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.     

The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy

Tumour development requires a combination of defects that allow nascent neoplastic cells to become self-sufficient for cell proliferation and insensitive to signals that normally restrain cell growth. Among the latter, evasion of programmed cell death (apoptosis) has proven to be critical for the development and sustained growth of many, perhaps all, cancers. Apoptotic cell death is regulated by complex interactions between pro-survival members and two subgroups of pro-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family. In this invited review article, we reminisce on the discovery of Bcl-2, the first regulator of cell death identified, we discuss the mechanisms that control apoptotic cell death, focussing on how defects in this process promote the development and sustained growth of tumours and also affect their responses to anticancer therapeutics and, finally, we describe how current knowledge of the regulatory networks of apoptosis is exploited to develop novel approaches for cancer therapy.     

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.     

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.     

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.     

细胞凋亡中的Bcl-2家族蛋白及其BH3结构域的功能研究

凋亡相关蛋白中的Bcl-2家族是细胞凋亡的关键调节分子,由抗凋亡和促凋亡成员组成,这些成员之间通过相互协同作用调节了线粒体结构与功能的稳定性,从而在线粒体水平发挥着细胞凋亡的“开关”作用.抗凋亡成员大都分布于线粒体的外膜,与促凋亡成员的BH3结构域相互作用对细胞凋亡发挥抵抗作用.促凋亡成员则主要分布于细胞浆中,细胞接受死亡信号刺激后,促凋亡成员自身受到一系列的调节,如典型的Bax构象改变、BAD和Bik的磷酸化调节以及Bid和Bim的蛋白裂解效应等,使得促凋亡成员在凋亡信号的刺激下整合于线粒体外膜,最终导致线粒体通透转换孔的开放,进而释放包括细胞色素c、凋亡诱导因子、Smac等重要的凋亡因子,随后caspase被激活进而断裂重要的细胞内结构蛋白与功能分子,执行细胞凋亡.     

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.     

Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays

Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein–protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein–protein interactions within the cell.     

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.     

Bcl-2/Beclin-1复合体在自噬中的调节作用

自噬(autophagy)是一种进化保守的溶酶体依赖性分解代谢途径,是细胞维持自稳态的重要机制之一,并参与多种疾病的发生. Beclin-1作为自噬体成核的关键分子之一,是1个调节自噬的关键靶点. Beclin-1有1个BH3结构域,Bcl-2、Bcl-XL等可以通过这个BH3结构域与Beclin-1结合而影响其活性. 抗凋亡Bcl-2家族蛋白和Beclin-1的表达水平、磷酸化、分子的亚细胞定位以及BH3-only蛋白等,均可调节Beclin-1蛋白和Bcl-2家族蛋白结合水平,进而调控自噬的发生,并可能对细胞最终走向自噬还是凋亡起着关键作用.     

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.     

BcL2蛋白质家族——定位与转位

Bcl-2蛋白质家族的抗凋亡和促凋亡成员,在线粒体水平上决定细胞的存活或死亡.在正常细胞中,这些成员呈现功能适应性的细胞内分布;抗凋亡成员主要定位于细胞内膜系特别是线粒体外膜上：但绝大多数促凋亡成员主要分布于细胞浆中.细胞接受死亡信号后,Bcl-2家族成员本身受到一系列的调节,如磷酸化、裂解、蛋白质-蛋白质相互作用等,结果之一是促凋亡成员发生细胞内定位的改变,从细胞浆转位于线粒体膜上,并引发线粒体功能异常及其内外膜间致凋亡因子的释放,最终导致细胞凋亡.     

唯BH3域蛋白与神经细胞凋亡

细胞凋亡在神经细胞的生理性和病理性死亡中起着重要作用。唯BH3域蛋白是Bcl-2家族中的一类仅含有BH3同源结构域的促凋亡分子,它们通过抑制Bcl-2抗凋亡成员的活性或激活Bax/Bak样促凋亡成员的活性来调节细胞凋亡。最近研究表明,唯BH3域蛋白在凋亡的启动及凋亡通路的沟通中发挥着极其重要的作用。     

BNips: a group of pro-apoptotic proteins in the Bcl-2 family

BNip (formerly known as Nip) proteins, including homologues isolated from human, mouse and Caenorhabditis. elegans, are a relatively new subgroup of the Bcl-2 family. These proteins are classified into this family based on limited sequence homology with the Bcl-2 homology domain 3 and carboxyl terminal transmembrane domain. BNip proteins were first discovered based on their interaction with the adenovirus E1B 19 kDa/Bcl-2 family protein and since then, their roles in cell death pathways have been actively studied. However, the precise mechanisms by which the BNip proteins induce apoptosis and/or necrosis remain to be determined. To advance our knowledge, we have provided a summary and review of current literature regarding BNip proteins including comparative sequence analysis, mutational mapping of the functional domains, and cell death mechanisms involving disruption of mitochondrial homeostasis. Since BNip proteins are expressed at high levels in the heart as compared to other organs, their roles in cardiomyocyte injury during hypoxia or viral infection is a focus of this review. Finally, we discuss potential directions for further study on this increasingly important group of pro-apoptotic proteins.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

Bcl-2 Antisense Therapy for Cancer: The Art of Persuading Tumour Cells to Commit Suicide

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis induced by numerous physiological and pathological stimuli, and uncontrolled cell survival due to Bcl-2 overexpression has been shown to contribute to tumour formation and the development of autoimmune diseases. The multifunctional action of Bcl-2 is thought to prevent activation of the ced3/caspase-3 subfamily of ICE proteases, resulting in suppression of the death effector machinery. Since most conventional anti-cancer agents act by triggering this suicide pathway, overexpression of Bcl-2 in cancer cells has also been associated with drug resistance. The antisense approach to inhibition of gene expression relies on the binding of small synthetic oligodeoxynucleotides to a complementary base sequence on a target mRNA. As a consequence, expression of the corresponding gene is downregulated due to endonuclease-mediated hydrolysis of the mRNA strand, or to translational arrest arising from sterie hindrance by the RNA:DNA heterodimer. Since these mechanisms of action differ from those exerted by conventional anticancer agents, antisense oligodeoxynucleotides designed to specifically inhibit bcl-2 gene expression hold great promise as agents that could overcome clinical drug resistance, and improve the treatment outcome of many hitherto incurable cancer diseases.     

Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists

Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.     

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release.