The substitution of the C‐terminus of bax by that of bcl‐xL does not affect its subcellular localization but abrogates its pro‐apoptotic properties

Oliver, Lisa; Priault, Muriel; Tremblais, Karine; LeCabellec, Marité; Méflah, Khaled; Manon, Stéphen; Vallette, François M.

doi:10.1016/s0014-5793(00)02330-9

Cited by 39 publications

(52 citation statements)

References 25 publications

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“…The results were interpreted as a requirement for a spatial conformation allowing the insertion of ␣9; however, they could as well be interpreted as a requirement for a conformation of ␣9 being able to move correctly or, alternatively, able to confer the adequate exposure to ␣5 and ␣6, which were recently demonstrated to be actually responsible for BAX insertion (38). In support to the latter hypothesis, a P168A or a P168V substitution induced a strong activation of BAX both in yeast and in BAX-deficient glioma cells, 2 suggesting that, in the absence of endogenous BAX, the conformational change induced by the change of the angle formed by Pro-168 allows the protein to reach a structure close to that able to be inserted into the membrane and to induce the release of cytochrome c. This work and the data from previous reports (20,31,32,35) support the hypothesis that not only is helix ␣9 not an addressing/insertion sequence but that its movement is also required to allow BAX to be addressed and inserted in the outer mitochondrial membrane, in a manner similar to the N-terminal ART sequence.…”

Section: Discussionsupporting

confidence: 61%

“…Finally, replacement of BAX ␣9-helix by the homologous sequence of BCL-x L (␣8, a true membrane targeting sequence) resulted in strong binding but no cytochrome c release. Similar data were reported in mammalian cells for the deletion and the replacement of BAX-␣9 by BCL-x L -␣8 (35), in contradiction with previous data obtained with BAX-GFP fusion protein.…”

Section: Resultssupporting

confidence: 53%

“…Less dramatic alterations were assayed in yeast (31); the replacement of ␣9 by an unstructured hydrophilic sequence resulted in a very active protein. On the other hand, its replacement by a true membrane anchor, that of BCL-x L , led to a still mitochondria-localized protein that had lost pro-apoptotic activity both in yeast (31) and mammalian cells (35).…”

Section: Discussionmentioning

confidence: 99%

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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

Arokium

Camougrand

Vallette

et al. 2004

Journal of Biological Chemistry

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The role of the two ends of the pro-apoptotic protein BAX in its interaction with mitochondria was challenged by assaying substituted mutants in yeast cells for the ability to bind and insert into the mitochondrial membrane and to promote the release of cytochrome c. Mutations at the N-terminal end confirmed the inhibitory function of this zone, known as apoptotic regulation of targeting (ART). On the other hand, mutations at the C-terminal end of the protein support the hypothesis that the hydrophobic helix ␣9 is not required for the insertion of BAX. In addition, three mutations (a T174D single substitution in the helix ␣9, a K189E/K190E double substitution at the end of the protein, and a P168A mutation in the loop before ␣9) exhibited a strong binding capacity, a strong insertion, as well as high ability to induce cytochrome c release. Considering the positions of these mutations and their potential effect on the movement of helix ␣9, we propose that the C-terminal end of the protein behaves like a second ART. Also, opposite to a mutation that changes the conformation of the N-terminal ART, the mutations in the C-terminal part of the protein impaired the inhibitory effect of antiapoptotic BCL-x L over BAX insertion, suggesting that the conformation of the ␣9-helix plays a significant role in BAX/BCL-x L interaction.Apoptosis, the main form of programmed cell death, is a highly regulated phenomenon. It is now largely accepted that mitochondria play a key role in regulating the apoptotic process by controlling the release of several proteins, termed apoptogenic factors, required for the acquisition of apoptotic hallmarks (for review see Refs. 1 and 2). This release strictly depends on the action of a family of proteins, termed the BCL-2 family, that modulates the permeability of the outer mitochondrial membrane to the apoptogenic factors (for review see Refs. 3 and 4). Moreover, the alteration of the bioenergetic capacity of mitochondria following the action of BCL-2 family members is thought to be further involved in the regulation of apoptosis by the production of reactive oxygen species (for review see Ref. 5) or the release of Ca 2ϩ (for review see Ref. 6).Among the BCL-2 family members, the pro-apoptotic protein BAX plays a crucial role (for review see Ref. 7). BAX is expressed at basal levels in nearly all mammalian cells. In healthy cells, it behaves as a soluble protein and is localized in the cytosol; following apoptotic induction, the protein is translocated to the mitochondrial outer membrane where it behaves as a membrane-inserted protein (8, 9) and actively participates in the release of apoptogenic factors. The molecular mechanisms governing the cytosol-to-mitochondria translocation of BAX therefore appear as a critical regulation in the implementation of apoptosis.The changes in the physical properties of BAX accompanying its translocation strongly support the hypothesis that profound conformational changes are the basis of the change in the localization of the protein. Analysis of the primary...

