Three-dimensional structure of Bax-mediated pores in membrane bilayers.

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ~3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.

CLIPR-59 regulates TNF-α-induced apoptosis by controlling ubiquitination of RIP1.

Tumor necrosis factor-α (TNF-α) has important roles in several immunological events by regulating apoptosis and transcriptional activation of cytokine genes. Intracellular signaling mediated by TNF-receptor-type 1 (TNFR1) is constituted by two sequential protein complexes: Complex-I containing the receptor and Complex-II-containing Caspase-8. Protein modifications, particularly ubiquitination, are associated with the regulation of the formation of these complexes. However, the underlying mechanisms remain poorly defined. Here, we identified CLIP-170-related 59 kDa protein (CLIPR-59) as a novel adaptor protein for TNFR1. Experimental reduction of CLIPR-59 levels prevented induction of apoptosis and activation of caspases in the context of TNF-α signaling. CLIPR-59 binds TNFR1 but dissociates in response to TNF-α stimulation. However, CLIPR-59 is also involved in and needed for the formation of Complex-II. Moreover, CLIPR-59 regulates TNF-α-induced ubiquitination of receptor-interacting protein 1 (RIP1) by its association with CYLD, a de-ubiquitinating enzyme. These findings suggest that CLIPR-59 modulates ubiquitination of RIP1, resulting in the formation of Complex-II and thus promoting Caspase-8 activation to induce apoptosis by TNF-α.

Targeting the BH3-interacting domain death agonist to develop mechanistically unique antidepressants.

The BH3-interacting domain death agonist (Bid) is a pro-apoptotic member of the B-cell lymphoma-2 (Bcl-2) protein family. Previous studies have shown that stress reduces levels of Bcl-2 in brain regions implicated in the pathophysiology of mood disorders, whereas antidepressants and mood stabilizers increase Bcl-2 levels. The Bcl-2 protein family has an essential role in cellular resilience as well as synaptic and neuronal plasticity and may influence mood and affective behaviors. This study inhibited Bid in mice using two pharmacological antagonists (BI-11A7 and BI-2A7); the selective serotonin reuptake inhibitor citalopram was used as a positive control. These agents were studied in several well-known rodent models of depression-the forced swim test (FST), the tail suspension test (TST), and the learned helplessness (LH) paradigm-as well as in the female urine sniffing test (FUST), a measure of sex-related reward-seeking behavior. Citalopram and BI-11A7 both significantly reduced immobility time in the FST and TST and attenuated escape latencies in mice that underwent the LH paradigm. In the FUST, both agents significantly improved duration of female urine sniffing in mice that had developed helplessness. LH induction increased the activation of apoptosis-inducing factor (AIF), a caspase-independent cell death constituent activated by Bid, and mitochondrial AIF expression was attenuated by chronic BI-11A7 infusion. Taken together, the results suggest that functional perturbation of apoptotic proteins such as Bid and, alternatively, enhancement of Bcl-2 function, is a putative strategy for developing novel therapeutics for mood disorders.

Co-chaperone BAG3 and adenovirus penton base protein partnership.

The BAG family of Hsp70/Hsc70 co-chaperones is characterised by the presence of a conserved BAG domain at the carboxyl-terminus. BAG3 protein is the only member of this family containing also the N-terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N-terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co-migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co-chaperone activity, suggesting that this interaction is not related to the classical BAG3 co-chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad-infected cells, suggesting that the interaction between virus penton base protein and cellular co-chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host-pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co-chaperone.

A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy.

We investigated the mRNA expression levels of all six antiapoptotic Bcl-2 subfamily members in 68 human cancer cell lines using qPCR techniques and measured the ability of known Bcl-2 inhibitors to induce cell death in 36 of the studied tumor cell lines. Our study reveals that Mcl-1 represents the anti-apoptotic Bcl-2 subfamily member with the highest mRNA levels in the lung, prostate, breast, ovarian, renal, and glioma cancer cell lines. In leukemia/lymphoma and melanoma cancer cell lines, Bcl-2 and Bfl-1 had the highest levels of mRNA, respectively. The observed correlation between the cell killing properties of known Bcl-2 inhibitors and the relative mRNA expression levels of anti-apoptotic Bcl-2 proteins provide critical insights into apoptosis-based anticancer strategies that target Bcl-2 proteins. Our data may explain current challenges of selective Bcl-2 inhibitors in the clinic, given that severe expression of Bcl-2 seems to be limited to leukemia cell lines. Furthermore, our data suggest that in most cancer types a strategy targeted to Mcl-1 inhibition, or combination of Bfl-1 and Mcl-1 inhibition for melanoma, may prove to be more successful than therapies targeting only Bcl-2.

TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-kappaB.

Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.

Neuron-specific overexpression of the co-chaperone Bcl-2-associated athanogene-1 in superoxide dismutase 1(G93A)-transgenic mice.

