Bak BH3

Characterization of an alternative BAK-binding site for BH3 peptides

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.

BCL-2 proteins and apoptosis: Recent insights and unknowns

Proteins of the B-cell lymphoma-2 (BCL-2) family control the intrinsic apoptosis pathway. The pro-apoptotic BCL-2 proteins BAX and BAK can commit a cell to its programmed death by permeabilizing the outer mitochondrial membrane (OMM) and subsequent initiation of the caspase cascade. Therefore, the activities of BAX and BAK are precisely controlled by a complex network of proteins inside and outside the BCL-2 family. Cells survive by constant control of dynamic translocation and retrotranslocation of BAX and BAK to the mitochondria and back into the cytosol. Recent insights into BAX/BAK shuttling, BCL-2 protein interactions, the role of BH3-only proteins in apoptosis signaling and the active BAX complex set the stage for the development of novel strategies in cancer therapy and the analysis of cellular predisposition to apoptosis.

Cell-Permeable Bak BH3 Peptide Induces Chemosensitization of Hematologic Malignant Cells

Hematologic malignancies such as leukemias and lymphomas are among the leading causes of pediatric cancer death worldwide, and although survival rates have improved with conventional treatments, the development of drug-resistant cancer cells may lead to patient relapse and limited possibilities of a cure. Drug-resistant cancer cells in these hematologic neoplasms are induced by overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein families, such as Bcl-_XL, Bcl-2, and Mcl-1. We have previously shown that peptides from the BH3 domain of the proapoptotic Bax protein that also belongs to the Bcl-2 family may antagonize the antiapoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. Furthermore, cell-permeable Bax BH3 peptides also elicit antitumor activity and extend survival in a murine xenograft model of human B non-Hodgkin's lymphoma. However, the activity of the BH3 peptides of the proapoptotic Bak protein of the Bcl-2 family against these hematologic malignant cells requires further characterization. In this study, we report the ability of the cell-permeable Bak BH3 peptide to restore apoptosis and induce chemosensitization of acute lymphoblastic leukemia and non-Hodgkin's lymphoma cell lines, and this event is enhanced with the coadministration of cell-permeable Bax BH3 peptide and represents an attractive approach to improve the patient outcomes with relapsed or refractory hematological malignant cells.

BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis

It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins' apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a "Membrane-mediated Permissive" model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.