Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P10415 (Bcl-2)
 33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bax, a member of the Bcl-2 family of proteins, has been shown to promote apoptosis while other members of the family, including Bcl-XL and Bcl-2, inhibit cell death induced by a variety of stimuli. The mechanism by which Bax promotes cell death is poorly understood. In the present report, we assessed the ability of Bax to antagonize the death repressor activity of Bcl-XL during chemotherapy-induced apoptosis in the lymphoid cell line, FL5.12. Expression of wild-type Bax countered the repressor activity of Bcl-XL against cell death mediated by VP-16 and cisplatin. We performed site-directed mutagenesis of the BH1, BH2, and BH3 homology regions in Bax to determine the ability of wild-type and mutant Bax to heterodimerize with Bcl-XL and to antagonize the protective effect of Bcl-XL against chemotherapy-induced apoptosis. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 in BH1, tryptophan 151 and 158 in BH2, and glycine 67 and aspartic acid 68 in BH3 retained their ability to promote chemotherapy-induced cell death that was inhibited by Bcl-XL and to form heterodimers with Bcl-XL. Bax proteins containing deletions of the most highly conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) maintained the ability of Bax to antagonize the death repressor activity of Bcl-XL and to associate with Bcl-XL. However, Bax with BH3 deleted did not form heterodimers with Bcl-XL, but retained its ability to counter the death repressor activity of Bcl-XL. These results demonstrate that the conserved BH3, but not BH1 or BH2, homology region of Bax is necessary for its interaction with Bcl-XL in mammalian cells. Furthermore, our results indicate that Bax does not require BH1, BH2, BH3, or heterodimerization with Bcl-XL to counter the death repressor activity of Bcl-XL. Therefore, Bax can antagonize Bcl-XL during VP-16 and, in a lesser degree, during cisplatin-induced cell death independent of its heterodimerization with Bcl-XL.
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PMID:Bax can antagonize Bcl-XL during etoposide and cisplatin-induced cell death independently of its heterodimerization with Bcl-XL. 879 52

Bax, a member of the Bcl-2 family of proteins, has been shown to accelerate apoptosis induced by growth factor withdrawal, gamma-irradiation, and the chemotherapeutic agent, etoposide. The mechanism by which Bax promotes apoptosis is poorly understood. Bax forms homodimers which have been suggested to act as accelerators or inducers of cell death. However, the requirement for homodimerization of Bax to promote cell death remains unclear. We performed site-directed mutagenesis of the BH1, BH2, and BH3 in Bax to determine the regions of Bax required for homodimerization and to define the role of Bax homodimers in cell death induced by chemotherapy drugs. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 (G108) in BH1, tryptophan 158 (W158) in BH2, and glycine 67 and aspartic acid 68 (GD67-68) in BH3 as well as deletion of the most conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) and deletion of BH3 (Delta63-71) maintained their ability to accelerate chemotherapy-induced cell death. Immunoprecipitation studies revealed that Bax with deletions in BH1 and BH2 still associated with wild-type Bax while deletion of BH3 disrupted Bax homodimerization. These results demonstrate that Bax does not require the conserved regions of homology, BH1, BH2, or BH3, to accelerate chemotherapy-induced cell death. Furthermore, our results established BH3 as a region required for Bax homodimerization in mammalian cells and demonstrate that monomeric forms of Bax are active in accelerating cell death induced by chemotherapy agents.
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PMID:Bax homodimerization is not required for Bax to accelerate chemotherapy-induced cell death. 894 58

