BH3 Domain Of Pro-apoptotic Proteins Research Articles

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains.

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins canmediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted keyresidues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

Abstract 418: Mitochondrial perturbations as a novel approach to personalized medicine

Abstract Recent progress in cancer research has been marked by the continued development of exciting drugs that selectively target vulnerabilities present only in cancer cells, so-called targeted therapies. Too often, however, we are lacking the proper predictive biomarkers to identify which patients will benefit most from individual targeted therapies. Since most chemotherapeutic agents ultimately function by inducing the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. Hence to address the unmet need for predictive biomarkers, we have developed a tool called Dynamic BH3 Profiling (DBP). In this approach, we briefly treat cancer cells (14h) from patients with targeted drugs and measure the apoptotic threshold of a cell “priming” by treatment with peptides derived from BH3 domains of pro-apoptotic proteins. Previous studies from our laboratory demonstrated that when a patient's cells exhibit robust death signaling in the DBP assay in response to a particular drug, it predicts a good clinical response to that drug in patients. Using xenotransplantation of primary AML samples into NSG mice (PDX models), we validated the utility of DBP to personalize acute myeloid leukemia therapy. We established 12 independent AML PDX models and determined the mitochondrial response of splenic myeloblasts from each PDX to 25 targeted agents. Ex-vivo DBP measurement demonstrated heterogeneous responses; while some drugs induced priming in all PDXs, others increased priming in only selective PDX models. For instance, JQ-1 (BRD-4 inhibitor) and birinapant (SMAC mimetic) demonstrated inverse correlation of their activity among 3 different PDXs. These suggested that mitochondrial priming can stratify AML PDX models according to predicted sensitivity to targeted agents. When we compared DBP results with cell death assay, we found that myeloblasts from most of the PDXs failed to sustain long-term culture (72h), a requirement for cytotoxicity measurements. However, myeloblasts from 3 of the PDXs with high starting viability showed strong correlation between mitochondrial priming and cell death, implying that cells with increased mitochondrial response are destined to commit apoptosis. By testing functional state of the apoptotic pathway in myeloblasts from spleen, bone-marrow and peripheral blood with BH3 profiling, we found that splenic blasts were relatively more primed for apoptosis in 8 out of 10 PDXs. Correlatively, drug induced priming also varied between myeloblasts from these 3 different sites. Finally, we are directly testing the performance of DBP as a predictive biomarker by treating a variety of PDX models with drugs, including drugs predicated to be active and drugs predicated to be inactive. Collectively, mitochondria based measurements can predict good and bad response to individual targeted agents and may serve as a powerful biomarker to choose patient therapy. Citation Format: shruti bhatt, david weinstock, Anthony Letai. Mitochondrial perturbations as a novel approach to personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 418.

Molecular Determinants of Bim(BH3) Peptide Binding to Pro-Survival Proteins

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.

Conformations and Interactions of BCL-2 Family Proteins: Implications For Apoptosis

The BCL-2 family proteins are major regulators of mitochondrion-dependent programmed cell death. They includes both pro-death and pro-life proteins, which exert their activities through physical interactions with each other with other non-homologous proteins, and with intracellular membranes. The BH3-only cytotoxic protein BID is activated by caspase-8 cleavage upon engagement of cell surface death receptors. The resulting C-terminal fragment, tBID, translocates to mitochondria, triggering the release of cytotoxic molecules and cell death. The activity of tBID is regulated by its interactions with pro-survival BCL-XL and pro-death BAX, both in the cytosol and at the mitochondrial membrane. Using NMR spectroscopy we show that full length BCL-XL is soluble and monomeric in aqueous solution. Its hydrophobic C-terminal tail, which is predicted to form a transmembrane helix in lipid membranes, folds back to interact with the hydrophobic pocket known to bind the BH3 domains of pro-apoptotic proteins. The presence of the C terminus reduces the binding affinity of BCL-XL for BH3 domains 4-fold, compared to the affinity of truncated BCL-XL. The activated pro-apoptotic protein tBID adopts an α-helical but dynamically disordered conformation in solution. However, its three-dimensional conformation is stabilized when tBID engages its BH3 domain in the BH3-binding hydrophobic groove of BCL-XL to form a stable heterodimeric complex. Studies in lipid micelles show that the proteins' conformations and interactions are dramatically different in the presence of lipids.[This research was supported by the National Institutes of Health (NIH), and utilized the Burnham Institute Structural Biology Facility supported by the NIH National Cancer Institute.]

