Network pharmacology approach to decipher signaling pathways associated with target proteins of NSAIDs against COVID-19.

Non-steroidal anti-inflammatory drugs (NSAIDs) showed promising clinical efficacy toward COVID-19 (Coronavirus disease 2019) patients as potent painkillers and anti-inflammatory agents. However, the prospective anti-COVID-19 mechanisms of NSAIDs are not evidently exposed. Therefore, we intended to decipher the most influential NSAIDs candidate(s) and its novel mechanism(s) against COVID-19 by network pharmacology. FDA (U.S. Food & Drug Administration) approved NSAIDs (19 active drugs and one prodrug) were used for this study. Target proteins related to selected NSAIDs and COVID-19 related target proteins were identified by the Similarity Ensemble Approach, Swiss Target Prediction, and PubChem databases, respectively. Venn diagram identified overlapping target proteins between NSAIDs and COVID-19 related target proteins. The interactive networking between NSAIDs and overlapping target proteins was analyzed by STRING. RStudio plotted the bubble chart of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of overlapping target proteins. Finally, the binding affinity of NSAIDs against target proteins was determined through molecular docking test (MDT). Geneset enrichment analysis exhibited 26 signaling pathways against COVID-19. Inhibition of proinflammatory stimuli of tissues and/or cells by inactivating the RAS signaling pathway was identified as the key anti-COVID-19 mechanism of NSAIDs. Besides, MAPK8, MAPK10, and BAD target proteins were explored as the associated target proteins of the RAS. Among twenty NSAIDs, 6MNA, Rofecoxib, and Indomethacin revealed promising binding affinity with the highest docking score against three identified target proteins, respectively. Overall, our proposed three NSAIDs (6MNA, Rofecoxib, and Indomethacin) might block the RAS by inactivating its associated target proteins, thus may alleviate excessive inflammation induced by SARS-CoV-2.

Computational analysis of binding free energies, hotspots and the binding mechanism of Bcl-xL/Bcl-2 binding to Bad/Bax.

The anti-apoptotic proteins B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma/leukemia-2 (Bcl-2) are members of the Bcl-2 protein family, and they play important roles in regulating apoptosis and cell cycle retardation. However, the binding mechanisms of Bcl-xL/Bcl-2 with their associated agonists, including Bcl-2-associated death promoter (Bad) and Bcl-2-associated X protein (Bax), are not well understood. In the present study, the recently developed interaction entropy approach was employed for the calculation of entropic contribution, and the computational alanine scanning method was used to identify the hot spot in the protein-protein interactions between Bcl-xL/Bcl-2 and Bad/Bax. The calculated binding free energies and their ranks for the four systems were in good agreement with the experimental results. Computational analysis shows that there are more hot-spot residues in the Bcl-xL/Bad complex than that in the Bcl-xL/Bax complex, leading to a stronger binding affinity in the former. It is interesting to find that the reason for the stronger binding affinity of Bcl-2 to Bad than to Bax is different for the Bcl-xL system. Although there are more hot-spot residues in the Bcl-2/Bax system than in the Bcl-2/Bad complex, there are also more negatively contributing residues in the Bcl-2/Bax. Our study identified Arg104, Tyr105, Leu116, and Leu134 to be the common key residues in the Bcl-xL complexes, and Arg107, Tyr108, Phe112, Gln118, Leu137, Arg146, and Tyr202 are common key residues in the Bcl-2 complexes. These results would provide valuable information for the design of potent inhibitors of Bcl-xL/Bcl-2.

Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Bag-1 stimulates Bad phosphorylation through activation of Akt and Raf kinases to mediate cell survival in breast cancer.

BACKGROUND: Bag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer. METHODS: Using immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry. RESULTS: We observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad's re-localization to the nucleus, and inhibits apoptosis in favor of cell survival. CONCLUSIONS: Overall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

SpBAG1 promotes the WSSV infection by inhibiting apoptosis in mud crab (Scylla paramamosain).

