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 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.

scholarly journals Preservation of Mitochondrial Structure and Function after Bid- or Bax-Mediated Cytochrome c Release

2000 ◽  
Vol 150 (5) ◽  
pp. 1027-1036 ◽  
Author(s):  
Oliver von Ahsen ◽  
Christian Renken ◽  
Guy Perkins ◽  
Ruth M. Kluck ◽  
Ella Bossy-Wetzel ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
And Function

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1–mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

scholarly journals Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

2007 ◽  
Vol 405 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Emily E. Franklin ◽  
John D. Robertson
Keyword(s):  
Cytochrome C ◽  
Genotoxic Stress ◽  
Jurkat Cells ◽  
Mitochondrial Outer Membrane ◽  
Cytochrome C Release ◽  
Drug Induced ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Caspase 2 ◽  
Induced Apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.

scholarly journals Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition

2004 ◽  
Vol 378 (1) ◽  
pp. 213-217 ◽  
Author(s):  
Vladimir GOGVADZE ◽  
John D. ROBERTSON ◽  
Mari ENOKSSON ◽  
Boris ZHIVOTOVSKY ◽  
Sten ORRENIUS
Keyword(s):  
Cytochrome C ◽  
In Vitro Model ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Mitochondrial Outer Membrane ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Mitochondrial Volume ◽  
Vitro Model

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active.

scholarly journals VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release

2001 ◽  
Vol 155 (6) ◽  
pp. 1003-1016 ◽  
Author(s):  
Muniswamy Madesh ◽  
György Hajnóczky
Keyword(s):  
Cytochrome C ◽  
Hepg2 Cells ◽  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Anion Channel ◽  
Permeability Transition ◽  
Mitochondrial Outer Membrane ◽  
Inner Mitochondrial Membrane ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release

Enhanced formation of reactive oxygen species (ROS), superoxide (O2·−), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2·− elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2·− and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2·−-induced CCR. Furthermore, O2·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2·−-induced CCR did not depend on Bax translocation to mitochondria. O2·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.

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.

scholarly journals Essential Role of Voltage-Dependent Anion Channel in Various Forms of Apoptosis in Mammalian Cells

2001 ◽  
Vol 152 (2) ◽  
pp. 237-250 ◽  
Author(s):  
Shigeomi Shimizu ◽  
Yosuke Matsuoka ◽  
Yasuo Shinohara ◽  
Yoshihiro Yoneda ◽  
Yoshihide Tsujimoto
Keyword(s):  
Cytochrome C ◽  
Mammalian Cells ◽  
Essential Role ◽  
Biological Significance ◽  
Anion Channel ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Isolated Mitochondria ◽  
Voltage Dependent ◽  
Induced Apoptosis

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Δψ), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

The warburg effect and tumour cell survival in human GBMs

2007 ◽  
Vol 30 (4) ◽  
pp. 97 ◽  
Author(s):  
A Wolf ◽  
J Mukherjee ◽  
A Guha
Keyword(s):  
Cytochrome C ◽  
Cell Survival ◽  
Expression Profile ◽  
Tumour Cell ◽  
Warburg Effect ◽  
Glycolytic Enzymes ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
The Warburg Effect

