Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

Apolipoprotein l6, a novel proapoptotic Bcl-2 homology 3-only protein, induces mitochondria-mediated apoptosis in cancer cells

Cancer cells frequently possess defects in the genetic and biochemical pathways of apoptosis. Members of the Bcl-2 family play pivotal roles in regulating apoptosis and possess at least one of four Bcl-2 homology (BH) domains, designated BH1 to BH4. The BH3 domain is the only one conserved in proapoptotic BH3-only proteins and plays an important role in protein-protein interactions in apoptosis by regulating homodimerization and heterodimerization of the Bcl-2 family members. To date, 10 BH3-only proapoptotic proteins have been identified and characterized in the human genome. The completion of the Human Genome Project and the availability of various public databases and sequence analysis algorithms allowed us to use the bioinformatic database-mining approach to identify one novel BH3-only protein, apolipoprotein L6 (ApoL6). The full-length cDNA of ApoL6 was identified, cloned, and functionally expressed in p53-null colorectal cancer cells (DLD-1). We found that overexpression of wild-type ApoL6 induced mitochondria-mediated apoptosis in DLD-1 cells characterized by release of cytochrome c and Smac/DIABLO from mitochondria and activation of caspase-9, whereas ApoL6 BH3 domain deletion allele did not. In addition, overexpression of ApoL6 also induced activation of caspase-8. Furthermore, we showed that adenovirus harboring the full-length cDNA of ApoL6 induced marked apoptosis in a variety of cancer cell types, and ApoL6 recruited and interacted with lipid/fatty acid components during the induction of apoptosis. To our knowledge, this is the first example that intracellular overproduction of an apolipoprotein induces marked apoptosis.     

BH3-only proteins in cell death initiation, malignant disease and anticancer therapy

Induction of apoptosis in tumour cells, either by direct activation of the death receptor pathway using agonistic antibodies or recombinant ligands, or direct triggering of the Bcl-2-regulated intrinsic apoptosis pathway by small molecule drugs, carries high hopes to overcome the shortcomings of current anticancer therapies. The latter therapy concept builds on a more detailed understanding of how Bcl-2-like molecules maintain mitochondrial integrity and how BH3-only proteins and Bax/Bak-like molecules can undermine it. Means to unleash the apoptotic potential of BH3-only proteins in tumour cells, or bypass the need for BH3-only proteins by blocking possible interactions of Bcl-2-like prosurvival molecules with Bax and/or Bak allowing their direct activation, constitute interesting options for the design of novel anticancer therapies.     

Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.     

腺病毒介导的bak基因诱发肿瘤细胞的凋亡

构建了含ｂａｋ基因的转移载体ｐＣＡ１３－ｂａｋ,并用该载体和含有Ａｄ－５（人腺病毒５型）大部分基因组的重组质凿ｐＢＨＧ１１共转染２９３细胞（转化人胚肾细胞系）,分离并提纯缺陷型病毒Ａｄ－ｂａｋ。用该病毒感染ＨｅＬａ细胞（人宫颈癌细胞系）,相差显微镜、扫描电子显微镜直接观察到了典型的细胞凋亡形态,联蛋埃Ａｎｎｅｘｉｎ）Ｖ－ＦＩＯＴ７凋亡试剂盒检测到原本位于细胞３膜内侧的磷脂酰丝氨酸向外播转,进一步证     

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。     

harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L).

Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans. The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell. Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis. The product of harakiri, Hrk, physically interacts with the death-repressor proteins Bcl-2 and Bcl-X(L), but not with death-promoting homologs, Bax or Bak. Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl-2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions. Expression of Hrk induces cell death which is inhibited by Bcl-2 and Bcl-X(L). Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl-2 and Bcl-X(L) in mammalian cells. Moreover, the killing activity of this mutant form of Hrk (Hrk deltaBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl-2 and Bcl-X(L). Because Hrk lacks conserved BH1 and BH2 domains that define Bcl-2 family members, we propose that Hrk and Bik/Nbk, another BH3-containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival-promoting Bcl-2 and Bcl-X(L).     

