Studies of the Interaction of Substituted Mutants of BAX with Yeast Mitochondria Reveal That the C-terminal Hydrophobic α-Helix Is a Second ART Sequence and Plays a Role in the Interaction with Anti-apoptotic BCL-xL

Arokium, Hubert; Camougrand, Nadine; Vallette, François M.; Manon, Stéphen

doi:10.1074/jbc.m408373200

Cited by 62 publications

(97 citation statements)

References 41 publications

(74 reference statements)

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“…The utilisation of yeast has provided new information concerning the molecular aspects and mechanisms of Bax pro-apoptotic function that have been further confirmed in mammalian cells [123][124][125][126]. Evidence accumulates that Bax lethality is tightly connected and might even be regulated by autophagic and in particular mitophagic processes.…”

Section: Discussionmentioning

confidence: 99%

The mitochondrial pathway in yeast apoptosis

et al. 2007

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Mitochondria are not only important for the energetic status of the cell, but are also the fatal organelles deciding about cellular life and death. Complex mitochondrial features decisive for cell death execution in mammals are present and functional in yeast: AIF and cytochrome c release to the cytosol, mitochondrial fragmentation as well as mitochondrial hyperpolarisation followed by an oxidative burst, and breakdown of mitochondrial membrane potential. The easy accessibility of mitochondrial manipulations such as repression of respiration by growing yeast on glucose or deletion of mitochondrial DNA (rho 0 ) on the one hand and the unique ability of yeast cells to grow on nonfermentable carbon sources by switching on mitochondrial respiration on the other hand have made yeast an excellent tool to delineate the necessity for mitochondria in cell death execution. Yeast research indicates that the connection between mitochondria and apoptosis is intricate, as abrogation of mitochondrial function can be either deleterious or beneficial for the cell depending on the specific context of the death scenario. Surprisingly, mitochondrion dependent yeast apoptosis currently helps to understand the aetiology (or the complex biology) of lethal cytoskeletal alterations, ageing and neurodegeneration. For example, mutation of mitochondrial superoxide dismutase or CDC48/VCP mutations, both T. Eisenberg · S. Büttner · F. Madeo ( ) Institute of Molecular Biosciences, Universitaetsplatz 2, University of Graz, 8010 Graz, Austria e-mail: frank.madeo@uni-graz.at G. Kroemer Apoptosis, Cancer, and Immunity Unit, Institut Gustave Roussy, Faculté Paris Sud-Université, Institut National de la Santé et de la Recherche Médicale, Paris XI, France implicated in several neurodegenerative disorders, are associated with mitochondrial impairment and apoptosis in yeast.

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Section: Discussionmentioning

confidence: 99%

The mitochondrial pathway in yeast apoptosis

et al. 2007

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“…The presence of the disruption cassette was verified by PCR with one primer located in the promoter of TOM22 and one located inside the gene kanMX4. W303D and W303D-Dtom22 were transformed with plasmids pYES3-BaxC and pYES3-Bax-T 174 D 38 and selected for growth in the absence of uracile and tryptophan, respectively. These plasmids expressed these active variants of Bax under the control of the regulatable promoter GAL1/10.…”

Section: Methodsmentioning

confidence: 99%

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

et al. 2006

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The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway. Apoptosis is a cell death program, which is central in many aspects to metazoan cell physiology and pathology. 1 Proteins of the Bcl-2 family are key players in the execution phase of apoptosis and their main functions is to control the release of apoptogenic proteins, such as cytochrome c (cyt c) and apoptosis inducing factor (AIF) from the mitochondria. 2 The Bcl-2 family is composed of antiapoptotic members such as Bcl-2 and proapoptotic proteins such as Bax or Bak, which share several domains of homology called BH. 1 In addition to these proteins, a third Bcl-2-related family of proteins have been identified as major amplifiers and/inducers of apoptosis. 2 The latter family has a homology to Bcl-2 limited to the BH3 domain and thus is called the BH3 only proteins. 2 The double knockout of Bax and Bak renders cells completely resistant to cell death 2 highlighting the essential role of these proteins during apoptosis.The determination of the solution structure of Bax showed its organization around nine a-helices (Ha1 to Ha9) 3 of which Ha2 contained most of the BH3 domain and Ha5 and Ha6, a putative pore forming domain that has been shown to be involved in the integration of Bax into the membrane. 4,5 Bax resides in an inactive state in the cytosol in many resting cells and is translocated to the mitochondria at the onset of apoptosis. 6 This translocation is associated with major conformational changes in Bax as both the amino-terminal end (N-T) and carboxy terminal end (C-T) become exposed facilitating its mitochondrial addressing and membrane insertion. 5,7 Bax mitochondrial membrane insertion and oligomerization is closely associated with the release into the cytosol of several intermembrane space proteins such as cyt c, second mitochondrial apoptotic factor (Smac) and AIF. 2 The nature of the different stimuli involved in the activation or in the inhibition of Bax is still largely unknown although this step remains one of the most critical points of control of the apoptotic program during which therapeutic interventions can be envisaged. 2 The mit...

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“…Some mutations in the hydrophobic groove of Bcl-2 were shown to prevent its binding to full-length Bax without affecting its binding to the sole BH3 domain of Bax (34). Moreover, regions distinct from that encompassing the Bax BH3 domain and from these forming the BH3 binding site of Bcl-2 homologues have recently been reported to be involved in the regulation of Bax binding to antiapoptotic Bcl-2 family members (35,36). The interaction between Bcl-2 and Bax might therefore be subject to more stringent conformational constraints than that between Bcl-2 and a sole BH3 domain, and HA14-1 might interact with regions within Bcl-2 that are more critical for its interaction with the Bax protein than for its binding to BH3 domains.…”

Section: Discussionmentioning

confidence: 99%

The Small Organic Compound HA14-1 Prevents Bcl-2 Interaction with Bax to Sensitize Malignant Glioma Cells to Induction of Cell Death

Manero¹,

Gautier²,

Gallenne³

et al. 2006

Cancer Research

125

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A functional imbalance between proapoptotic Bax and antiapoptotic Bcl-2 is likely to participate in the resistance of cancer cells to therapy. We show here that ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a small organic compound recently proposed to function as an inhibitor of Bcl-2, increases the sensitivity of human glioblastoma cells to radiotherapy and chemotherapy. This sensitizing effect is lost if Bcl-2 expression, but not Bcl-xL expression, is knocked down or if cells only express a mutant of Bax that does not interact with Bcl-2. This points to a specific Bcl-2 inhibitory function of HA14-1 and implies that it selectively involves hindrance of Bcl-2 binding to Bax, which HA14-1 inhibits in cell-free assays and in cells in receipt of an apoptotic stimulation. Moreover, HA14-1, in combination with a cytotoxic treatment, slows down the growth of glioblastoma in vivo. Thus, the inhibition of Bcl-2 achieved by HA14-1 might improve treatment outcome. (Cancer Res 2006; 66(5): 2757-64)

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Studies of the Interaction of Substituted Mutants of BAX with Yeast Mitochondria Reveal That the C-terminal Hydrophobic α-Helix Is a Second ART Sequence and Plays a Role in the Interaction with Anti-apoptotic BCL-xL

Cited by 62 publications

References 41 publications

The mitochondrial pathway in yeast apoptosis

The mitochondrial pathway in yeast apoptosis

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

The Small Organic Compound HA14-1 Prevents Bcl-2 Interaction with Bax to Sensitize Malignant Glioma Cells to Induction of Cell Death

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