirectly: G, S et Cp G = -RT ln. K G = H - T S Cp = d. H/d. T 2 nd Annual European Micro. Cal Meeting

Penetratin affinity for anionic and zwitterionic lipids DSPG LUVs DSPC H = -0. 38 Kcal/mol KD = 21 M n = 6. 3 - There is no interaction with the zwitterionic lipid interaction is necessary for the P/L recognition; - The role of electrostatic interactions was confirmed by the considerable increase in affinity observed when the experience was performed in absence of salt; DSPG DSPC, an electrostatic H = - 0. 4 Kcal/mol Kd = 0. 2 M n = 14 Maniti O, Alves I, Trugnan G, Ayala-Sanmartin J (2010) Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids. Plos. ONE 5, e 15819. 2 nd Annual European Micro. Cal Meeting

Enthalpy variation with temperature - Cp Cp > 0 – typical of electrostatic interactions – « enthalpy favorable » Cp < 0 – typical of hydrophobic interactions – « entropy driven » – transfert of a hydrophobic molecule from polar to non polar environment; 20 25 30 35 40 45 50 55 60 65 70 -0. 3 �H (Kcal/mol) -0. 35 -0. 45 -0. 55 Temperature (° C) Cp = +3 cal/mol – electrostatic interaction 2 nd Annual European Micro. Cal Meeting

ITC- penetratin interaction with living cells, on the role of cell membrane carbohydrates CELL LINES - CHO K 1 (wild type -black) - CHO 745 (no GAGs - gris) - CHO lec 2 (no sialic acid- blanc) Cell suspension of 1. 5 106 CHO cells at 37 °C are placed in ITC cell with stirring. 50 nmol penetratin solution is added. - GAGs greatly contribute to the binding of penetratin to the cell surface; - Sialic acids are not important for penetratin binding; Alves ID, Bechara C, Walrant A, Zaltsman Y, Jiao CY, Sagan S. (2011) Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study. PLo. S One. 6(9): e 24096. 2 nd Annual European Micro. Cal Meeting

CPP interaction with GAGs: role of peptide charge and number of Trp Peptides with variable charge and number of Trp residus GAGs with variable size and charge GAGs M PEPTIDE M 2 nd Annual European Micro. Cal Meeting

CPP interaction with GAGs Affinity: - no correlation with number of charged residus but directly proportional to the number of Trp; Enthalpy: - favorable and larger for larger GAGs; - no correlation with number of charged residus but directly proportional to the number of Trp; Entropy: - unfavorable; - no correlation with number of charged residus but directly proportional to the number of Trp; 2 nd Annual European Micro. Cal Meeting

CPP internalisation efficacy: Role of charge and Trp residues - Internalisation efficacy is higher for peptides with more Trp - Stoichiometry GAG/CPP is close to electroneutrality. Bechara C, Pallerla M, Zaltsman Y, Burlina F, Alves ID, Lequin O, Sagan S. (2012) Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis. FASEB J. 27, 738 -49. 2 nd Annual European Micro. Cal Meeting

Conclusions - Electrostatic interactions between the peptide and the cel membrane (lipid or GAG) play an important role. - Hydrophobic interactions are also important, Trp residues play important role in internalisation – GAG clustering. 2 nd Annual European Micro. Cal Meeting

Acknowledgments Paris 6 - UMR 7203 - Astrid WALRANT - Cherine BECHARA - Sandrine SAGAN - Gérard CHASSAING - Solange LAVIELLE Financial support - ANR Prob. DOM - Interdisciplinaire project from CNRS « Soutien à prise de risque » THANKS MUCH FOR YOUR ATTENTION! 2 nd Annual European Micro. Cal Meeting