Bcl-2-associated athanogene-1 (BAG1) binds heat-shock protein 70 (Hsp70)/Hsc70, increases intracellular chaperone activity in neurons and proved to be protective in several models for neurodegeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. A common property shared by all mutant SOD1 (mtSOD1) species is abnormal protein folding and the propensity to form aggregates. Toxicity and aggregate formation of mutant SOD1 can be overcome by enhanced chaperone function in vitro. Moreover, expression of mtSOD1 decreases BAG1 levels in a motoneuronal cell line. Thus, several lines of evidence suggested a protective role of BAG1 in mtSOD1-mediated motoneuron degeneration. To explore the therapeutic potential of BAG1 in a model for ALS, we generated SOD1G93A/BAG1 double transgenic mice expressing BAG1 in a neuron-specific pattern. Surprisingly, substantially increased BAG1 protein levels in spinal cord neurons did not significantly alter the phenotype of SOD1G93A-transgenic mice. Hence, expression of BAG1 is not sufficient to protect against mtSOD1-induced motor dysfunction in vivo. Our work shows that, in contrast to the in vitro situation, modulation of multiple cellular functions in addition to enhanced expression of a single chaperone is required to protect against SOD1 toxicity, highlighting the necessity of combined treatment strategies for ALS.

Bcl-2 family proteins and cancer.

BCL-2 was the first anti-death gene discovered, a milestone with far reaching implications for tumor biology. Multiple members of the human Bcl-2 family of apoptosis-regulating proteins have been identified, including six antiapoptotic, three structurally similar proapoptotic proteins and several structurally diverse proapoptotic interacting proteins that operate as upstream agonists or antagonists. These proteins, in turn, are regulated through myriad post-translational modifications and interactions with other proteins. Bcl-2-family proteins regulate all major types of cell death, including apoptosis, necrosis and autophagy, thus operating as nodal points at the convergence of multiple pathways with broad relevance to oncology. Experimental therapies targeting Bcl-2-family mRNAs or proteins are currently in clinical testing, raising hopes that a new class of anticancer drugs may soon be available.

IL-18-producing Salmonella inhibit tumor growth.

Previous studies have shown that intravenously applied bacteria can accumulate in tumors and lead to sporadic tumor regression. Recently, systemic administration of attenuated Salmonella typhimurium was demonstrated to generate no significant side effects in humans, but also no antitumor responses. We report the enhanced antitumor activity in preclinical mouse cancer models of nonvirulent S. typhimurium engineered to synthesize the cytokine Interleukin-18 (IL-18). IL-18-producing bacteria (but not control bacteria) inhibit the growth of primary subcutaneous tumors as well as pulmonary metastases in immunocompetent mice challenged with syngeneic multidrug-resistant clones of murine carcinoma cell lines, without overt toxicity to normal tissues. Antitumor activity was associated with increased accumulation of T-lymphocytes and NK cells in tumors, and massive infiltration of granulocytes, as well as increased intra-tumoral production of several cytokines. In summary, these findings provide evidence of promising preclinical antitumor activity of IL-18-expressing, attenuated S. typhimurium, suggesting a novel strategy for cancer immunotherapy.

Reciprocal negative regulation between S100A7/psoriasin and beta-catenin signaling plays an important role in tumor progression of squamous cell carcinoma of oral cavity.

Overexpression of S100A7 (psoriasin), a small calcium-binding protein, has been associated with the development of psoriasis and carcinomas in different types of epithelia, but its precise functions are still unknown. Using human tissue specimens, cultured cell lines, and a mouse model, we found that S100A7 is highly expressed in preinvasive, well-differentiated and early staged human squamous cell carcinoma of the oral cavity (SCCOC), but little or no expression was found in poorly differentiated, later-staged invasive tumors. Interestingly, our results showed that S100A7 inhibits both SCCOC cell proliferation in vitro and tumor growth/invasion in vivo. Furthermore, we demonstrated that S100A7 is associated with the beta-catenin complex, and inhibits beta-catenin signaling by targeting beta-catenin degradation via a noncanonical mechanism that is independent of GSK3beta-mediated phosphorylation. More importantly, our results also indicated that beta-catenin signaling negatively regulates S100A7 expression. Thus, this reciprocal negative regulation between S100A7 and beta-catenin signaling implies their important roles in tumor development and progression. Despite its high levels of expression in early stage SCCOC tumorigenesis, S100A7 actually inhibits SCCOC tumor growth/invasion as well as tumor progression. Downregulation of S100A7 in later stages of tumorigenesis increases beta-catenin signaling, leading to promotion of tumor growth and tumor progression.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms, physiological roles, and therapeutic opportunities.

Bcl-2-family proteins are central regulators of cell life and death. At least three major classes of Bcl-2-family proteins have been delineated, including proapoptotic proteins that contain several conserved regions of sequence similarity (termed 'multidomain'). In mammals, the multidomain proteins (MDPs) of the Bcl-2 family include Bax, Bak, and Bok. The founding member of the MDP group of Bcl-2-family proteins was discovered by Stanley Korsmeyer and co-workers, initiating an exciting area of cell death research. The status of current knowledge about the mechanisms and functions of MDPs is reviewed here, and some areas for future research are outlined. Therapeutic opportunities emerging from a growing understanding of MDPs with respect to their three-dimensional structures, biochemical actions, and roles in disease raise hopes that the foundation of basic research laid by Korsmeyer and others will eventually be translated into clinical benefits, leaving a legacy that benefits the world for many decades.

Thymidine-phosphorothioate oligonucleotides induce activation and apoptosis of CLL cells independently of CpG motifs or BCL-2 gene interference.

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt.

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways.

The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.