scholarly journals Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6

2004 ◽  
Vol 378 (1) ◽  
pp. 247-255 ◽  
Author(s):  
Gerd HEIMLICH ◽  
Alastair D. McKINNON ◽  
Katussevani BERNARDO ◽  
Dieter BRDICZKA ◽  
John C. REED ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
Deletion Mutants ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Cytochrome C Release ◽  
Alpha Helices ◽  
Bh3 Domain ◽  
Apoptotic Protein

The pro-apoptotic protein Bax plays a key role in the mitochondrial signalling pathway. Upon induction of apoptosis, Bax undergoes a conformational change and translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol. However, the domains of Bax that are essential for the induction of cytochrome c release are still elusive. Therefore various Bax deletion mutants were generated and expressed in Escherichia coli. The proteins were then purified in order to delineate the function of the transmembrane domain, the BH3 (Bcl-2 homology 3) domain and the putative pore-forming α-helices-5 and -6. These proteins were used to analyse the mechanism of Bax-induced cytochrome c release from mitochondria. None of the Bax proteins caused cytochrome c release merely through physical perturbation of the mitochondrial outer membrane. The α-helices-5 and -6 of Bax were shown to mediate the insertion of the protein into mitochondrial membranes and to be essential for the cytochrome c-releasing activity of Bax. In contrast, neither the transmembrane domain nor a functional BH3 domain is required for the Bax-mediated release of cytochrome c from mitochondria.

The Molecular Basis for BCL-2 Oncogene Addiction in CLL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5008-5008
Author(s):  
Anthony Letai ◽  
Michael Certo ◽  
Jennifer R. Brown ◽  
Victoria Moore
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Protein Function ◽  
Ex Vivo ◽  
Single Agent ◽  
Myeloma Cell ◽  
Cytochrome C Release ◽  
Bh3 Domain ◽  
Apoptotic Protein

Abstract CLL cells consistently express BCL-2 at a high level. Using a compound demonstrated to antagonize BCL-2 function, ABT-737, we show that CLL cells in short-term primary culture are uniformly sensitive to single-agent BCL-2 antagonism, with EC50’s in the 10 nM range. To understand the mechanism of CLL sensitivity to this compound, we studied mitochondria from these primary CLL cells. We employed a panel of peptides derived from the BH3 domain of pro-death BH3-only proteins, certain of which selectively inhibit BCL-2 function in vitro. We demonstrate that those with activity against BCL-2 in vitro induce cytochrome c release, a hallmark of the mitochondrial dysfunction during apoptosis. A non-malignant cellular model of BCL-2 dependence revealed that binding of the pro-apoptotic protein BIM to BCL-2 correlated with sensitivity to BCL-2 inhibitors. We likewise discovered that BCL-2 binds and sequesters BIM in CLL cells. In contrast to the BCL-2 dependent CLL cells, we examined myeloma cell lines with defined MCL-1 dependence and relative BCL-2 independence. These cell lines were 100 fold less sensitive to ABT-737. Furthermore, in mitochondrial assays only BH3 peptides which interact with MCL-1 induce cytochrome c release. These experiments reveal that cellular requirement for an anti-apoptotic protein function can be deduced from a functional mitochondrial requirement determined using our panel of BH3 peptides. In CLL, and perhaps other malignancies, it appears that BCL-2 maintains survival by sequestering pro-apoptotic proteins like BIM. Inhibition of this sequestration provokes cell death ex vivo, even with a single agent BCL-2 inhibitor. Reliance on BCL-2 for survival may be an Achilles heel for CLL and other cancers.

scholarly journals The Mitochondrial TOM Complex Is Required for tBid/Bax-induced Cytochrome c Release

2007 ◽  
Vol 282 (38) ◽  
pp. 27633-27639 ◽  
Author(s):  
Martin Ott ◽  
Erik Norberg ◽  
Katharina M. Walter ◽  
Patrick Schreiner ◽  
Christian Kemper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Mitochondrial Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Unknown Mechanism

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.

scholarly journals The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

1999 ◽  
Vol 147 (4) ◽  
pp. 809-822 ◽  
Author(s):  
Ruth M. Kluck ◽  
Mauro Degli Esposti ◽  
Guy Perkins ◽  
Christian Renken ◽  
Tomomi Kuwana ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Cytochrome C Release ◽  
Free System ◽  
Xenopus Egg ◽  
Apoptotic Proteins ◽  
Outer Membrane Permeability

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

scholarly journals Bid-Cardiolipin Interaction at Mitochondrial Contact Site Contributes to Mitochondrial Cristae Reorganization and Cytochrome c Release

2004 ◽  
Vol 15 (7) ◽  
pp. 3061-3072 ◽  
Author(s):  
Tae-Hyoung Kim ◽  
Yongge Zhao ◽  
Wen-Xing Ding ◽  
Jin Na Shin ◽  
Xi He ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Intermembrane Space ◽  
Mitochondrial Localization ◽  
Contact Site ◽  
Helical Structures ◽  
Cytochrome C Release ◽  
Outer Membranes ◽  
Bh3 Domain ◽  
Family Protein ◽  
Mitochondrial Intermembrane Space

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release. We found that Bid binding to the mitochondria required only three of its eight helical structures (α4-α6), but not the BH3 domain, and the binding could not be inhibited by the antideath molecule Bcl-xL. Membrane fractionations indicated that tBid bound to mitochondrial outer membranes at both contact and noncontact sites. Bid could interact with specific cardiolipin species on intact mitochondria as identified by mass spectrometry. Like the binding to the mitochondria, this interaction could not be blocked by the mutation in the BH3 domain or by Bcl-xL. However, a cardiolipin-specific dye, 10-N-nonyl acridine orange, could preferentially suppress Bid binding to the mitochondrial contact site and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release. These findings thus suggest that interactions of Bid with mitochondrial cardiolipin at the contact site can contribute significantly to its functions.

