BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X_L bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-X_L/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-X_L-selective inhibitors such as {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540"}}S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.Subject terms: Cancer metabolism, Lipid signalling     

Venetoclax imparts distinct cell death sensitivity and adaptivity patterns in T cells

BH3 mimetics are increasingly used as anti-cancer therapeutics either alone or in conjunction with other chemotherapies. However, mounting evidence has also demonstrated that BH3 mimetics modulate varied amounts of apoptotic signaling in healthy immune populations. In order to maximize their clinical potential, it will be essential to understand how BH3 mimetics affect discrete immune populations and to determine how BH3 mimetic pressure causes immune system adaptation. Here we focus on the BCL-2 specific inhibitor venetoclax (ABT-199) and its effects following short-term and long-term BCL-2 blockade on T cell subsets. Seven day “short-term” ex vivo and in vivo BCL-2 inhibition led to divergent cell death sensitivity patterns in CD8⁺ T cells, CD4⁺ T cells, and Tregs resulting in shifting of global T cell populations towards a more memory T cell state with increased expression of BCL-2, BCL-X_L, and MCL-1. However, twenty-eight day “long-term” BCL-2 blockade following T cell-depleted bone marrow transplantation did not lead to changes in the global T cell landscape. Despite the lack of changes in T cell proportions, animals treated with venetoclax developed CD8⁺ and CD4⁺ T cells with high levels of BCL-2 and were more resistant to apoptotic stimuli following expansion post-transplant. Further, we demonstrate through RNA profiling that T cells adapt while under BCL-2 blockade post-transplant and develop a more activated genotype. Taken together, these data emphasize the importance of evaluating how BH3 mimetics affect the immune system in different treatment modalities and disease contexts and suggest that venetoclax should be further explored as an immunomodulatory compound.Subject terms: Bone marrow transplantation, Immune cell death     

BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-X_L with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.     

BH3-Only Proteins and BH3 Mimetics Induce Autophagy by Competitively Disrupting the Interaction between Beclin 1 and Bcl-2/Bcl-XL

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X_L) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X_L. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X_L. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X_L can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X_L complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X_L complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X_L at the level of the endoplasmic reticulum.

Addendum to:

Functional and Physical Interaction Between Bcl-X_L and a BH3-Like Domain in Beclin-1

M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. Kroemer

EMBO J 2007; In press     

Targeting γ-Herpesvirus 68 Bcl-2-mediated Down-regulation of Autophagy

γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine γHV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-X_L, a cellular Bcl-2 homolog, and the murine γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-X_L dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-X_L by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-X_L, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-X_L, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-X_L. The x-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-X_L-mediated, down-regulation of autophagy.     

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x_L, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x_L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x_L. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x_L. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x_L and Beclin 1.     

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).     

Exploring the anti-apoptotic role of HAX-1 versus BCL-XL in cytokine-dependent bone marrow-derived cells from mice

HS-1-associated protein X-1 (HAX-1) is a multi-functional protein that has been implicated in the regulation of apoptosis, cell motility and calcium homeostasis. In the present study, we set out to assess the postulated functional resemblance of HAX-1 to the BCL-2 family of anti-apoptotic proteins using non-transformed, cytokine-dependent murine bone marrow cells as a model system. BCL-X_L, but not HAX-1 protected against cytokine withdrawal-induced apoptosis while HAX-1 and BCL-X_L significantly reduced thapsigargin-triggered (calcium-dependent) apoptosis. The data argue in favor of cell type- and stimulus-specific roles of HAX-1 in regulation of cell survival.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-X_L over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-X_L expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- X_L upregulation in AML cells.     

Mutagenesis of the BH3 Domain of BAX Identifies Residues Critical for Dimerization and Killing

The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45–49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic α helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-X_L, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX’s killing function reflects mechanisms beyond its binding to BCL-2 or BCL-X_L to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

Programmed cell death and its morphologic equivalent, apoptosis, are orchestrated by a distinct genetic pathway that is apparently possessed by all multicellular organisms (22). Moreover, the biochemical details of how encoded proteins function are beginning to emerge. The BCL-2 family of proteins constitutes a central decisional point within the common portion of the apoptotic pathway. This family possesses both proapoptotic (BAX, BAK, BCL-X_S, BAD, BIK, BID, HRK, and BIM) and antiapoptotic (BCL-2, BCL-X_L, MCL-1, and A1) molecules (5, 11). The ratio of antiapoptotic to proapoptotic molecules such as BCL-2/BAX determines the response to a proximal apoptotic signal (14). A striking characteristic of many family members is their propensity to form homo- and heterodimers (16, 19). The BCL-2 family has homology clustered principally within four conserved domains called BH1, BH2, BH3, and BH4 (5, 11). The multidimensional nuclear magnetic resonance (NMR) and X-ray crystallographic structure of a BCL-X_L monomer indicates that the BH1-4 domains correspond to α helices 1 to 7. Notably, the BH1, -2, and -3 domains are in close proximity and create a hydrophobic pocket presumably involved in interactions with other BCL-2 family members (13). The NMR analysis of a BCL-X_L-BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-X_L pocket and a BH3 amphipathic α-helical peptide from BAK (17).Prior mutagenesis studies of BCL-2 and BCL-X_L revealed the importance of BH1 and BH2 domains for both their antiapoptotic function and the capacity to heterodimerize with proapoptotic molecules like BAX or BAK (2, 19, 26). In general, most mutations that disrupt heterodimerization with BAX also lose their death repressor function. However, exceptions do exist; some mutants of BCL-X_L fail to bind BAX or BAK but still repress cell death, suggesting that these functions can be separated for antiapoptotic molecules (2). Moreover, a genetic approach with Bcl-2-deficient and Bax-deficient mice also suggested that BCL-2 and BAX could function independently of one another (10).Deletion studies of the death agonist BAK first implicated the BH3 domain as having the capacity to bind BCL-X_L and promote apoptosis (3). However, the functional significance of BH3 in BAX is uncertain as indicated in the literature. Three deletion analyses indicated the necessity of the BH3 domain in BAX to promote cell death as well as to heterodimerize with BCL-2 (3, 9, 28). Yet, two recent studies reported that BAX functions as a death activator independent of its heterodimerization (21, 27). Moreover, substitution mutants within the BH3 domain showed conflicting specificities of heterodimerization (20, 21, 27).Our initial screen of yeast two-hybrid libraries with BCL-2 as bait yielded multiple clones that possess only the NH₂ terminus of BAX, bearing the BH3 but not the BH1 or the BH2 domains. A similar set of isolates was obtained when BCL-2 (G145A) was used as bait (15). We also noted by deletion analysis and assessment of minimal domains of BAX that the BH3 domain was required for both homodimerization and heterodimerization. Consequently, we undertook an extensive site-directed mutagenesis of the BH3 domain of BAX. These studies demonstrate the importance of the hydrophobic face of the amphipathic α helix of BH3 for the dimerization and cell death activities of BAX. Furthermore, analysis of a BAX mutant indicates that its retained conformation as a cross-linkable dimer at mitochondrial membranes correlates with its intact apoptotic function.     

Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL

Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2

inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which Death

Associated Protein Kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thus

promotes Beclin 1 dissociation from Bcl-X_L and autophagy induction.¹ Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, in

line with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-XL proteins. Our results reveal a new phosphorylation-based

mechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X(L) bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound,Z18

Inhibitors of Bcl-X_L/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-X_L/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis. Unexpectedly, the inhibition of Beclin 1 and phosphatidylinositol 3-kinase have no obvious effect on Z18-induced autophagy in HeLa cells, implying that it is a non-canonical Beclin 1-independent autophagy. Meanwhile, the accumulation of autophagosomes is positively correlated with Z18-induced cell death and the full flux of autophagy is not necessary.     

Atypical BH3-domains of BNIP3 and BNIP3L lead to autophagy in hypoxia

Normal and tumor cells subjected to a hypoxic microenvironment show evidence of autophagy. We hypothesize that cells will sense hypoxia as a warning signal to upcoming drastic microenvironmental conditions and that autophagy, acting as a survival mechanism, will provide time for cells to adapt. This work demonstrates for the first time that the atypical BH3-domain of BNIP3 and BNIP3L, two HIF-target genes, can compete with Beclin1-Bcl-2 and Beclin1-Bcl-X_L complexes, releasing Beclin 1 from the complex and then enhancing autophagy. We thus revealed a new role for BH3-only proteins in the cellular response to hypoxia.     

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X_L and Bcl-2. A structural model of the Bcl-X_L/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X_L/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X_L. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.