Bcl-2 Protein Interplay on the Outer Mitochondrial Membrane

Abstract

Apoptosis is a fundamental process in all multicellular organisms; it removes cells that are less fi t, superfl uous, infected, or damaged (Clavería et al. 2013 ; Sulston and Horvitz 1977 ; Zychlinsky et al. 1992) and it provides the most prominent defense against tumor development. Cells that undergo apoptosis become fragmented into membrane-bound vesicles termed " apoptotic bodies, " which are then completely eliminated by phagocytosis (Kerr et al. 1972). This form of cell death protects the neighboring cells from potentially harmful contents of damaged cells and is essen-tial for tissue homeostasis, immunity, and development. Mitochondrial apoptosis signaling is the most common form of programmed death (Hotchkiss et al. 2009) and involves proteins of the B-cell lymphoma-2 (Bcl-2) family. Bcl-2 has been found to be overexpressed following the common t(14; 18) chromosome translocation in B-cell follicular lymphoma cells (Tsujimoto et al. 1985) and later has been shown to protect cells from programmed cell death (Vaux et al. 1988). Bcl-2 proteins have been classifi ed into two functional classes accord-ing to their activities in programmed cell death (Youle and Strasser 2008). The pro-apoptotic family members, Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer 1 (Bak), can commit cells to apoptosis (Oltvai et al. 1993 ; Shimizu et al. 1999). Their activation leads to the permeabilization of the outer mitochondrial 70 membrane (OMM) and subsequent release of proteins such as cytochrome c from the intermembrane space (IMS) into the cytoplasm (Eskes et al. 1998). Release of IMS proteins by Bax and/or Bak results in mitochondrial dysfunction and initiates a cascade of cysteinyl aspartate proteases (caspases) that ultimately leads to com-plete dismantling of the cell (Bratton and Cohen 2001 ; Green and Kroemer 2004). Therefore, the activation of Bax and Bak is the fi rst irreversible step in intrinsic apoptosis signaling, leading to full activation of caspases. However, some neuronal cells and cardiomyocytes can survive mitochondrial outer membrane permeabiliza-tion (MOMP), when caspase activation is inhibited or reduced (Martinou et al. 1999 ; Potts et al. 2005). Moreover, overexpression of glyceraldehyde-3-phosphate dehydrogenase has been found to protect cells downstream of MOMP , through stimulation of autophagy (Colell and Green 2009). Therefore, some cell types could have additional layers of protection from regulated suicide. Bax and Bak share a high degree of functional redundancy (Lindsten et al. 2000). While each single gene knockout has only a mild phenotype in the mouse, the dou-ble knockout of both pro-apoptotic genes results in high embryonic lethality. Recent observations suggest that the Bcl-2-related ovarian killer, Bok, could have a similar function to Bax and Bak (D.R. Green and A. Strasser, personal communications). However, the function of this protein remains controversial, in particular because Bok-defi cient mice are normal and deletion of Bok in a Bax and Bak double KO strain does not further modify the phenotype (Ke et al. 2013). In addition, Bok has been shown to localize to the ER and the GOLGI (Echeverry et al. 2013), which may argue against a role of Bok in mitochondrial apoptosis signaling. Pro-apoptotic Bcl-2 proteins are antagonized by their pro-survival counterparts, namely Bcl-2, B-cell lymphoma-extra large (Bcl-x L), Bcl-2-like protein 2 (Bcl-w), Bcl-2-like protein 10 (Bcl-B), myeloid cell leukemia 1 (Mcl-1), and Bcl-2-related gene A1 (A1). All Bcl-2 family proteins contain four conserved motifs known as Bcl-2 homology (BH) domains 1-4 (BH1-4). They also share a globular protein fold with bundled α helices surrounding a central hydrophobic α helix (Fig. 4.1 ; Muchmore et al. 1996). This fold generates a hydrophobic surface groove that is occupied in the cytosolic forms of Bax, Bcl-w, and Bcl-x L by the hydrophobic C-terminal membrane anchor (MA) domain (Hinds et al. 2003 ; Suzuki et al. 2000). Of note, other Bcl-2 protein structures have been obtained using C-terminal protein truncations and thus intramolecular interactions between MA and hydrophobic groove in these proteins have not been characterized. The hydrophobic groove can also mediate intermolecular interactions with the BH3 domain of other Bcl-2 pro-teins, as originally shown for the binding of the Bak BH3 peptide to Bcl-x L (Sattler et al. 1997). The central importance of this interaction in the regulation of pro-apoptotic and pro-survival Bcl-2 proteins and thus mitochondrial apoptosis signal-ing has been demonstrated by single amino acid substitutions in both the hydrophobic groove and the BH3 domains and by the use of low molecular weight mimetics of the BH3 domain (van Delft et al. 2006 ; Desagher et al. 1999 ; Dlugosz et al. 2006 ; Fletcher et al. 2008 ; Kvansakul et al. 2007 ; Sedlak et al. 1995). From these studies, the structural insights into Bcl-2 protein complexes have been deduced from inter-actions between short peptides and truncated Bcl-2 proteins, and it would be inter-F. Edlich and J.-C. Martinou