ABT-737 Synergizes with Chemotherapy to Kill Head and Neck Squamous Cell Carcinoma Cells via a Noxa-Mediated Pathway

Overexpression of Bcl-X(L), an antiapoptotic Bcl-2 family member, occurs in a majority of head and neck squamous cell carcinomas (HNSCCs) and correlates with chemotherapy resistance in this disease. Overexpression of Bcl-2 is also observed in HNSCC, albeit less frequently. We have previously shown that peptides derived from the BH3 domains of proapoptotic proteins can be used to target Bcl-X(L) and Bcl-2 in HNSCC cells, promoting apoptosis. In this report, we examined the impact of ABT-737 (for structure, see Nature 435: 677-681, 2005 ), a potent small-molecule inhibitor of Bcl-X(L) and Bcl-2, on HNSCC cells. As a single agent, ABT-737 was largely ineffective at promoting HNSCC cell death. By contrast, ABT-737 strongly synergized with the chemotherapy drugs cisplatin and etoposide to promote HNSCC cell death and loss of clonogenic survival. Synergism between ABT-737 and chemotherapy was associated with synergistic activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. Treatment with ABT-737 plus chemotherapy resulted in dramatic up-regulation of proapoptotic Noxa protein, and small interfering RNA (siRNA)-mediated inhibition of Noxa up-regulation partially attenuated cell death by the synergistic combination. Treatment with cisplatin or etoposide, alone or in combination with ABT-737, resulted in substantial down-regulation of Mcl-1L, a known inhibitor of ABT-737 action. Further down-regulation of Mcl-1L using siRNA failed to enhance killing by the cisplatin/ABT-737 synergistic combination, indicating that chemotherapy treatment of HNSCC cells is sufficient to remove this impediment to ABT-737. Together, our results demonstrate potent synergy between ABT-737 and chemotherapy drugs in the killing of HNSCC cells and reveal an important role for Noxa in mediating synergism by these agents.

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.

A conserved hydrophobic core at Bcl-x L 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.

In vitro Effects of the BH3 Mimetic, (−)-Gossypol, on Head and Neck Squamous Cell Carcinoma Cells

Bcl-xL overexpression is common in head and neck squamous cell carcinomas (HNSCC) and correlates with resistance to chemotherapy. Thus, a nonpeptidic, cell-permeable small molecule that mimics the BH3 domain of proapoptotic proteins may inhibit Bcl-xL function and have therapeutic potential for HNSCC by overcoming drug-resistance. (-)-Gossypol, the levorotatory isomer of a natural product isolated from cottonseeds and roots, was recently discovered to bind to the BH3 binding groove of Bcl-xL and Bcl-2. We investigated the in vitro effects of (-)-gossypol on HNSCC cell lines as well as on fibroblast and keratinocyte cultures by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell survival assays and assessed the results with respect to Bcl-2 family protein expression. We observed dose-dependent growth inhibition of 10 HNSCC cell lines at biologically achievable doses (2.5-10 micromol/L). (-)-Gossypol doses required to inhibit the growth of human fibroblast cell lines by 50% were 2- to 10-fold higher than for HNSCC cell lines. To inhibit human oral keratinocyte growth by 50%, (-)-gossypol concentrations were 2-to 3-fold higher than for HNSCC cell lines. There is a direct correlation between Bcl-xL-to-Bcl-xS ratios and sensitivity to (-)-gossypol. This agent induced apoptosis in a much higher proportion of cells with wild-type p53. Importantly, cell lines resistant to cisplatin were very sensitive to (-)-gossypol. These results demonstrate that (-)-gossypol has potent antitumor activity in HNSCC in vitro. This agent may be developed as a novel therapeutic agent for HNSCC, either alone or in combination with existing chemotherapeutic agents.

Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-XL inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-XL with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH2 terminal to the core. In addition, circular dichroism spectroscopy revealed that high alpha-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.