Unravelling the Bcl-2 Apoptosis Code with a Simple Model System

Using defined lipids and purified proteins, Billen et al. offer a new model to reconcile the two currently opposing models for how Bcl-2 family member interactions regulate cell death

BIM and tBID are not mechanistically equivalent when assisting BAX to permeabilize bilayer membranes.

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-X(L), BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency

BFL1 Modulates Apoptosis at the Membrane Level through a Bifunctional And Multimodal Mechanism Showing Key Differences With BCLXL

BFL1 is a relatively understudied member of the BCL2 protein family which has been implicated in the pathogenesis andchemoresistance of a variety of human cancers, including hematological malignancies and solid tumours. BFL1 is generallyconsidered to have an antiapoptotic function, although its precise mode of action remains unclear. By quantitativelyanalyzing BFL1 action in synthetic membrane models and in cells, we found that BFL1 inhibits apoptosis through threedistinct mechanisms which are similar but not identical to those of BCLXL, the paradigmatic antiapoptotic BCL2 familyprotein. Strikingly, alterations in lipid composition during apoptosis activate a prodeath function of BFL1 that is based onnoncanonical oligomerization of the protein and breaching of the permeability barrier of the outer mitochondrial membrane(OMM). This lipid-triggered prodeath function of BFL1 is absent in BCLXL and also differs from that of the apoptoticeffector BAX, which sets it apart from other BCL2 family members. Ourfindings support a new model in which BFL1modulates apoptosis through a bifunctional and multimodal mode of action that is distinctly regulated by OMM lipidscompared to BCLXL.This work was supported by Grants BFU2011-28566 from the Ministerio de Economia y Competitividad and IT838-13 from Gobierno Vasco. HFR is a recipient of a predoctoral fellowship from the Ministerio de Educacion (Spain). We also thank to LE facility technician in the Achucarro Basque Center for Neuroscience for the support in STED experiments. Finally, we thank Dr. Frank Essmann and Prof. Klaus Schulze-Osthoff for providing the HCT116 BAX/BAK DKO cells and Prof. Jean Claude Martinou for HCT116 CL KO cells

Mutante de bak, método asociado para la identificación de sustancias moduladoras de bak y péptido inhibidor de la actividad bak

[EN] The invention relates to: a truncated form ofhuman BAK protein, which retains the transmembrane segment and the function; a method for identifYing BAK-activity-modulating substances, using the truncated form; a BAK-activity-inhibiting peptide; and a kit comprising said truncated form.[ES] La presente invención se refiere a una forma truncada de la proteína BAK humana, que conserva el segmento transmembrana y la función, a un método de identificación de sustancias moduladoras de la actividad de BAK que emplea dicha forma truncada, a un péptido con actividad inhibitoria de la actividad BAK y a un kit que comprende dicha forma truncada.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad del País Vasco.A1 Solicitud de patente con informe sobre el estado de la técnic

A new view of the lethal apoptotic pore.

Cell death by apoptosis is indispensable for proper development and tissue homeostasis in all multicellular organisms, and its deregulation plays a key role in cancer and many other diseases. A crucial event in apoptosis is the formation of protein-permeable pores in the outer mitochondrial membrane that release cytochrome c and other apoptosis-promoting factors into the cytosol. Research efforts over the past two decades have established that apoptotic pores require BCL-2 family proteins, with the proapoptotic BAX-type proteins being direct effectors of pore formation. Accumulating evidence indicates that other cellular components also cooperate with BCL-2 family members to regulate the apoptotic pore. Despite this knowledge, the molecular pathway leading to apoptotic pore formation at the outer mitochondrial membrane and the precise nature of this outer membrane pore remain enigmatic. In this issue of PLOS Biology, Kushnareva and colleagues describe a novel kinetic analysis of the dynamics of BAX-dependent apoptotic pore formation recapitulated in native mitochondrial outer membranes. Their study reveals the existence of a hitherto unknown outer mitochondrial membrane factor that is critical for BAX-mediated apoptotic pore formation, and challenges the currently popular view that the apoptotic pore is a purely proteinaceous multimeric assembly of BAX proteins. It also supports the notion that membrane remodeling events are implicated in the formation of a lipid-containing apoptotic pore

Putative Conformations of Bcl-XL with a-Helices 2 (BH3 Domain), 5–6 (Central Hairpin), and 9 (C-Terminal Hydrophobic Tail) Depicted in Orange, Green, and Red, Respectively

<p>(A) Structure of Bcl-XL in solution. (B) Membrane-integrated conformation of Bcl-XL in which the C-terminal tail is inserted into the membrane in a transmembrane orientation, while the remainder of the protein maintains the solution structure. In this conformation, the BH3-binding pocket of Bcl-XL is expected to be available to engage in inhibitory groove–BH3 interactions with pro-death Bcl-2 family proteins, such as tBid and Bax, as is seen in solution-based binding studies. (C) Membrane-integrated conformation of Bcl-XL in which the C-terminal tail and the central helical hairpin are inserted into the membrane. In this conformation, the BH3-binding pocket of soluble Bcl-XL is expected to be substantially modified or destroyed. However, Bcl-XL may still engage in neutralizing interactions with Bax, as previously proposed for Bcl-2 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060154#pbio-0060154-b020" target="_blank">20</a>]. Alternatively, this conformation may correspond to functionally silent Bcl-XL, pro-apoptotic Bcl-XL, or a yet unknown function of Bcl-XL. (D) Membrane-integrated conformation of Bcl-XL, in which many different helical regions of the protein are inserted in the membrane, but with different orientations and at different penetration depths. In this conformation, the BH3-binding pocket of soluble Bcl-XL no longer exists. Nevertheless, it is conceivable that Bcl-XL inhibits pro-apoptotic proteins through interaction surfaces other than the groove and/or via interactions with membrane lipids. Alternatively, this conformation may correspond to functionally silent, pro-apoptotic, or a yet unknown function of Bcl-XL. We postulate that the topology adopted by Bcl-2-type proteins in the membrane can be modulated by changes in membrane lipid composition.</p