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 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.     

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.     

Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-X_L antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.     

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.     

Bcl—2家族蛋白与细胞凋亡

Bcl 2家族蛋白是在细胞凋亡过程中起关键性作用的一类蛋白质。在线粒体上 ,Bcl 2家族蛋白通过与其他凋亡蛋白的协同作用 ,调控线粒体结构与功能的稳定性 ,发挥着细胞凋亡“主开关”的作用。Bcl 2家族包括两类蛋白质 :一类是抗凋亡蛋白 ,另一类是促凋亡蛋白。在细胞凋亡时 ,Bcl 2家族中的促凋亡蛋白成员发生蛋白质的加工修饰 ,易位到线粒体的外膜上 ,引起细胞色素c、凋亡诱导因子等其他促凋亡因子的释放 ,导致细胞凋亡 ;而平时被隔离在线粒体等细胞器内的该家族的抗凋亡蛋白成员则抑制细胞色素c和凋亡诱导因子等促凋亡因子的释放 ,具有抑制细胞凋亡的功能。但一旦这类抗凋亡蛋白成员与激活的促凋亡蛋白发生相互作用后 ,便丧失了对细胞凋亡的抑制作用 ,造成线粒体等细胞器的功能丧失和细胞器内促凋亡因子的释放 ,导致细胞凋亡。现以Bcl 2家族调控细胞凋亡的最新研究进展为基础 ,对Bcl 2家族成员及其蛋白质结构、分布和调控细胞凋亡的分子机制进行综述。     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.     

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.     

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X_L and Bcl-2. A structural model of the Bcl-X_L/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X_L/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X_L. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.     

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.     

Anti-tumor pyrimidinylpiperazines bind to the prosurvival Bcl-2 protein family member Bcl-XL

Overexpression of prosurvival or underexpression of pro-death Bcl-2 family proteins can lead to cancer cell resistance to chemotherapy and radiation treatment. Inhibition of the prosurvival Bcl-2 family proteins has become a strategy for cancer therapy and inhibitors are currently being evaluated in the clinic both as single agents and in combination with established drugs. Here we describe the design, synthesis, and evaluation of pyrimidylpiperazines that were discovered to be inhibitors of the prosurvival Bcl-2 protein family member Bcl-XL. This study identified compound 21 which demonstrated a GI₅₀ value of 8.4 μM against A549 lung adenocarcinoma cells and a binding affinity K_i value for Bcl-XL of 127 nM.     

Caspase family proteases and apoptosis

Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin- 1 ~-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regu- lated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain, and Ca^2＋. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.     

Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells

Many laboratory studies and epidemiological observations confirm that nematodes prevent some immune-mediated diseases. The development of immunologically well-defined laboratory models of intestinal nematode infection has allowed significant advances to be made in understanding the immunological basis of effector mechanisms operating during infection under controlled laboratory conditions. The Heligmosomoides polygyrus- mouse system is used for studies of parasite immunomodulation. H. polygyrus causes a chronic, asymptomatic intestinal infection and effectively maintains both local and systemic tolerance to reduce allergic and autoimmune inflammation. However, exposure of mice to H. polygyrus antigen reduced spontaneous and glucocorticoid-induced apoptosis of CD4- positive T cells in mesenteric lymph node (MLN). In this study we evaluate the proliferation, cytokine secretion, cell cycle progression and expression of apoptosis related genes in MLN CD4 T cells of uninfected and H. polygyrus infected mice ex vivo and in vitro after restimulation with parasite excretory secretory antigen (ESAg), somatic antigen (SAg) and fraction 9 (F9Ag) of somatic antigen. For the first time we explain the influence of H. polygyrus antigens on the intrinsic pathway of apoptosis. We found that the proliferation provoked by fraction 9 and inhibition of apoptosis was dependent on a low Bax/Bcl-2 ratio, dramatical upregulation of survivin, D1 cyclin, P-glycoprotein, and loss of p27Kip1 protein with inhibition of active caspase-3 but not caspase- 8.     

Bcl-2/Bax protein expression in heart,slow-twitch and fast-twitch muscles in young rats growing under chronic hypoxia conditions

We have studied the magnitude of apoptosis in heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius) of rats exposed to 3 weeks in vivo chronic hypoxia. Apoptosis was evaluated biochemically by DNA laddering and by TUNEL and annexin V-staining. The expression of Bax and Bcl-2 proteins was determined by immunohistochemistry and Western blotting.Western blot analysis revealed only a slight difference in Bax expression among the different tissues under normoxic and hypoxic conditions; therefore we can consider that Bax protein is constitutively expressed in muscle tissues. However a singular pattern of Bcl-2 expression was observed among the different tissues under normoxic conditions. Bcl-2 protein was more expressed in fast-twitch glycolytic muscles than in slow-twitch or oxidative muscles with a highest value found in gastrocnemius (4926 ± 280 AU), followed by soleus (2138 ± 200 AU) and a very low expression was displayed in the heart muscle (543 ± 50 AU). After exposure to hypoxia for 21 days (10% O2), Bcl-2 protein expression markedly increased, (44%) in gastrocnemius, (323%) in soleus and (1178%) in heart, with significant differences (p < 0.05 student t-test), reaching a similar threshold of expression in both types of muscles. Furthermore, no sign of apoptosis was detected by TUNEL assay, annexin V-binding assay or DNA electrophoresis analysis. The latter suggested some indiscriminate fragmentations of DNA without apoptosis. In conclusion, we postulate that these protein modifications could represent a adaptative mechanism allowing a better protection against the lack of oxygen in oxidative muscles by preventing apoptosis.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.