The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Membrane Permeability ; Cytochrome c Group ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

Pore-formation and protein-protein interactions are considered to play critical roles in the regulation of apoptosis by Bcl-2 family proteins. During the initiation of apoptosis, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax form different pores to regulate the permeability of mitochondrial outer membrane, playing their opposite functions. Overexpression of Bcl-2 has been found in various cancer cells, therefore it is gaining widespread interest to discover small molecules to compromise Bcl-2 function for anti-cancer treatment. Since Bax binds to Bcl-2's hydrophobic groove via its BH3 domain (composed of helices 2 and 3), by which their functions are inhibited each other, the H2-H3 peptide that contains the functional BH3 domain of Bax has been considered as a potential Bcl-2 antagonist. We recently reported that Bax peptide H2-H3 promotes cell death by inducing Bax-mediated cytochrome c release and by antagonizing Bcl-2's inhibitory effect on Bax. However, the mechanism of how H2-H3 inhibits the anti-apoptotic activity of Bcl-2 remains poorly understood. To address this question, we reconstituted the Bcl-2 or Bax pore-forming process in vitro. We found that H2-H3 inhibited Bcl-2's pore formation and neutralized Bcl-2's inhibitory effect on Bax pore formation in the membrane, whereas the mutant H2-H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2's activities. Thus, inhibiting Bcl-2's pore-forming and anti-Bax activities in the membrane is strongly correlated with H2-H3's pro-apoptosis function in cells.
Apoptosis ; physiology ; BH3 Interacting Domain Death Agonist Protein ; chemistry ; Humans ; Membrane Proteins ; chemistry ; metabolism ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Membranes ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; chemistry ; bcl-2 Homologous Antagonist-Killer Protein ; chemistry ; bcl-2-Associated X Protein ; chemistry

The three dimensional structures of both pro-apoptotic Bax and anti-apoptotic Bcl-2 are strikingly similar to that of pore-forming domains of diphtheria toxin and E. coli colicins. Consistent with the structural similarity, both Bax and Bcl-2 have been shown to possess pore-forming property in the membrane. However, these pore-forming proteins form pores via different mechanisms. While Bax and diphtheria toxin form pores via oligomerization, the colicin pore is formed only by colicin monomers. Although the oligomers of Bcl-2 proteins have been found in the mitochondria of both healthy and apoptotic cells, it is unknown whether or not oligomerization is involved in the pore formation. To determine the mechanism of Bcl-2 pore formation, we reconstituted the pore-forming process of Bcl-2 using purified proteins and liposomes. We found that Bcl-2 pore size depended on Bcl-2 concentration, and the release of smaller entrapped molecules was faster than that of larger ones from liposomes at a given Bcl-2 concentration. Moreover, the rate of dye release mediated by pre-formed wild-type Bcl-2 oligomers or by the mutant Bcl-2 monomers with a higher homo-association affinity was much higher than that by wild-type Bcl-2 monomers. Together, it is suggested that oligomerization is likely involved in Bcl-2 pore formation.
Apoptosis ; physiology ; Cytosol ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; Protein Multimerization ; Proto-Oncogene Proteins c-bcl-2 ; metabolism