Mtd, a Novel Bcl-2 Family Member Activates Apoptosis in the Absence of Heterodimerization with Bcl-2 and Bcl-XL

Abstract

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-X L . Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-X L . A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-X L . Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-X L .Apoptosis, a morphologically distinguished form of programmed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases (1). Several regulatory components of the apoptotic pathway have been identified in various living organisms, including man (1, 2). bcl-2, the first member of an evolutionary conserved family of apoptosis regulatory genes, was initially isolated from the t(14;18) chromosomal translocation found in human B-cell follicular lymphomas and was subsequently shown to repress cell death triggered by a diverse array of stimuli (3-6). Several members of the family, including Bcl-2 and Bcl-X L , share conserved regions termed Bcl-2 homology domains 1, 2, 3 and 4 (BH1, BH2, BH3, and BH4) and function by repressing apoptosis (for review, see Ref. 7). The biochemical process by which Bcl-2 family members regulate cell death are poorly understood. The crystal structures of human and rat Bcl-X L have revealed a significant similarity to the pore-forming domains of diphtheria toxin and bacterial colicins, suggesting that Bcl-2 family members could function as channels for ions, proteins, or both (8, 9). Experiments with synthetic membranes have shown that recombinant Bcl-2, Bcl-X L , and Bax exhibit ion channel activity, suggesting that Bcl-2-related proteins could regulate apoptosis by regulating trafficking of molecules through intracellular membranes (10 -12). However, the significance of these latter findings is unclear as there is no evidence that Bcl-2 or related proteins form ion-channels in vivo.Analyses of the nematode cell death regulators CED-3, CED-4, and CED-9 has provided important insight into the biochemical mechanism ...

Intracellular localization of the BCL-2 family member BOK and functional implications

“…The low extent of cell death seen in the absence of BAX/BAK could therefore be due to additional, caspase-independent death pathways that, in fact, have been suggested in early reports on BOK. 16,17,40 Importantly, Reprobing of the membranes with antibodies to Porin or cytochrome c (mitochondria), calnexin (ER), GM130 (Golgi) and GAPDH (cytosol) served as controls for the purity of the subcellular fractions. Heavy P 10 and light P 100 membrane fractions from SV40 WT MEF (d) and mouse primary hepatocytes (e) were extracted directly with detergent (total) or first treated with sodium carbonate solution (pH 12) to remove loosely attached membrane proteins.…”

“…16,17 A BH3-domain deletion mutant of BOK was unable to interact with MCL-1 and BFL1/ A1 but, surprisingly, still retained its killing activity. 23 Although we did not detect interactions between BOK and other BCL-2 family members, we found that HA-BOK and FLAG-BOK co-immunoprecipitated in a BH3 domain-dependent manner, as the interaction was disrupted by the BOK(D76A) point mutation.…”

“…This may indicate that BOK does not exert a major role overlapping with those of BAX and BAK. To investigate the function of BOK further, and given that overexpression of BOK was previously reported to induce apoptosis, [16][17][18][21][22][23] we examined whether BOKinduced apoptosis depends on the presence of BAX/BAK.…”

“…2,[10][11][12][13][14][15] BOK (BCL-2-related ovarian killer) was identified in a yeast two-hybrid screen using MCL-1 as bait and was shown to selectively interact with MCL-1 and BFL-1/A1 but not with BCL-2 or BCL-X L . 16,17 Among the BCL-2 family members, BOK shows the highest evolutionary conservation. 18,19 Bok expression was first reported to be restricted to reproductive tissues.…”

“…18,21,22 Several studies, largely relying on protein overexpression, have shown that BOK can induce cell death; however, the exact mechanisms are poorly understood and may involve both caspase-dependent and -independent pathways. 16,17,23 Based on the high sequence similarity of BOK with BAX and BAK (particularly in the BH1-3 domains), it has been assumed that BOK might function similar to BAX/BAK. 16,17,[20][21][22]24 However, the high resistance of cells lacking both BAX and BAK towards possibly all intrinsic apoptotic stimuli, 25,26 together with detectable BOK levels in those cells, raises the question whether BOK can substitute for the function of BAX and BAK.…”