Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.     

Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.     

Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL.

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrated that the protein not only could abrogate the protective capacity of coexpressed Bcl-XL but could accelerate the apoptotic response to a death signal when it was expressed in the absence of exogenous Bcl-XL. Using deletion analysis, we have identified the minimal domain in the murine Bad protein that can dimerize with Bcl-xL. A 26-amino-acid peptide within this domain, which showed significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, was found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminated the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retained the ability to impart protection against cell death regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions.     

Molecular basis of Bcl-xL's target recognition versatility revealed by the structure of Bcl-xL in complex with the BH3 domain of Beclin-1

Beclin-1, originally identified as a Bcl-2 binding protein, is an evolutionarily conserved protein required for autophagy. The direct interaction between Beclin-1 and Bcl-2 or Bcl-xL provides a potential convergence point for apoptosis and autophagy, two programmed cell death processes. Given the functional significance of the interaction between Beclin-1 and Bcl-2/Bcl-xL, we performed detailed biochemical and structural characterizations of this interaction. We demonstrated that the Bcl-xL-binding domain of Beclin-1 contains a BH3 domain. Therefore, Beclin-1 is a new member of the BH3-only family proteins. The structure of Bcl-xL in complex with the Beclin-1 BH3 domain was determined at high resolution by NMR spectroscopy. Although similar to other known BH3 domains, the Beclin-1 BH3 domain displays its own distinct features in the complex with Bcl-xL. Systematic analysis of all known Bcl-xL/BH3 domain complexes helped us to identify the molecular basis underlying the capacity of Bcl-xL to recognize diverse target sequences.     

Deciphering the antitumoral activity of quinacrine: Binding to and inhibition of Bcl-xL

From the screening of a unique collection of 880 off-patent small organic molecules, we have found that quinacrine inhibits the interaction between a BH3 domain-derived peptide and the antiapoptotic protein Bcl-xL. Nuclear magnetic resonance spectroscopy confirmed that quinacrine binds to the hydrophobic groove that Bcl-xL uses for interacting with the BH3 domain of proapoptotic proteins. This activity can contribute to the anticancer activity of quinacrine.     

Modeling backbone flexibility to achieve sequence diversity: the design of novel alpha-helical ligands for Bcl-xL

Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. Here, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested 17 designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL.     

Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins

The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.     

Novel Bcl-2 Homology-3 Domain-like Sequences Identified from Screening Randomized Peptide Libraries for Inhibitors of the Pro-survival Bcl-2 Proteins

Interactions between Bcl-2 homology-3 (BH3)-only proteins and their pro-survival Bcl-2 family binding partners initiate the intrinsic apoptosis pathway. These interactions are mediated by a short helical motif, the BH3 domain, on the BH3-only protein, which inserts into a hydrophobic groove on the pro-survival molecule. To identify novel peptidic ligands that bind Mcl-1, a pro-survival protein relative of Bcl-2, both human and mouse Mcl-1 were screened against large randomized phage-displayed peptide libraries. We identified a number of 16-mer peptides with sub-micromolar affinity that were highly selective for Mcl-1, as well as being somewhat selective for the species of Mcl-1 (human or mouse) against which the library was panned. Interestingly, these sequences all strongly resembled natural BH3 domain sequences. By switching residues within the best of the human Mcl-1-binding sequences, or extending beyond the core sequence identified, we were able to alter the pro-survival protein interaction profile of this peptide such that it now bound all members tightly and was a potent killer when introduced into cells. Introduction of an amide lock constraint within this sequence also increased its helicity and binding to pro-survival proteins. These data provide new insights into the determinants of BH3 domain:pro-survival protein affinity and selectivity.     

