Selective involvement of BH3-only proteins and differential targets of Noxa in diverse apoptotic pathways

The BH3-only proteins of the Bcl-2 family are known to mediate mitochondrial dysfunction during apoptosis. However, the identity of the critical BH3-only proteins and the mechanism of their action following treatment by diverse apoptotic stimuli remain to be fully resolved. We therefore used RNAi to screen the entire Bcl-2 family for their involvement in three major apoptotic pathways in HeLa cells. We found that Bcl-xL and Mcl-1 are major inhibitors of apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), endoplasmic reticulum (ER) stress, and proteasome inhibition. Among the 10 BH3-only proteins, Bid and Noxa were found to be critically involved in TRAIL-induced apoptosis, in which Noxa participates by constitutively binding to Mcl-1. Bim and Noxa were found to be necessary for ER stress-induced apoptosis, in which Noxa assisted Bim function by sequestering Mcl-1 and binding to Bcl-xL. As a critical BH3-only protein, Noxa was strongly upregulated and became associated with both Mcl-1 and Bcl-xL during apoptosis induced by proteasome inhibition. In addition, we found that Noxa became 'Mcl-1 free' following treatment by ER stress and proteasome inhibition, but not after TRAIL treatment. These results defined the critical Bcl-2 network during apoptosis and suggested that Noxa participated in triggering mitochondrial dysfunction in multiple apoptotic pathways through distinct mechanisms.     

A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bim(S)2A, which is highly selective for Mcl-1. Unlike Noxa, Bim(S)2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bim(S)2A promotes cell killing only when Bcl-x(L) is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: characteristics of broad-spectrum protein binding and its effects on anti-tumor activity

On the basis of the comparison of the structure of the Bim BH3: Bcl-x(L) complex and that of the ABT-737: Bcl-x(L) complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3's broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x(L), Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x(L), Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.     

Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function

Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-xL, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.     

BH3 domains other than Bim and Bid can directly activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.     

Mouse Noxa uses only the C-terminal BH3-domain to inactivate Mcl-1

Noxa is a member of the pro-apoptotic BH3-only group of Bcl-2 proteins that is known to bind specifically to anti-apoptotic Mcl-1 and A1, antagonizing their function. Mcl-1 has been reported to have a short half-life, and Noxa up-regulation accelerates Mcl-1 degradation by the proteasome. Unlike human Noxa, mouse Noxa has two BH3-domains, which both have affinity for Mcl-1. We here investigate two aspects of the molecular function of Noxa, namely the requirements for the two BH3-domains in mouse Noxa and the role of Noxa in Mcl-1-degradation. We found that only the C-terminal BH3-domain of mouse Noxa is active in neutralizing Mcl-1. This was the result of the targeting of Noxa to the outer mitochondrial membrane through its C-terminal alpha-helix, which allowed Mcl-1-neutralization only when the BH3-domain was immediately N-terminal of the membrane anchor. However, the N-terminal BH3-domain enhanced interaction with Mcl-1 and A1. The Noxa-dependent degradation of Mcl-1 was independent of the kinase GSK3 and the deubiquitinase Usp9x in mouse embryonic fibroblasts. These data show that Noxa is targeted to the mitochondrial membrane where it neutralises Mcl-1 via its C-terminal BH3-domain and suggest that Noxa is co-degraded with Noxa, in a way independent of ubiquitin-modifying enzymes described for Mcl-1.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists

Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.     

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.     

Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK)

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK), a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK) may trigger apoptosis.For efficient overexpression, Bcl-x(AK) was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK) appeared as an essential and initial step. Bcl-x(AK) was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L). A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.     

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.Cell Death and Differentiation (2008) 15, 1609-1618; doi:10.1038/cdd.2008.86; published online 20 June 2008.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Structure of the BH3 domains from the p53-inducible BH3-only proteins Noxa and Puma in complex with Mcl-1

Pro-survival proteins in the B-cell lymphoma-2 (Bcl-2) family have a defined specificity profile for their cell death-inducing BH3-only antagonists. Solution structures of myeloid cell leukaemia-1 (Mcl-1) in complex with the BH3 domains from Noxa and Puma, two proteins regulated by the tumour suppressor p53, show that they bind as amphipathic α-helices in the same hydrophobic groove of Mcl-1, using conserved residues for binding. Thermodynamic parameters for the interaction of Noxa, Puma and the related BH3 domains of Bmf, Bim, Bid and Bak with Mcl-1 were determined by calorimetry. These unstructured BH3 domains bind Mcl-1 with affinities that span 3 orders of magnitude, and binding is an enthalpically driven and entropy-enthalpy-compensated process. Alanine scanning analysis of Noxa demonstrated that only a subset of residues is required for interaction with Mcl-1, and these residues are localised to a short highly conserved sequence motif that defines the BH3 domain. Chemical shift mapping of Mcl-1:BH3 complexes showed that Mcl-1 engages all BH3 ligands in a similar way and that, in addition to changes in the immediate vicinity of the binding site, small molecule-wide structural adjustments accommodate ligand binding. Our studies show that unstructured peptides, such as the BH3 domains, behave like their structured counterparts and can bind tightly and selectively in an enthalpically driven process.     

Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization,induction of cytochrome c release,and activation of cell death

Overexpression of Bcl-2, an anti-apoptotic oncoprotein, is commonly observed in a variety of human malignancies and is associated with resistance to chemotherapy and radiotherapy. Although the precise mechanism of Bcl-2 action remains elusive, current evidence indicates that Bcl-2 inhibits apoptosis by binding and inhibiting pro-apoptotic molecules such as Bax. Therefore, agents that disrupt the ability of Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value. Several studies have shown that the BH3 domains of Bcl-2 and Bax are critically important for Bax/Bcl-2 heterodimerization. In this report, we designed and synthesized peptides based on the BH3 domains of three distinct Bcl-2 family members, Bcl-2, Bax and Bad. In vitro interaction assays were used to compare the abilities of the different peptides to inhibit Bax/Bcl-2 and Bax/Bcl-x(L) heterodimerization, as well as Bcl-2 and Bax homodimerization. Bax BH3 peptide (20-amino acids) potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions, exhibiting IC(50) values of 15 and 9.5 microM, respectively. The Bad BH3 peptide (21 amino acids) was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-x(L) but failed to disrupt Bax/Bcl-2. Bcl-2 BH3 peptide (20-amino acids) was inactive toward Bax/Bcl-2 and had only a weak inhibitory effect on Bax/Bcl-x(L) heterodimerization. All three BH3 peptides failed to significantly inhibit homodimerization of Bcl-2 or Bax. Consistent with its ability to disrupt Bax/Bcl-2 heterodimerization, Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c release from mitochondria of Bcl-2-overexpressing Jurkat T leukemic cells. Bad BH3 peptide, while potently inducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2 overexpression. Bcl-2 BH3 failed to induce cytochrome c release, even in wild-type cells. Delivery of the Bax BH3 and Bad BH3 peptides into wild-type Jurkat cells induced comparable levels of cell death. In cells overexpressing Bcl-2, the potency of Bax BH3 peptide was similar to that seen in wild-type cells, while the efficacy of Bad BH3 peptide was reduced. By contrast, in Bcl-x(L)-overexpressing cells, Bad BH3 exhibited greater cell-killing activity than Bax BH3. The Bcl-2 BH3 peptide and a mutant Bax BH3 peptide had no appreciable effect on Jurkat cells. Together, our data suggest that agents based on the Bax BH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on the BH3 domain of Bad may be more useful for tumors overexpressing Bcl-x(L).     

Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa

Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation.