Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

Tatulian, Suren A.; Garg, Pranav; Nemec, Kathleen N.; Chen, Bin; Khaled, Annette R.

doi:10.1021/bi301195f

Cited by 19 publications

(16 citation statements)

References 67 publications

(187 reference statements)

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“…In addition, we examined whether the α9 helix (or the α9:α9 interface) might be necessary for the step of pore formation, as α9 appears to be the only region that traverses the MOM before and after pore formation, [21][22][23] and α9 peptides have been proposed to be membranolytic with antitumor activity. 43,58 Thus, understanding α9 membrane topology may reveal novel insight for cancer therapy.…”

Section: Discussionmentioning

confidence: 99%

“…Single amphipathic helices such as melittin can form relatively stable 'lipidic' pores, 64 and hydrophobic peptides based on Bak or Bax α9 can also permeabilize mitochondria (Supplementary Figures S5a and b) 65 and vesicles. 41,43,46,47 However, green fluorescent protein (GFP) fused to Bax or Bak α9 was not reported to kill cells, 28,32,34,66 and we know of no instance in which a C-terminal transmembrane anchor in a full-length protein takes part directly in forming the pore. We note also that a Bak C terminus is not essential for the step of pore formation, as recombinant Bak with a hexahistidine tag in lieu of its C terminus can bind and permeabilize nickelincorporated liposomes.…”

Section: Discussionmentioning

confidence: 99%

“…The peptides adopt a predominantly α-helical secondary structure, 40-43 with orientation affected by lipid composition. 42,44,45 The peptides could also permeabilize lipid vesicles, 41,43,46,47 suggesting that the …”

mentioning

confidence: 99%

“…To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure, [40][41][42][43] with orientation affected by lipid composition. 42,44,45 The peptides could also permeabilize lipid vesicles, 41,43,46,47 suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.…”

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confidence: 99%

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Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

et al. 2015

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Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins. 1-3 Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices. 4,5 They are activated by BH3-only proteins binding to the α2-α5 surface groove, 6-12 or for Bax, to the α1/α6 'rear pocket'. 13 Binding triggers dissociation of the latch domain (α6-α8) from the core domain (α2-α5), together with exposure of N-terminal epitopes and the BH3 domain. 6,7,14-16 The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

et al. 2015

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“…One strategy is to derive peptides from previously characterized, endogenous cellular proteins. This approach takes advantage of the information available on the localization and function of the protein from which the peptide is derived and could inform of the potential [4][5][6]11] molecular target and action of the peptide. Recently, we reported that a 20 amino acid -helical peptide developed from the 9 transmembrane domain of the pro-apoptotic protein, Bax, has cytotoxic actions for cancer cells [4] .…”

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confidence: 99%

The CT20 peptide: More than a Piece of Bax

Lee¹,

Bassiouni

Iketani

et al. 2014

Can Cell Microenviron

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Use of cytotoxic peptides for cancer treatment is an emerging therapeutic direction with promising potential; however, in many instances the intracellular actions of these peptides remain unknown. Finding ways to systematically decipher the intercellular targets and functions of rationally designed or endogenously sourced therapeutic peptides to better define clinical use is a significant challenge that needs to be addressed. Here we describe our recent results outlining the activity of CT20p, a peptide derived from the pro-apoptotic protein Bax, which specifically kills metastatic cancer cells but not normal epithelial cells. Using breast cancer as a model system to test the peptide, we found that CT20p can be delivered to cells by polymeric hyper branched nanoparticles and that, once intracellular, have profound effects on cytosolic proteins and organelles in a cancerspecific fashion that are different from the parent protein. In cancer cells, CT20p localizes to the mitochondria, forms pores in mitochondrial-like membranes, alters mitochondrial movement and membrane polarization, and impairs cytoskeletal reorganization, leading to cell detachment and death. Further, we discuss our strategy for determining the molecular actions of CT20p that could help inform the activities of other currently available antimicrobial and anticancer therapeutic peptides.

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Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

et al. 2021

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Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

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Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

Cited by 19 publications

References 67 publications

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

The CT20 peptide: More than a Piece of Bax

Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

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