Glucocorticoid hormones are known to enhance gonadotropin/cAMP-induced steroidogenesis in rat and human granulosa cells. As glucocorticoids induce apoptosis in numerous cell types, we investigated the role of glucocorticoids in the control of apoptosis in immortalized human granulosa cells (HO-23) transfected with a temperature-sensitive mutant of p53 (Val(135)). When HO-23 were incubated with forskolin in the presence or absence of dexamethasone (Dex) at 32 or 37 C, progesterone production was higher by 4- and 8-fold in the presence of Dex at 37 or 32 C, respectively (P: < 0. 01). The expression of adrenodoxin (ADX), which is an intrinsic part of the cytochrome P450 side-chain cleavage enzyme system, remained the same in the presence or absence of Dex in forskolin-stimulated cells. Dex reduced apoptosis (to 33% of control) in cultures after activation of p53 by shifting the temperature from 37 to 32 C. Moreover, Dex suppressed apoptosis induced by serum deprivation (to 40% of control) or forskolin stimulation (to 28% and 40% at 37 and at 32 C, respectively). The protective effect of Dex on cAMP-, p53-, and serum deprivation-induced apoptosis was confirmed by both 4',6-diamido-2-phenylindole hydrochloride DNA staining and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling with an ED(50) of 7 nM Dex. Hydrocortisone showed a similar antiapoptotic effect. The protective effect of glucocorticoids against apoptosis was completely abolished by RU486 when cells were coincubated with 10 nM Dex and 10-100 nM RU486. The protection against apoptosis by glucocorticoid involved a sharp elevation in intracellular levels of Bcl-2 (3-7.6 fold; P: < 0.01). In contrast to the effect of Dex in the prevention of apoptosis in HO-23 granulosa cells, Dex dramatically stimulated apoptosis by 3-fold in LTR-6 myeloid leukemia cells expressing the same temperature-sensitive mutant (Val(135) p53) and the same amount of glucocorticoid receptor-alpha. Forskolin did not stimulate apoptosis when incubated with these cells. However, it augmented by 1.2-fold the p53-induced apoptosis in cells shifted from 37 to 32 C. Dex further enhanced apoptosis by 1.9-fold in p53-activated cultures (32 C). Incubation of the cells with Dex dramatically reduced Bcl-2 levels to 15% of control at 37 C (P: < 0.01) or 32 C in the presence or absence of forskolin (P: < 0.01). Our data suggest that glucocorticoids exert a protective effect against induced apoptosis in immortalized granulosa cells and a stimulatory effect on apoptosis in myeloid leukemia cells. Moreover, modulation of Bcl-2 levels plays an important role in mediating the glucocorticoid effect on cell survival. The opposite effect of glucocorticoids on Bcl-2 levels in the two cell lines may be due to the different ontogeneses of the two cell types: epithelial for granulosa cells vs. mesenchymal for myeloid cells studied in the present work.
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PMID:Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2. 1115 53

HTLV-I is etiologically implicated with tropical spastic paraparesis/HTLV-I associated myelopathy, adult T-cell leukemia and certain other diseases. However, after infection the virus enters into a dormant state, whereas the characteristics of the HTLV-I related diseases indicate that their genesis requires activation of the dormant virus by a Tax-independent mechanism. In the present study we demonstrate that a variety of stress-inducing agents (TPA, cisplatin, etoposide, taxol, and 3-methylcholanthrene) are capable of Tax-independent activation of HTLV-I LTR and that this activation is detected mainly in cells that are undergoing through the apoptotic process. Furthermore, it is demonstrated that both apoptosis induction and HTLV-I LTR activation are inhibited by Bcl-2 and by PKC, indicating that these two processes are mechanistically cross-linked. In addition, using an HTLV-I producing human T-cell line which permanently express the negatively transdominant tax mutant, Delta58tax, under the Tet-Off control system, we prove that the virally encoded Tax protein protects the host cells from apoptosis. Together, these data suggest that activation of the dormant virus in the carriers' infected T-cells by certain stress-inducing conditions and protecting these cells from the consequent apoptotic death by the viral Tax protein emerging after this activation, might be the basis for switching the virus from latency to a pathogenic phase.
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PMID:Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I-infected T-cells from apoptosis by the viral tax protein. 1169 93

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.
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PMID:Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax. 1209 70

The sequence of Bcl-2 homology domains, BH1 and BH2, is known to be conserved among anti- and pro-apoptotic members of Bcl-2 family proteins. But structural conservation of these domains with respect to functionally active residues playing role in heterodimerization-mediated regulation of apoptosis has never been elucidated. Here, we have suggested the formation of an active site by structurally conserved residues in BH1 (glycine, arginine) and BH2 (tryptophan) domains of Bcl-2 family members, which also accounts for the functional effect of known mutations in BH1 (G145A, G145E) and BH2 (W188A) domains of Bcl-2.
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PMID:Structural conservation of residues in BH1 and BH2 domains of Bcl-2 family proteins. 1594 1

Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization from the cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurements were performed in the absence and presence of lipid vesicles with Bcl-xL. A pH-dependent model is proposed for the solution to membrane conformational change that consists of three stable conformations: a solution conformation, a conformation similar to the solution conformation but anchored to the membrane by its C-terminal transmembrane domain, and a membrane conformation that is fully associated with the membrane. This model predicts that the solution to membrane conformational change is independent of the C-terminal transmembrane domain, which is experimentally demonstrated. The conformational change is associated with changes in secondary and, especially, tertiary structure of the protein, as measured by far and near-UV circular dichroism spectroscopy, respectively. Membrane insertion was distinguished from peripheral association with the membrane by quenching of intrinsic tryptophan fluorescence by acrylamide and brominated lipids. For the cytosolic domain, the free energy of insertion (DeltaG degrees x) into lipid vesicles was determined to be -6.5 kcal mol(-1) at pH 4.9 by vesicle binding experiments. To test whether electrostatic interactions were significant to this process, the salt dependence of this conformational change was measured and analyzed in terms of Gouy-Chapman theory to estimate an electrostatic contribution of DeltaG degrees el approximately -2.5 kcal mol(-1) and a non-electrostatic contribution of DeltaG degrees nel approximately -4.0 kcal mol(-1) to the free energy of insertion, DeltaG degrees x. Calcium, which blocks ion channel activity of Bcl-xL, did not affect the solution to membrane conformational change more than predicted by these electrostatic considerations. The lipid cardiolipin, that is enriched at mitochondrial contact sites and reported to be important for the localization of Bcl-2 proteins, did not affect the solution to membrane conformational change of the cytosolic domain, suggesting that this lipid is not involved in the localization of Bcl-xL in vivo. Collectively, these data suggest the solution to membrane conformational change is controlled by an electrostatic mechanism. Given the distinct biological activities of these conformations, the possibility that this conformational change might be a regulatory checkpoint for apoptosis is discussed.
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PMID:Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism. 1665 Aug 55

Novel trinuclear complexes C23H31N6O6CuSn2Cl5 [1], C23H31N6O6CuZr2Cl5 [2], C23H31N6O6ZnSn2Cl5 [3], and C23H31N6O6ZnZr2Cl5 [4] were synthesized and characterized by spectroscopic (IR, 1H, 13C, 2D COSY, and 119Sn NMR, EPR, UV-vis, ESI-MS) and analytical methods. In complexes 1-4, the geometry of copper and zinc metal ions were described as square-based pyramidal with l-tryptophan coordinated to copper/zinc via carboxylate group while Sn/Zr was present in the hexacoordinate environment. The interaction of 1 and 2 with calf thymus DNA in Tris buffer was studied by electronic absorption titration, luminescence titration, cyclic voltammetry, circular dichroism, and viscometric measurements. The emission quenching of these complexes by [Fe(CN)6]4- depressed greatly when bound to DNA. Observed changes in the circular dichoric spectra of DNA in presence of 1 and 2 support the strong binding of complexes with DNA. The relative specific viscosity of DNA bound to 1 and 2 decreased, indicating that the complexes bind to DNA via covalent binding. The results reveal that the extent of DNA binding of 1 was greater than that of 2. To evaluate the mechanistic pathway of DNA inhibition, counting experiments and MTT assay were employed to assess the induction of apoptosis by 1. Western blot analysis of whole cell lysates and mitochondrial fractions with Bcl-2 and p-53 family proteins and caspase-3 colorimetry assay were also carried out on a human neuroblastoma cell line SY5Y.
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PMID:DNA binding studies of novel Copper(II) complexes containing L-tryptophan as chiral auxiliary: in vitro antitumor activity of Cu-Sn2 complex in human neuroblastoma cells. 1737 49