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h-24 h, but down-regulated from 48 h-72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

Reverse Chemical Proteomics Identifies an Unanticipated Human Target of the Antimalarial Artesunate.

Artemisinins are the most potent and safe antimalarials available. Despite their clinical potential, no human target for the artemisinins is known. The unbiased interrogation of several human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of artesunate; the intrinsically disordered Bcl-2 antagonist of cell death promoter (BAD). We show that artesunate inhibits the phosphorylation of BAD, thereby promoting the formation of the proapoptotic BAD/Bcl-xL complex and the subsequent intrinsic apoptotic cascade involving cytochrome c release, PARP cleavage, caspase activation, and ultimately cell death. This unanticipated role of BAD as a possible drug target of artesunate points to direct clinical exploitation of artemisinins in the Bcl-xL life/death switch and that artesunate's anticancer activity is, at least in part, independent of reactive oxygen species.

PIM2 survival kinase is upregulated in a p53-dependent manner in cells treated with camptothecin or co-treated with actinomycin D and nutlin-3a.

The p53 protein is an inducer of apoptosis, acting as a transcriptional regulator of apoptotic genes. In a previous study, we found that actinomycin D and nutlin-3a (A + N) synergistically activate p53. To better understand the molecular consequences of this synergism, we incubated arrays of antibodies against apoptotic proteins with extracts of A549 cells in which p53 had been activated. We found that strong activation of p53, marked by serine 46 and 392 phosphorylation, was associated with inactivating phosphorylation of proapoptotic BAD protein on serine 136. Investigation of the source of this phosphorylation revealed that activation of p53 was associated with accumulation of PIM2, a survival kinase. The accumulation of PIM2 following treatment with A + N was suppressed in p53-knockdown cells. Others discovered that PIM2 was activated by cooperatively acting p53 molecules. Our results are consistent with this finding. Moreover, we found that in A549 cells, the treatment with A + N stimulated in p53-dependent fashion the expression of other high cooperativity p53 target genes, DRAXIN and H19. Activation of antiapoptotic H19 can mechanistically explain relatively low rate of apoptosis of A549 cells exposed to A + N. We conclude that PIM2, DRAXIN and H19 are efficiently stimulated by strongly activated p53 molecules, probably acting cooperatively.

Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic ß-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency.

Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8-14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells.

This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent.

ISAV infection promotes apoptosis of SHK-1 cells through a ROS/p38 MAPK/Bad signaling pathway.

The impact that the infectious salmon anemia virus (ISAV) has on the immune response of Salmo salar, from the perspective of activating/inactivating cellular processes, is currently unknown. Therefore, the present study evaluated this interaction and found that SHK-1 cells infected with ISAV resulted in respiratory burst activation and the induction of a strong pro-apoptotic imbalance through an increased expression of the Bad protein and decreased transcripts of Bcl-xl. Interestingly, the pharmacological inhibition of the p38 MAPK protein through SB203580 blocked the production of reactive oxygen species, the activity of caspase 3, and the formation of apoptotic nuclei in SHK-1 cells. Additionally, when the NADPH oxidase complex, a producer of superoxide anions, was blocked through apocynin, decreased apoptotic activity was observed in infected cells without significant modifications to viral amplification. These results, together with bioinformatics analysis performed for the Bad gene of fugu, suggest that the ISA virus triggers a strong production of oxygen radicals capable of activating transduction signaling pathways and mediating the expression and activation of pro-apoptotic proteins through the p38 MAPK pathway, all of which results in the apoptosis of ISAV infected cells.

Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation.

The IκB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-κB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-κB activation suppresses TNFα-induced apoptosis. TNFα-treated Ikkß(-/-) mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFα-induced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFα-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-κB and inactivation of the proapoptotic BH3-only BAD protein.

Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET.