Solution structure of prosurvival Mcl-1 and characterization of its binding by proapoptotic BH3-only ligands

The B cell lymphoma-2 (Bcl-2) homologs myeloid cell leukemia-1 (Mcl-1) and A1 are prosurvival factors that selectively bind a subset of proapoptotic Bcl homology (BH) 3-only proteins. To investigate the molecular basis of the selectivity, we determined the solution structure of the C-terminal Bcl-2-like domain of Mcl-1. This domain shares features expected of a prosurvival Bcl-2 protein, having a helical fold centered on a core hydrophobic helix and a surface-exposed hydrophobic groove for binding its cognate partners. A number of residues in the binding groove differentiate Mcl-1 from its homologs, and in contrast to other Bcl-2 homologs, Mcl-1 has a binding groove in a conformation intermediate between the open structures characterized by peptide complexes and the closed state observed in unliganded structures. Mutagenesis of potential binding site residues was used to probe the contributions of groove residues to the binding properties of Mcl-1. Although mutations in Mcl-1 had little impact on binding, a single mutation in the BH3-only ligand Bad enabled it to bind both Mcl-1 and A1 while retaining its binding to Bcl-2, Bcl-xL, and Bcl-w. Elucidating the selective action of certain BH3-only ligands is required for delineating their mode of action and will aid the search for effective BH3-mimetic drugs.     

Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL

To study the role of the BH3 domain in mediating pro-apoptotic and anti-apoptotic activities of Bcl-2 family members, we identified a series of novel small molecules (BH3Is) that inhibit the binding of the Bak BH3 peptide to Bcl-xL. NMR analyses revealed that BH3Is target the BH3-binding pocket of Bcl-xL. Inhibitors specifically block the BH3-domain-mediated heterodimerization between Bcl-2 family members in vitro and in vivo and induce apoptosis. Our results indicate that BH3-dependent heterodimerization is the key function of anti-apoptotic Bcl-2 family members and is required for the maintenance of cellular homeostasis.     

Nuclear Magnetic Resonance study of protein–protein interactions involving apoptosis regulator Diva (Boo) and the BH3 domain of proapoptotic Bcl‐2 members

According to biochemical assays, the Bcl‐2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease‐activating factor Apaf‐1. In typical Bcl‐2 heterodimers, peptide fragments comprising the Bcl‐2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High‐resolution structural studies have revealed that the BH3 peptide forms an α‐helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein. Because Diva shows mutations in conserved residues within this area, it has been proposed to have a different interacting surface. However, we showed previously that Diva binds through the canonical groove the BH3 peptide of the human Bcl‐2 killing member Harakiri. To further test Diva's binding capabilities, here we show Nuclear Magnetic Resonance (NMR) data, indicating that Diva binds peptides derived from the BH3 domain of several other proapoptotic Bcl‐2 proteins, including mouse Harakiri, Bid, Bak and Bmf. We have measured the binding affinities of the heterodimers, which show significant variability. Structural models of the protein–peptide complexes based on NMR chemical shift perturbation data indicate that the binding surface is analogous. These models do not rely on NMR NOE (Nuclear Overhauser Effect) data, and thus our results can only suggest that the complexes share similar intermolecular interactions. However, the observed affinity differences correlate with the α‐helical population of the BH3‐peptides obtained from circular dichroism experiments, which highlights a role of conformational selection in the binding mechanism. Altogether, our results shed light on important factors governing Diva‐BH3 peptide molecular recognition mode. Copyright © 2012 John Wiley & Sons, Ltd.     

Tissue transglutaminase is a multifunctional BH3-only protein

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.     

Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members

It is still unclear whether the BH3-only protein Puma (p53 up-regulated modulator of apoptosis) can prime cells to death and render antiapoptotic BH3-binding Bcl-2 homologues necessary for survival through its ability to directly interact with proapoptotic Bax and activate it. In this study, we provide further evidence, using cell-free assays, that the BH3 domain of Puma binds Bax at an activation site that comprises the first helix of Bax. We also show that, in yeast, Puma interacts with Bax and triggers its killing activity when Bcl-2 homologues are absent but not when Bcl-xL is expressed. Finally, endogenous Puma is involved in the apoptotic response of human colorectal cancer cells to the Bcl-2/Bcl-xL inhibitor ABT-737, even in conditions where the expression of Mcl-1 is down-regulated. Thus, Puma is competent to trigger Bax activity by itself, thereby promoting cellular dependence on prosurvival Bcl-2 family members.     

Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax

Mitochondria act as a focal point for upstream apoptosis signals by releasing cytochrome c into the cytosol, leading to the activation of caspases and subsequent cell death. Members of the Bcl-2 protein family regulate this phenomenon by heterodimerization via the BH3 domain of proapoptotic members opposing their pro- and antiapoptotic functions. The mechanism of cytochrome c release from mitochondria and of its regulation remains controversial. In vitro binding studies of purified and biologically active proteins should help in understanding the molecular mechanism of interactions and protein functions. In this work, the Bcl-2-related antiapoptotic chicken protein Nr-13 was overexpressed as a highly soluble recombinant protein which showed correct folding as judged by circular dichroism and fluorescence spectroscopy. Purified Nr-13 inhibits caspase-3 activation in a Xenopus egg-derived cell-free system, and neutralizes the proapoptotic activity of a synthetic peptide containing the BH3 domain of Bax. The latter effect correlates with the high-affinity binding of the BH3 peptide to Nr-13 as monitored by the intrinsic tryptophan fluorescence. On the basis of the structural similarity with Bcl-x(L), putative residues involved in this interaction were identified. Nr-13 exhibits a high-affinity interaction with cytochrome c which is prevented by preincubation with the BH3-Bax peptide. These findings are discussed with respect to a model for the regulation of apoptosis in which a direct interaction between the antiapoptotic protein and cytochrome c may prevent the apoptosis.     

A conserved hydrophobic core at Bcl-xL mediates its structural stability and binding affinity with BH3-domain peptide of pro-apoptotic protein

Bcl-2 family proteins regulate apoptosis through their homo- and heterodimerization. By protein sequence analysis and structural comparison, we have identified a conserved hydrophobic core at the BH1 and BH2 domains of Bcl-2 family proteins. The hydrophobic core is stabilized by hydrophobic interactions among the residues of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 in Bcl-x_L. Destabilization of the hydrophobic core can promote the protein unfolding and pore formation in synthetic lipid vesicles. Interestingly, though the hydrophobic core does not participate in binding with BH3 domain of pro-apoptotic proteins, disruption of the hydrophobic core can reduce the affinity of Bcl-x_L with BH3-domain peptide by changing the conformation of Bcl-x_L C-terminal residues that are involved in the peptide interaction. The BH3-domain peptide binding affinity and pore forming propensity of Bcl-x_L were correlated to its death-repressor activity, which provides new information to help study the regulatory mechanism of anti-apoptotic proteins. Meanwhile, as the tryptophans are conserved in the hydrophobic core, in vitro binding assay based on FRET of “Trp → AEDANS” can be devised to screen for new modulators targeting anti-apoptotic proteins as well as “multi-BH domains” pro-apoptotic proteins.     

Binding modes of Bcl-2 homology 3 (BH3) peptides with anti-apoptotic protein A1 and redesign of peptide inhibitors: a computational study

The interaction between protein and peptide ligand is a challenging problem in molecular biology and drug design. The binding of the Bcl-2 homology 3 (BH3) peptide to the anti-apoptotic protein A1 was revealed as a critical step in the regulation of apoptosis. These BH3 peptides hold high structural similarity, but are diverse in their regulation abilities. Based on molecular simulations and MM-P(G)BSA methods, this work presented a detailed analysis on binding mechanism of the BH3 peptides derived from PUMA and BMF. Residue-level energy decomposition showed that the core regions of BH3 peptides maintain in stable helical conformations and the four conserved hydrophobic residues together with an invariant aspartic acid contribute the major driving force for binding, whereas their two terminal segments exhibit obvious flexibility and various binding modes. Such kind of behavior was suggested as the reason for binding diversity and selectivity of BH3 peptides. As a further step, several BH3-mimetic peptides have been redesigned by computational mutation. Those new peptides showed not only stronger affinities when binding to protein A1, as well transferable binding patterns at some specific positions. A long-range coupling effect was disclosed for BH3 peptides, side-chain orientation and binding contribution of terminal residues were even affected by mutations at large sequence interval. Overall, this work reports that the binding modes of BH3 peptides are primarily dependent on its two terminal segments. The computational methods applied herein are also demonstrated to be of great assistance in the rational design of peptide inhibitors.     

A new assay based on fluorescence resonance energy transfer to determine the binding affinity of Bcl-xL inhibitors

We developed a new assay of Bcl-xL inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-xL. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-xL inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.     

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.     

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications.     

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.