Peptides Derived from Apoptotic Bax and Bid Reproduce the Poration Activity of the Parent Full-Length Proteins

Abstract

Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

“…This difference in the selectivity of colicin channels in neutral and anionic membranes is also consistent with a model of direct involvement of lipids in the pore wall (90). In fact, the ion selectivity of alamethicin (87) and OmpF (87, 91) channels can be modulated by mutation of charged amino acid residues located in the pore walls, in agreement with the local electrostatics.…”

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model

“…Hence, the central helices (a5-a6) of Bax carry the minimal structural information to form pores in lipid membranes, similar to amphipathic peptide antibiotics. [63][64][65][66][67] We previously established that a 29-residue peptide (Poro1) corresponding to the extended fifth helix of Bax can disrupt the mitochondrial membrane, inducing DJm loss and cytochrome c release. 43 This Bax-derived peptide was more efficient than (KLAKLAK) 2 or the BH3 peptidic domain of Bax in inducing apoptosis in tumor cells in vitro.…”

Bid: a Bax-like BH3 protein

“…Glycosylation mapping suggested that helix 6, or the combination of helices 6 and 7, could insert into membranes (Garcia-Saez et al, 2004). Furthermore, expression of various Bid fragments, either alone or as fusion proteins, strongly suggested the involvement of helix 6 in binding to membranes (Lutter et al, 2000;Hu et al, 2003;Kim et al, 2004;Garcia-Saez et al, 2005). Determination of the structure of membrane-bound tBid by electron paramagnetic resonance (EPR), NMR and CD spectroscopy indicated that although helices 6, 7 and the short C-terminal helix 8 all insert into the membrane, none is membrane-spanning and only a portion of each helix inserts into the bilayer (Gong et al, 2004;Oh et al, 2005).…”