Increased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initiation, progression and resistance to traditional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to chemotherapy. Recently, we applied fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pattern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the corresponding BimEL mutant showed no decrease in binding to these anti-apoptotic proteins. Therefore, in MCF-7 cells, Bim has unique binding properties compared with other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics.

Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems.

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.

Molecular dynamics simulations of pro-apoptotic BH3 peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein Bcl-X(L).

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the intrinsic pathway of apoptosis. Interactions between specific anti- and pro-apoptotic Bcl-2 proteins determine the fate of a cell. Anti-apoptotic Bcl-2 proteins have been shown to be over-expressed in certain cancers and they are attractive targets for developing anti-cancer drugs. Peptides from the BH3 region of pro-apoptotic proteins have been shown to interact with anti-apoptotic Bcl-2 proteins and induce biological activity similar to that observed in parent proteins. However, the specificity of BH3 peptides derived from different pro-apoptotic proteins differ for different anti-apoptotic Bcl-2 proteins. In this study, we have investigated the relationship between the stable helical nature of BH3 peptides and their affinities to Bcl-X(L), an anti-apoptotic Bcl-2 protein. We have carried out molecular dynamics simulations of six BH3 peptides derived from Bak, Bad and Bim pro-apoptotic proteins for a period of 50 ns each in aqueous medium. Due to the amphipathic nature of BH3 peptides, the hydrophobic residues on the hydrophobic face tend to cluster together in all BH3 peptides. While this process resulted in a complete loss of helical structure in 16-mer Bak and 16-mer Bad wild type peptides, stabilizing interactions in the hydrophilic face of the BH3 peptides and capping interactions helped to maintain partial helical character in 16-mer Bad mutant and 16-mer Bim peptides. The latter two 16-mer peptides exhibit higher affinity for Bcl-X(L). Similarly the longer BH3 peptides, 25-mer Bad and 33-mer Bim, also resulted in smaller and stable helical fragments and their helical conformation is stabilized by interactions between residues in the solvent-exposed hydrophilic half of the peptide. The stable nature of helical segment in a BH3 peptide can be directly correlated to its binding affinity and the helical region encompassed the highly conserved Leu residue. We propose that upon approaching the hydrophobic groove of anti-apoptotic proteins, a longer helix will be induced in high affinity BH3 peptides by extending the smaller stable helical segments around the conserved Leu residue in both N- and C-terminal regions. The results reported in this study will have implications in developing peptide-based inhibitors for anti-apoptotic Bcl-2 proteins.

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

Activation of a non-genomic Pim-1/Bad-Pser75 module is required for an efficient pro-survival effect of Bcl-xL induced by androgen in LNCaP cells.

The present report investigated the pathway(s) involved in the inhibition of apoptosis by the synthetic androgen, R1881 in serum-starved LNCaP cells exposed to the pi3K inhibitor, LY294002. R1881 blocked LY294002-induced apoptosis through the inhibition of Bak activation via an increase in Bcl-xL transcription and protein expression. In addition, R1881 treatment enhanced the stability of the Pim-1 kinase, resulting in the inhibition of the activation of the BH3-only protein Bad through its phosphorylation at ser75. Pharmacological inhibition of the Pim-1 kinase activity with quercetagetin, a highly selective Pim-1 inhibitor, prevented R1881-mediated increase in Bad phosphorylation and restored cell sensitivity to LY294002-induced apoptosis despite the increase in Bcl-xL expression. These results demonstrate for the first time that the inhibition of LY294002-induced apoptosis by androgen is a function of an androgen receptor-dependent genomic signaling pathway leading to an increase in Bcl-xL expression as well as a non-genomic, Pim-1-dependent, signaling pathway mediated via phosphorylation of Bad at ser75.

Pore-forming activity of BAD is regulated by specific phosphorylation and structural transitions of the C-terminal part.

