Laurent M. Dejean

A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2

Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.

Mitochondrial ion channels in cancer transformation

Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process.

MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax / Bak oligomers

Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax and Bak oligomers in the mitochondrial outer membrane. iMACs exert comparable effects on channel conductance of Bax or Bak and similarly affect cytochrome c release from Bax or Bak-containing mitochondria. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. Western blot analysis showed that wild type mitochondria had lower steady state levels of Bak in the absence of apoptotic stimulation.

A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer.

New Insights on the Regulation of Programmed Cell Death by Bcl-2 Family Proteins at the Mitochondria: Physiological and Pathophysiological Implications

Apoptosis is the major form of programmed cell death in animals, playing a crucial role in development and tissue homeostasis. Alterations of apoptosis are involved in major pathologies such as cancers and neurodegenerative diseases. The group of proteins known as Bcl-2 family members are at the core of the implementation of this process, to the point that any cancer study almost compulsorily follows the expression of these proteins. For long, the literature about the molecular aspects underlying their function has often been contradictory, if not overly simplified. However, recent breakthroughs have been obtained with structural and imaging methods, offering the opportunity to shed light on many elusive and hardly understandable observations made in the last 20 years, starting when investigators recognized the connection between Bcl-2 family members and mitochondria. This survey does not intend to be an exhaustive review of the large amount of literature on Bcl-2 family members. Rather, we would like to highlight issues that have been somewhat overlooked in the mainstream literature and that might deserve more attention in the light of recent advances. As mitochondriologists have moved toward the field of apoptosis, our views might be different from the ones of many of our colleagues who have done the reverse journey. Our wish is that some of our readers will find them interesting enough to embark upon studying this exciting and fascinating family of proteins.