NR-13, Mcl-1, and BCL-X(L), are conserved anti-apoptotic proteins that belong to the anti-apoptotic Bcl-2 sub-family, which inhibits cell death by preventing mitochondrial membrane permeabilization (MMP). Given the anti-apoptotic functions of these proteins in vertebrates (e.g. human, mouse, and zebrafish) and the involvement of apoptotic regulation in immune responses, we studied the sequences of these genes and their transcript expression in Atlantic cod (Gadus morhua) during innate immune responses to viral and bacterial stimuli. Based on previously generated Atlantic cod expressed sequence tags (ESTs), we identified partial cDNA sequences of putative orthologues of Atlantic cod NR-13, Mcl-1, and Bcl-X, and obtained the full-length cDNA, genomic, and promoter region sequences for these genes. The analyses of Atlantic cod cDNA sequences, and comparisons of the cod deduced amino acid sequences to putative orthologues in other species, revealed the presence of highly conserved Bcl-2 homology (BH) and transmembrane (TM) domains in the Atlantic cod sequences. Analysis of gene structure revealed conserved intron/exon boundaries within the coding regions of human and Atlantic cod putative orthologues. We found that an intron/exon boundary immediately following the codon for the 8th residue (tryptophan) of the BH2 domain exists in all anti-apoptotic Bcl-2 sub-family genes regardless of vast evolutionary distance. We also identified a non-coding exon in the Atlantic cod NR-13-like gene, which appears to be absent in its putative mammalian orthologues. Quantitative reverse transcription-polymerase chain reaction (QPCR) was used to study constitutive gene expression in six tissues (blood, brain, gill, head kidney, pyloric caecum, and spleen) of non-stressed juvenile cod; NR-13 and Bcl-X2 were most highly expressed in gill, whereas Mcl-1 and Bcl-X1 were most highly expressed in blood. In cod challenged with intraperitoneal (IP) injections of the viral mimic polyriboinosinic polyribocytidylic acid (pIC), (1) NR-13 mRNA expression was significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6h post-injection and in head kidney at both 6 and 24h post-injection (HPI), and (2) both Mcl-1 and Bcl-X2 were significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6 HPI. QPCR was used to show that, in cod challenged with IP injections of formalin-killed, atypical Aeromonas salmonicida (ASAL), only NR-13 appeared to be responsive (significantly up-regulated in spleen at 6 HPI compared to 0h pre-injection controls). Interestingly, QPCR showed that saline injection had a mild (less than 3-fold) but significant inductive effect (compared to 0h pre-injection controls) on both NR-13 and Mcl-1 transcript expression in spleen at 2 HPI. Although we only obtained partial cDNA and genomic sequences for Bcl-X2, sufficient evidence was accumulated to show that two Bcl-X paralogues exist in Atlantic cod, possibly due to the teleost-specific genome duplication event. Promoter regions for NR-13, Mcl-1, and Bcl-X1 were obtained and analyzed for the first time in fish, and potential regulatory sites (e.g. putative NF-kappaB binding sites) that were found in the promoter regions of NR-13 and Mcl-1 may account for their transcriptional activation by pIC.
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PMID:Characterization and expression analyses of anti-apoptotic Bcl-2-like genes NR-13, Mcl-1, Bcl-X1, and Bcl-X2 in Atlantic cod (Gadus morhua). 1992 1

5-Hydroxytryptophan (5HTP), an analogue of tryptophan, is a precursor of serotonin that also has effective antioxidative and anti-apoptotic properties (1) . However, the cellular mechanisms underlying these properties of 5HTP have not been explored. In this study, we tested the hypothesis that 5HTP exerts its antioxidative action against oxidative stress and inflammation by suppressing the activation of the key pro-inflammatory transcriptional pathways, p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kappaB (NF-kappaB). The study was carried out using human fibroblast cells that were challenged by tert-butylhydroperoxide (t-BHP)-induced oxidative damage. Results show that 5HTP significantly reduced t-BHP-induced oxidative damage in human fibroblast cells, as determined by cell cytotoxicity, intracellular reactive species (RS) and peroxynitrite (ONOO(-)) generation, and inducible nitric oxide synthase expression. Moreover, 5HTP protected human fibroblast cells against t-BHP-induced oxidative DNA damage, as determined by 4,6-diamidino-2-phenlylindole (DAPI) staining. Pretreatment of human fibroblast cells with 5HTP also dose-dependently inhibited glutathione (GSH) depletion, indicating that it protects cells against t-BHP-induced oxidative damage. Western blot analysis revealed that 5HTP also markedly increased Bcl-2 expression and suppressed both p38MAPK and NF-kappaB activation in the t-BHP-treated human fibroblast cells. When these results are taken together, they strongly indicate that 5HTP has beneficial and protective effects against t-BHP-induced cell death in vitro, as demonstrated by its antioxidative and anti-inflammatory actions. Data further showed that the protective mechanisms underlying the actions of 5HTP against oxidative stress-induced damage are associated with RS/ONOO(-) scavenging and the inhibition of lipid peroxidation and GSH depletion.
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PMID:5-Hydroxytrytophan inhibits tert-butylhydroperoxide (t-BHP)-induced oxidative damage via the suppression of reactive species (RS) and nuclear factor-kappaB (NF-kappaB) activation on human fibroblast. 2041 19


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