BACKGROUND: BAD protein (Bcl-2 antagonist of cell death) belongs to the BH3-only subfamily of proapoptotic proteins and is proposed to function as the sentinel of the cellular health status. Physiological activity of BAD is regulated by phosphorylation, association with 14-3-3 proteins, binding to membrane lipids and pore formation. Since the functional role of the BAD C-terminal part has not been considered so far, we have investigated here the interplay of the structure and function of this region. METHODS: The structure of the regulatory C-terminal part of human BAD was analyzed by CD spectroscopy. The channel-forming activity of full-length BAD and BAD peptides was carried out by lipid bilayer measurements. Interactions between proteins and peptides were monitored by the surface plasmon resonance technique. In aqueous solution, C-terminal part of BAD exhibits a well-ordered structure and stable conformation. In a lipid environment, the helical propensity considerably increases. The interaction of the C-terminal segment of BAD with the isolated BH3 domain results in the formation of permanently open pores whereby the phosphorylation of serine 118 within the BH3 domain is necessary for effective pore formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. C-terminal part of BAD controls BAD function by structural transitions, lipid binding and phosphorylation. Conformational changes of this region upon membrane interaction in conjunction with phosphorylation of the BH3 domain suggest a novel mechanism for regulation of BAD. GENERAL SIGNIFICANCE: Multiple signaling pathways mediate inhibition and activation of cell death via BAD.

Ligand-independent antiapoptotic function of estrogen receptor-beta in lung cancer cells.

Recent studies have demonstrated the presence of estrogen receptor (ER)beta in the mitochondria in various cell types and tissues, but the exact function of this localization remains unclear. In this study, we have examined the function of mitochondrial ERbeta in non-small-cell lung cancer (NSCLC) cells. Down-regulation of ERbeta by short hairpin RNA constructs sensitized NSCLC cells to various apoptosis-inducing agents such as cisplatin, taxol, and etoposide. The increased growth inhibition and induction of apoptosis in ERbeta-knockdown cells was observed irrespective of estrogen treatment, suggesting a ligand-independent role of ERbeta in regulating the intrinsic apoptotic pathway. Further, ERbeta from the mitochondrial fraction physically interacted with the proapoptotic protein Bad, in a ligand-independent manner. Glutathione-S-transferase pull-down assays and molecular modeling studies revealed that the DNA-binding domain and hinge region of ERbeta, and the BH3 domain of Bad were involved in these interactions. Further investigations revealed that ERbeta inhibited Bad function by disrupting Bad-Bcl-X(L) and Bad-Bcl-2 interactions. Reintroduction of ERbeta in the mitochondria of ERbeta knockdown cells reversed their sensitivity to cisplatin. Overall, our results demonstrate a ligand-independent role of ERbeta in regulating apoptosis, revealing a novel function for ERbeta in the mitochondria.

Molecular modeling of BAD complex resided in a mitochondrion integrating glycolysis and apoptosis.

BAD (Bcl-2 antagonist of cell death) and GK (glucokinase) reside in a mitochondrial complex together with PKA and PP1 catalytic units (PKAc and PP1c) and WAVE-1 that integrates glycolysis and apoptosis. Our research results reveal that BAD is phosphorylated and inactivated on Ser 75 in a BAD-Bcl-xL complex by PKA (targeted to mitochondria through association with WAVE1), resulting in the dissociation of BAD and its binding to GK. Moreover, GK can interact with PP1c and also distinguish WAVE1. On the other hand, BAD is dephosphorylated and activated on Ser75 by PP1c, leading to the separation of PKAc and its binding to the regulatory (R) subunit of PKA which by the dimerization domain of its R subunit connects with WAVE1 linked with GK of the complex. This may be the reason of the complex existing in liver mitochondria, regardless of phosphorylated and dephosphorylated BAD. Additionally, GK like PKA may also prevent Bcl-xL from rebinding to BAD by phosphorylating BAD at Ser 118. The BAD complex model reveals that BAD and GK play key roles because of BAD as a substrate for the PKA-PP1 pair and by BH3 domain directly interacting with GK. This is helpful for our development and research of the molecular mechanism of BAD integrating glycolysis and apoptosis.