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           Search results for: Recombinant Human Bcl-2 -BH3 Proteins    

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#28807790   2017/08/15 Save this To Up

The functional domains for Bax∆2 aggregate-mediated caspase 8-dependent cell death.

Bax∆2 is a functional pro-apoptotic Bax isoform having alterations in its N-terminus, but sharing the rest of its sequence with Baxα. Bax∆2 is unable to target mitochondria due to the loss of helix α1. Instead, it forms cytosolic aggregates and activates caspase 8. However, the functional domain(s) responsible for BaxΔ2 behavior have remained elusive. Here we show that disruption of helix α1 makes Baxα mimic the behavior of Bax∆2. However, the other alterations in the Bax∆2 N-terminus have no significant impact on aggregation or cell death. We found that the hallmark BH3 domain is necessary but not sufficient for aggregation-mediated cell death. We also noted that the core region shared by Baxα and Bax∆2 is required for the formation of large aggregates, which is essential for BaxΔ2 cytotoxicity. However, aggregation by itself is unable to trigger cell death without the C-terminus. Interestingly, the C-terminal helical conformation, not its primary sequence, appears to be critical for caspase 8 recruitment and activation. As Bax∆2 shares core and C-terminal sequences with most Bax isoforms, our results not only reveal a structural basis for Bax∆2-induced cell death, but also imply an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members.

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#26858413   2016/02/24 Save this To Up

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

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#26494789   2015/10/23 Save this To Up

Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells.

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

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#25314294   2014/12/23 Save this To Up

Lipid-dependent bimodal MCL1 membrane activity.

Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors. Contrary to currently popular models, MCL1ΔNΔC did not require becoming embedded into the membrane to inhibit membrane permeabilization, and interaction with cBID was more productive for MCL1ΔNΔC inhibitory activity than interaction with BAX. We also report that membranes rich in cardiolipin (CL), but not phosphatidylinositol (PI), trigger a profound conformational change in MCL1ΔNΔC leading to membrane integration and unleashment of an intrinsic lipidic pore-forming activity of the molecule. Cholesterol (CHOL) reduces both the conformational change and the lipidic pore-forming activity of MCL1ΔNΔC in CL-rich membranes, but it does not affect the interaction of MCL1ΔNΔC with proapoptotic partners in MOM-like liposomes. In addition, we identified MCL1α5 as the minimal domain of the protein responsible for its membrane-permeabilizing function both in model membranes and at the mitochondrial level. Our results provide novel mechanistic insight into MCL1 function in the context of a membrane milieu and add significantly to a growing body of evidence supporting an active role of mitochondrial membrane lipids in BCL2 protein function.

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#25211642   2014/12/23 Save this To Up

Direct binding of Bcl-2 family proteins by quercetin triggers its pro-apoptotic activity.

Bcl-2 family proteins are important regulators of apoptosis and its antiapoptotic members, which are overexpressed in many types of cancer, are of high prognostic significance, establishing them as attractive therapeutic targets. Quercetin, a natural flavonoid, has drawn much attention because it exerts anticancer effects, while sparing normal cells. A multidisciplinary approach has been employed herein, in an effort to reveal its mode of action including dose-response antiproliferative activity and induced apoptosis effect, biochemical and physicochemical assays, and computational calculations. It may be concluded that, quercetin binds directly to the BH3 domain of Bcl-2 and Bcl-xL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis.

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#24316551   2013/12/09 Save this To Up

Combining the antimesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cells.

