Bcl-2 family member Bcl-G is not a proapoptotic protein

The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.     

Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies

Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8⁺ conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein

Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.     

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.     

Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2′, it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2′, Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.     

Anti-autophagic Bcl-2

Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell’s quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a ‘double hit’ of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.     

The Bcl-2 family in autoimmune and degenerative disorders

Members of the Bcl-2 family are essential regulators of programmed cell death and thus play a major role in the development and function of many tissues. The balance between pro-survival and pro-apoptotic members of the family decides whether a cell will live or die. This mechanism allows organisms to get rid of cells that are no longer needed or have become dangerous. Deregulation of apoptosis is a major contributing factor in the development of many diseases. A deeper understanding of how the Bcl-2 family proteins orchestrate death in normal and pathologic conditions is thus relevant not only for disease etiology, but also to try to prevent these various disorders. Experiments with transgenic and gene-ablated mice have helped elucidate the function of the different members of the Bcl-2 family and their physiological roles. The present review highlights the role of Bcl-2 family members in autoimmune and degenerative disorders, with a particular focus on the mouse models that have been used to study their function.     

Apoptotic blocks and chemotherapy resistance: strategies to identify Bcl-2 protein signatures.

Acquired or innate resistance to chemotherapy is a major drawback of cancer therapeutics, which is frequently seen in epithelial cancers. However, the molecular mechanisms underlying chemotherapy resistance remain poorly understood. The mitochondrial pathway is a critical death pathway common to many different types of chemotherapy. Aberrations in this pathway can result in resistance to chemotherapy. The Bcl-2 family of proteins control commitment to programmed cell death by mitochondrial apoptosis. In this review, we will summarize the strategies in determining the components of apoptotic defects responsible for chemotherapy resistance, mainly focused on Bcl-2 protein network.     

TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death

Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Deltaloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Deltaloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Deltaloop occurs at 37 degrees C but not at 4 degrees C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Deltaloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.     

p38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2

INTRODUCTION: 2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. METHODS: 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. RESULTS: 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. CONCLUSION: Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Anti-autophagic Bcl-2: Not just an innocent bystander

Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell's quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a 'double hit' of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.     

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions.     

Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic leukemia cells

Malignant cell accumulation in B-cell chronic lymphocytic leukemia (B-CLL) is primarily caused by defective apoptosis rather than increased proliferation. To further understand the role of Bcl-2 family members, known regulators of apoptosis, in the abnormal B-CLL survival, we have measured their mRNA levels in fresh B-CLL cells and in cultures undergoing spontaneous apoptosis. Using RNA protection assays we found constitutive expression of most bcl-2 members with high levels of bcl2, bcl-w, bad, bak, bax, and the bcl-2/bax ratio, compared to normal PBL. Spontaneous apoptosis of B-CLL cells by in vitro culture resulted in decreased bcl-2, bcl-w, bfl-1, mcl-1, bak, bax, and bcl-2/bax expression. The pro-apoptotic member bik was only expressed in 5/19 cases and was not modulated during apoptosis, suggesting that bik is not involved in this process. Thus, several Bcl-2 family genes are regulated during B-CLL spontaneous apoptosis and their relative levels may contribute to in vivo progression of the disease.     

Yeast techniques for modeling drugs targeting Bcl-2 and caspase family members

Development of drugs targeting Bcl-2 relatives and caspases, for treating diseases including cancer and inflammatory disorders, often involves measuring interactions with recombinant target molecules, and/or monitoring cancer cell killing in vitro. Here, we present yeast-based methods for evaluating drug-mediated inhibition of Bcl-2 relatives or caspases. Active Bax and caspases kill Saccharomyces cerevisiae, and pro-survival Bcl-2 proteins can inhibit Bax-induced yeast death. By measuring the growth or adenosine triphosphate content of transformants co-expressing Bax with pro-survival Bcl-2 relatives, we found that the Bcl-2 antagonist drugs ABT-737 or ABT-263 abolished Bcl-2 or Bcl-x_L function and reduced Bcl-w activity, but failed to inhibit Mcl-1, A1 or the poxvirus orthologs DPV022 and SPPV14. Using this technique, we also demonstrated that adenoviral E1B19K was resistant to these agents. The caspase inhibitor Q-VD-OPh suppressed yeast death induced by caspases 1 and 3. Yeast engineered to express human apoptotic regulators enable simple, automatable assessment of the activity and specificity of candidate drugs targeting Bcl-2 relatives or caspases.