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Section: Discussionsupporting

confidence: 61%

Section: Resultssupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

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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

Arokium

Camougrand

Vallette

et al. 2004

Journal of Biological Chemistry

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“…22,23 The C-terminus of Bax appears to play an essential role in apoptosis as deletion of this segment abrogates its mitochondrial insertion, 24 and substitution of Bax Cterminus with the Bcl-xL C-terminus abrogates its proapoptotic activity. 25 Indeed, a conformational change in Bax occurs to allow pore formation in cellular membranes. 26 Pro-apoptotic Bad, however, lacks the C-terminus transmembrane domain and is localised in the cytosol, but becomes membrane associated when complexed with Bcl-xL and Bcl-2.…”

Section: Introductionmentioning

confidence: 99%

High Bcr-Abl expression prevents the translocation of Bax and Bad to the mitochondrion

2002

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Bcr-Abl is a constitutively active tyrosine kinase involved in the development and progression of chronic myeloid leukaemia (CML). It has been demonstrated that Bcr-Abl-positive cells can be uniquely resistant to apoptosis induced by different types of stimuli, but the mechanism by which this is achieved is not defined. In this study we have investigated how cells expressing high expression levels of Bcr-Abl may gain resistance to cytotoxic drugs. We have established cell lines expressing low and high expression levels of Bcr-Abl. Cells expressing elevated Bcr-Abl are resistant to cytotoxic drugs. In drug-sensitive 32D-parental and low Bcr-Abl expressing cells, pro-apoptotic Bcl-2 family members, Bax and Bad translocate from the cytosol to the mitochondrion following a cytotoxic insult. In contrast, high Bcr-Abl expression prevents the early translocation of these pro-apoptotic proteins to the mitochondrion, mitochondrial membrane potential is retained and caspases are inactive. We also demonstrate that IL-3 can contribute to drug resistance in low Bcr-Abl expressing cells, however, independent inhibition of IL-3 activated pathways (PI3K/AKT and Jak/STAT) does not sensitise cells to apoptosis. This study demonstrates that the subcellular translocation of Bax and Bad can be regulated by elevated Bcr-Abl expression and this may be a key event in the abrogation of an apoptotic response following a cytotoxic insult.

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“…The 28 kDa band may correspond to Mcl-1S whereas the 26 kDa band could be the result of a cleavage of Mcl-1 (Snowden et al, 2003;Herrant et al, 2004;Michels et al, 2004). Indeed, MDN cells treated by melphalan during 8 h presented a significant down-regulation of Mcl-1L, an accumulation of the 26 kDa band and the appearance of a weak 16 kDa band, suggesting that (Oliver et al, 2000). An amount of 50 mg mitochondrial or cytosolic fraction (S100) were separated in 15% SDS-PAGE.…”

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confidence: 98%

Melphalan-induced apoptosis in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim complex

et al. 2005

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Multiple myeloma (MM) is a rapidly fatal plasma-cell malignancy that evolves mainly in the bone marrow. Melphalan is widely used to treat patients with MM but as yet its mechanisms of action are poorly documented. In the current study, we demonstrate that melphalan induces a drastic downregulation of Mcl-1L, Bcl-x L and BimEL in human melphalan-sensitive myeloma cells while the most potent proapoptotic isoforms, BimL and S, are affected to a lesser extent. Moreover, Mcl-1L and BimEL disappearance is associated with the generation of proapoptotic cleaved forms generated by a caspase cleavage. In myeloma cells, we have previously shown that Mcl-1 neutralizes the proapoptotic function of Bim and therefore, prevents the activation of death effectors. In this study, we demonstrate that melphalan disrupts the Mcl-1/ Bim complex whereas the Bcl-2/Bim complex is not modified. The disappearance of full length Mcl-1 allows the release of Bim isoforms, particularly L and S, which can exert their proapoptotic function and leads to Bax activation and cytochrome c release. Thus, we can hypothesize that the cleaved 26 kDa proapoptotic Mcl-1 and the 19 and 12 kDa of Bim, generated during melphalan treatment could contribute to the amplification loop of apoptosis.

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The substitution of the C‐terminus of bax by that of bcl‐xL does not affect its subcellular localization but abrogates its pro‐apoptotic properties

Cited by 39 publications

References 25 publications

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

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

High Bcr-Abl expression prevents the translocation of Bax and Bad to the mitochondrion

Melphalan-induced apoptosis in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim complex

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