SS1P is an antimesothelin recombinant immunotoxin (RIT). Pancreatic ductal adenocarcinoma (PDAC) cell lines are resistant to SS1P, despite high mesothelin expression. The aim of this study is to examine whether combining SS1P and BH3-mimetic ABT-737 induces cell death in a panel of PDAC cell lines. ABT-737 binds and neutralizes several antiapoptotic BCL2 family proteins, but has a low affinity for the short-lived MCL1 and BCL2A1. SS1P inhibits protein synthesis, which has shown to downregulate MCL1. PDAC cell lines KLM-1, BxPc-3, and Panc 3.014 were resistant to SS1P or ABT-737 alone. Combining both compounds led to a significant increase in cell death. After 48 hours of treatment, cell death was observed in 92% of KLM-1, 55% of BxPc-3, and 23% of Panc 3.014 cells. Panc 3.014 had the highest number of mesothelin-binding sites (92×10(3)), followed by KLM-1 (58×10(3)) and BxPc-3 (3×10(3)). ABT-737 had no effect on SS1P internalization, but enhanced SS1P-induced protein synthesis inhibition significantly in KLM-1, to a lesser extent in BxPc-3, and very little in Panc 3.014. SS1P alone or in combination with ABT-737 downregulated MCL1 in KLM-1 and BxPc-3, but not in Panc 3.014. Similar observations were made for BCL2A1, which had the highest levels in Panc 3.014. Compared with KLM-1, Panc 3.014, and BxPc-3 also had lower proapoptotic BAK and a trend toward higher MCL1. Proapoptotic BAX was similar in KLM-1 and BxPc-3, but lower in Panc 3.014. In conclusion, combining SS1P with ABT-737 overcomes SS1P-resistance in PDAC, although to a variable extent. The efficacy of the combination is mainly associated with the RIT-associated inhibition of protein synthesis and the ability to downregulate MCL1 and BCL2A1, while levels of other key apoptotic proteins may also be important. Our data support the combination of an RIT and a BH3-mimetic, and identify factors that potentially limit the efficacy of such therapeutic approach.

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#23996493   2013/09/02 Save this To Up

Biophysical basis of the promiscuous binding of B-cell lymphoma protein 2 apoptotic repressor to BH3 ligands.

B-cell lymphoma protein 2 (Bcl2) apoptotic repressor carries out its function by virtue of its ability to bind to BH3 domains of various pro-apoptotic regulators in a highly promiscuous manner. Herein, we investigate the biophysical basis of such promiscuity of Bcl2 toward its cognate BH3 ligands. Our data show that although the BH3 ligands harboring the LXXXAD motif bind to Bcl2 with submicromolar affinity, those with the LXXX[G/S]D motif afford weak interactions. This implies that the replacement of alanine at the fourth position (A + 4)-relative to the N-terminal leucine (L0) within the LXXXAD motif-to glycine/serine results in the loss of free energy of binding. Consistent with this notion, the A + 4 residue within the BH3 ligands harboring the LXXXAD motif engages in key intermolecular van der Waals contacts with A149 lining the ligand binding groove within Bcl2, whereas A + 4G/S substitution results in the disruption of such favorable binding interactions. Of particular interest is the observation that although increasing ionic strength has little or negligible effect on the binding of high-affinity BH3 ligands harboring the LXXXAD motif, the binding of those with the LXXX[G/S]D motif in general experiences a varying degree of enhancement. This salient observation is indicative of the fact that hydrophobic forces not only play a dominant but also a universal role in driving the Bcl2-BH3 interactions. Taken together, our study sheds light on the molecular basis of the factors governing the promiscuous binding of Bcl2 to pro-apoptotic regulators and thus bears important consequences on the development of rational therapeutic approaches.

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#22174825   2011/12/16 Save this To Up

Latent Epstein-Barr virus can inhibit apoptosis in B cells by blocking the induction of NOXA expression.

Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt's lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency-associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents--ionomycin and staurosporine--and show that these act by a distinct pathway that involves a p53-independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus--that encodes the BCL2-homologue BHRF1 and three microRNAs--partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV-mediated block to NOXA accumulation.

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#20942963   2010/10/26 Save this To Up

The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein.

Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood.

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#20704564   2010/10/12 Save this To Up

Identification of the BCL2/adenovirus E1B-19K protein-interacting protein 2 (BNIP-2) as a granzyme B target during human natural killer cell-mediated killing.

Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin-mediated delivery of pro-apoptotic serine proteases known as granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. A simple bioinformatics strategy identified a candidate granzyme B cleavage site in the widely expressed BNIP-2 (BCL2/adenovirus E1B-19K protein-interacting protein 2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK (natural killer) cell-mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full-length BNIP-2 or a truncated molecule mimicking the granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID (BH3-interacting domain death agonist) expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximizes the chances of immune-mediated clearance of infected cells or tumour cells.

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