scholarly journals Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein

2002 ◽  
Vol 368 (3) ◽  
pp. 915-921 ◽  
Author(s):  
Xavier ROUCOU ◽  
Sylvie MONTESSUIT ◽  
Bruno ANTONSSON ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Plasma Membranes ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Caspase 8 ◽  
Mitochondrial Membranes ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Isolated Mitochondria

In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required.

scholarly journals Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli

2002 ◽  
Vol 159 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Damien Arnoult ◽  
Philippe Parone ◽  
Jean-Claude Martinou ◽  
Bruno Antonsson ◽  
Jérôme Estaquier ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Actinomycin D ◽  
Membrane Permeabilization ◽  
Mitochondrial Outer Membrane ◽  
Simple Explanation ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Mitochondrial Inner Membrane ◽  
Apoptosis Inducing Factor

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

scholarly journals Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

2001 ◽  
Vol 194 (9) ◽  
pp. 1325-1338 ◽  
Author(s):  
Gui-Qiang Wang ◽  
Eva Wieckowski ◽  
Leslie A. Goldstein ◽  
Brian R. Gastman ◽  
Asaf Rabinovitz ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Knockout Mice ◽  
Granzyme B ◽  
Membrane Permeabilization ◽  
Mitochondrial Outer Membrane ◽  
Target Cells ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
S 100

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP

2007 ◽  
Vol 292 (4) ◽  
pp. C1388-C1397 ◽  
Author(s):  
Wenzhi Tan ◽  
Johnathan C. Lai ◽  
Paul Miller ◽  
C. A. Stein ◽  
Marco Colombini
Keyword(s):  
Cytochrome C ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Cytochrome C Release ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Vdac Channels ◽  
Outer Membrane Permeability ◽  
Apoptotic Cascade

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane ( 21 ). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a Ki between 0.2 and 0.5 μM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 μM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 μM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

scholarly journals The Effects of a Mitochondrial Targeted Peptide (Elamipretide/SS31) on BAX Recruitment and Activation During Apoptosis

2021 ◽  
Author(s):  
Joshua Grosser ◽  
Rachel Fehrman ◽  
Dennis Keefe ◽  
Martin Redmon ◽  
Robert Nickells
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Outer Membrane ◽  
Intrinsic Apoptosis ◽  
Mitochondrial Fragmentation ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Bax Protein ◽  
Induced Apoptosis ◽  
Kinetics Of ◽  
Peptide Treatment

Abstract Objective: Elamipretide (SS31) is a mitochondria-targeted peptide that has reported functions of stabilizing mitochondrial cristae structure and improving mitochondrial bioenergetics. Several studies have documented cell protective features of this peptide, including impairment of intrinsic apoptosis by inhibiting the recruitment and activation of the pro-apoptotic BAX protein. We used live-cell imaging of ARPE-19 cells expressing fluorescently labeled BAX, cytochrome c, and a mitochondrial marker to investigate the effect of elamipretide on the kinetics of BAX recruitment, mitochondrial outer membrane permeabilization (as a function of cytochrome c release), and mitochondrial fragmentation, respectively. Result: In nucleofected and plated ARPE-19 cells, elamipretide accelerated the formation of larger mitochondria. In the presence of the apoptotic stimulator, staurosporine, cells treated with elamipretide exhibited moderately slower rates of BAX recruitment. Peptide treatment, however, did not significantly delay the onset of BAX recruitment or the final total amount of BAX that was recruited. Additionally, elamipretide showed no impairment or delay of cytochrome c release or mitochondrial fragmentation, two events associated with normal BAX activation during cell death. These results indicate that the protective effect of elamipretide is not at the level of BAX activity to induce pro-apoptotic mitochondrial dysfunction after the initiation of staurosporine-induced apoptosis.

scholarly journals Biphasic translocation of Bax to mitochondria

2002 ◽  
Vol 367 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Michela CAPANO ◽  
Martin CROMPTON
Keyword(s):  
Green Fluorescent Protein ◽  
Cytochrome C ◽  
Fluorescent Protein ◽  
Adenine Nucleotide ◽  
Phase 1 ◽  
Mitochondrial Outer Membrane ◽  
Second Phase ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Green Fluorescent

Using green fluorescent protein-tagged Bax, we demonstrate that Bax is sequestered from the cytosol of cardiomyocytes in two distinct phases following the induction of apoptosis with staurosporine. In the first phase, lasting several hours, Bax removal from the cytosol was relatively small. In the second phase, Bax was very largely removed from the cytosol and sequestered into large aggregates associated with the mitochondria. To test which of the phases involved cytochrome c release, cells were transfected with a red fluorescent protein—cytochrome c fusion. The cytochrome c fusion protein was accumulated by mitochondria of healthy cells and was released by staurosporine in phase 1. When green fluorescent protein—Bax was immunoprecipitated from extracts of cells in phase 1 and phase 2, the voltage-dependent anion channel (mitochondrial outer membrane) and the adenine nucleotide translocase (mitochondrial inner membrane) were also precipitated. These data support a two-phase model of Bax translocation in which Bax targets the mitochondrial intermembrane contact sites and releases cytochrome c in the first phase, and is then packaged into large aggregates on mitochondria in the second.

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