Introduction: GBMs are resistant to apoptosis induced by the hypoxic microenvironment and standard therapies including radiation and chemotherapy. We postulate that the Warburg effect, a preferential glycolytic phenotype of tumor cells even under aerobic conditions, plays a role in these aberrant pro-survival signals. In this study we quantitatively examined the expression profile of hypoxia-related glycolytic genes within pathologically- and MRI-defined “centre” and “periphery” of GBMs. We hypothesize that expression of hypoxia-induced glycolytic genes, particularly hexokinase 2 (HK2), favours cell survival and modulates resistance to tumour cell apoptosis by inhibiting the intrinsic mitochondrial apoptotic pathway. Methods: GBM patients underwent conventional T1-weighted contrast-enhanced MRI and MR spectroscopy studies on a 3.0T GE scanner, prior to stereotactic sampling (formalin and frozen) from regions which were T1-Gad enhancing (“centre”) and T2-positive, T1-Gad negative (“periphery”). Real-time qRT-PCR was performed to quantify regional gene expression of glycolytic genes including HK2. In vitro functional studies were performed in U87 and U373 GBM cell lines grown in normoxic (21% pO2) and hypoxic (< 1%pO2) conditions, transfected with HK2 siRNA followed by measurement of cell proliferation (BrdU), apoptosis (activated caspase 3/7, TUNEL, cytochrome c release) and viability (MTS assay). Results: There exists a differential expression profile of glycolytic enzymes between the hypoxic center and relatively normoxic periphery of GBMs. Under hypoxic conditions, there is increased expression of HK2 at the mitochondrial membrane in GBM cells. In vitro HK2 knockdown led to decreased cell survival and increased apoptosis via the intrinsic mitochondrial pathway, as seen by increased mitochondrial release of cytochrome-C. Conclusions: Increased expression of HK2 in the centre of GBMs promotes cell survival and confers resistance to apoptosis, as confirmed by in vitro studies. In vivo intracranial xenograft studies with injection of HK2-shRNA are currently being performed. HK2 and possibly other glycolytic enzymes may provide a target for enhanced therapeutic responsiveness thereby improving prognosis of patients with GBMs.

scholarly journals Phosphorylation of the mitochondrial triphosphate tunnel metalloenzyme TTM1 regulates programmed cell death in senescence

2020 ◽  
Author(s):  
Purva Karia ◽  
Keiko Yoshioka ◽  
Wolfgang Moeder
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Map Kinases ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Mitogen Activated Protein ◽  
Animal Growth ◽  
Triphosphate Tunnel Metalloenzyme

ABSTRACTThe role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein kinases (MAPKs) regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (S10, S437, and S490), which are phosphorylated upon perception of senescence cues such as ABA and prolonged darkness. S437 is phosphorylated by the MAP kinases MPK3 and MPK4, and S437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), phosphorylate S10 and S490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-related PCD, revealing a novel link between mitochondria and PCD.SummaryEmail addresses: [email protected]

scholarly journals BIOGENESIS OF MITOCHONDRIA

1969 ◽  
Vol 42 (2) ◽  
pp. 377-391 ◽  
Author(s):  
G. M. Kellerman ◽  
D. R. Biggs ◽  
Anthony W. Linnane
Keyword(s):  
Unsaturated Fatty Acids ◽  
Mitochondrial Protein ◽  
Mitochondrial Membranes ◽  
Phylogenetic Implications ◽  
Obligate Aerobic ◽  
Biogenesis Of Mitochondria ◽  
Citric Acid Cycle Enzymes ◽  
Definition Of

Growth under conditions of oxygen restriction results in a generalized decrease in the definition of the mitochondrial membranes, a decrease in the mitochondrial cytochromes, and a decrease in citric acid cycle enzymes of the obligate aerobic yeast Candida parapsilosis. Addition of unsaturated fatty acids and ergosterol to cultures exposed to limited oxygen results in improved definition of the mitochondrial membranes and an increase in the total mitochondrial cytochrome content of the cells. Euflavine completely inhibits mitochondrial protein synthesis in vitro. Its in vivo effect is to cause the formation of giant mitochondrial profiles with apparently intact outer membranes and modified internal membranes; the cristae (in-folds) appear only as apparently disorganized remnants while the remainder of the inner membrane seems intact. Cytochromes a, a3, b, and c1 are not synthesized by the cells in the presence of euflavine. Ethidium appears to have effects identical to those of euflavine, whereas chloramphenicol, lincomycin, and erythromycin have similar effects in principle but they are less marked. The effects of all the inhibitors are freely reversible after removal of the drugs. The results are discussed in terms of a functionally three-membrane model of the mitochondrion. In addition, the phylogenetic implications of the observed differences between this organism and the facultative anaerobic yeasts are considered.

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