Cloning of zebrafish BAD,a BH3-only proapoptotic protein,whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.     

Proapoptotic BH3-only Bcl-2 family member Bik/Blk/Nbk is expressed in hemopoietic and endothelial cells but is redundant for their programmed death

The BH3-only members of the Bcl-2 protein family are essential for initiation of programmed cell death and stress-induced apoptosis. We have determined the expression pattern in mice of the BH3-only protein Bik, also called Blk or Nbk, and examined its physiological function by gene targeting. We found that Bik is expressed widely in the hematopoietic compartment and in endothelial cells of the venous but not arterial lineages. Nevertheless, its loss did not increase the numbers of such cells in mice or protect hematopoietic cells in vitro from apoptosis induced by cytokine withdrawal or diverse other cytotoxic stimuli. Moreover, whereas loss of the BH3-only protein Bim rescued mice lacking the prosurvival protein Bcl-2 from fatal polycystic kidney disease and lymphopenia, loss of Bik did not. These results indicate that any function of Bik in programmed cell death and stress-induced apoptosis must overlap that of other BH3-only proteins.     

Three novel Bid proteins generated by alternative splicing of the human Bid gene

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.     

The cellular protein PRA1 modulates the anti-apoptotic activity of Epstein-Barr virus BHRF1, a homologue of Bcl-2, through direct interaction

The Epstein-Barr virus-encoded early protein, BHRF1, is a structural and functional homologue of the anti-apoptotic protein, Bcl-2. There is accumulating evidence that BHRF1 protects a variety of cell types from apoptosis induced by various external stimuli. To identify specific proteins from normal epithelial cells that interact with BHRF1 and that might promote or inhibit its anti-apoptotic activity, we screened a yeast two-hybrid cDNA library derived from human normal foreskin keratinocytes and identified a cellular gene encoding human prenylated rab acceptor 1 (hPRA1). The interaction of hPRA1 with BHRF1 was confirmed using glutathione S-transferase pull-down assays, confocal laser scanning microscopy, and co-immunoprecipitation. Two regions of PRA1, amino acids 30-53 and the carboxyl-terminal 21 residues, are important for BHRF1 interactions and two regions of BHRF1, amino acids 1-18 and 89-142, including the Bcl-2 homology domains BH4 and BH1, respectively, are crucial for PRA1 interactions. PRA1 expression interferes with the anti-apoptotic activity of BHRF1, although not of Bcl-2. These results indicate that the PRA1 interacts selectively with BHRF1 to reduce its anti-apoptotic activity and might play a role in the impeding completion of virus maturation.     

Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family.

Beginning with a mouse gene mTSARG3, which was related to apoptosis of spermatogenic cells, bioinformatics was applied and a predicted novel rat gene full-length cDNA sequence was attained. Gene-specific primers were designed for PCR in rat testis cDNA library. A new gene Tsarg1 (GenBank Accession No. AY380804) was cloned, which is related to apoptosis in rat spermatogenic cells. The gene whose full cDNA length is 1176 bp containing 8 exons and 7 introns is located in rat chromosome 1q32-1q33, which encoded a protein containing 316 amino acid residues and being a new member of HSP40 protein family since the sequence contains the highly conserved J domain, which is present in all DnaJ-like proteins and is supported to have a critical role in DnaJ-DnaK protein-protein interactions. The results of RT-PCR and Northern blot analysis showed that Tsarg1 was specifically expressed in rat testis, which probably inhibits rat testis spermatogenic cell apoptosis.     

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.     

Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax.     

