Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipin.
BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices alphaH6 and alphaH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the alphaH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the alphaH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of alphaH6 into the phospholipid monolayer. The modification of two charged amino acid residues in alphaH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and alphaH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that alphaH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via alphaH6.
Institut Cochin, INSERM U567/CNRS UMR8104, Université Paris Descartes, France.
Patrice X
PX
Petit
Pauline
P
Dupaigne
Fabrizio
F
Pariselli
François
F
Gonzalvez
François
F
Etienne
Carole
C
Rameau
Sophie
S
Bernard
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry*; D002308:Cardiolipins / Q000737:chemistry*; D004734:Energy Metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary
D051023:BH3 Interacting Domain Death Agonist Protein; D002308:Cardiolipins