Mouse keratinocytes express c98, a novel gene homologous to bcl-2, that is stimulated by insulin-like growth factor 1 and prevents dexamethasone-induced apoptosis

Many studies have been undertaken to investigate the mechanisms of skin differentiation. In particular, growth factors and hormones are believed to play important roles in skin proliferation, differentiation and survival. Insulin-like growth factor-1 (IGF-1) has been identified as a survival factor in many tissues including the skin, but the molecular mechanism of IGF-1 in epidermal differentiation is not completely understood. Neonatal mouse skin is useful for studying changes in gene expression, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display (DD), a 357-nt message that is specifically expressed in the epidermal keratinocytes of IGF-1-injected newborn mice but not in controls, has been identified. Confirmation of expression of this gene by ribonuclease protection assay (RPA) showed that its mRNA expression in the epidermal keratinocytes is induced by IGF-1. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-5'-RACE), we have successfully isolated a 3473-bp full-length gene, c98, that has 97% sequence homology to a bcl-2-like gene, bcl-w. The latter has been identified as a proto-oncogene in several murine myeloid cell lines. Amino acid sequence analysis of the c98 showed that it has 97% sequence identity to the bcl-w protein and possesses bcl-2 homology domains (BH) 1, 2 and 3. Immunoblotting data revealed similar increases of c98 protein expression to its mRNA expression in the keratinocytes of IGF-1-injected animals. Weak expression of other bcl-2 family member proteins, bax, bcl-2 and bcl-xL, were also found in the immunoblots. Additionally, IGF-1 was found to be able to protect epidermal keratinocytes from dexamethasone (DEX)-induced apoptosis, based on the findings that after the cells were treated with DEX, DNA laddering was present in the control mice but not in those injected with IGF-1. Further, using a photometric enzyme-linked immunoassay to quantitate keratinocyte death, we found that after addition of DEX, the amounts of cytoplasmic histone-associated DNA fragments were not significantly (P>0.05) different in IGF-1-treated cells compared with untreated control cells during the high mitotic stage of skin epidermis. To assess the role of c98 in these anti-apoptotic processes, we have generated a recombinant plasmid that contains an expression vector and c98 and transfected this plasmid into the keratinocytes from mice without IGF-1-treatment. Expression of the c98 protein was found to completely (P>0.05) block DEX-induced apoptosis after cell transfection. Taken together, our current data demonstrated that IGF-1 plays an anti-apoptotic role in the DEX-induced apoptosis in epidermal keratinocytes and this, at least in part, may be mediated through expression of c98.     

Transmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis,but not cytochrome c release in the pathway of inostamycin-induced apoptosis

Bcl-2 protein plays important roles in the regulation of apoptosis. However, the exact mechanism by which Bcl-2 blocks apoptosis is still unclear. In the present study, we found that overexpression of Bcl-2 in human small cell lung carcinoma Ms-1 cells inhibited not only the release of cytochrome c from mitochondria into cytosol but also de novo ceramide synthesis induced by inostamycin, a phosphatidylinositol turnover inhibitor. To investigate the correlation between the structure of Bcl-2 and its inhibitory function in inostamycin-induced apoptosis, Ms-1 cells that stably overexpress domain-deletional mutants of Bcl-2 were established. Transmembrane domain-deleted Bcl-2 failed to inhibit inostamycin-induced de novo ceramide synthesis, whereas it inhibited inostamycin-induced cytochrome c release, indicating that anchoring of Bcl-2 to membrane was a requirement for its inhibitory effect on inostamycin-induced ceramide synthesis, but not cytochrome c release. Thus, the deletion mutant of tarnsmembrane domain of Bcl-2 can suppress inostamycin-induced apoptosis by inhibiting cytochrome c release, a downstream event of ceramide synthesis in the pathway of inostamycin-induced apoptosis. We also found that the BH3 and BH4 domains of Bcl-2 were necessary for inhibition of inostamycin-induced apoptosis, and deletion of BH1 or BH2 did not affect the inhibitory effect of Bcl-2 to inostamycin-induced apoptotic events.