Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor (original) (raw)
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Bcl-2-Protein Family as Modulators of IP3Receptors and Other Organellar Ca2+Channels
Cold Spring Harbor Perspectives in Biology, 2019
The pro-and antiapoptotic proteins belonging to the B-cell lymphoma-2 (Bcl-2) family exert a critical control over cell-death processes by enabling or counteracting mitochondrial outer membrane permeabilization. Beyond this mitochondrial function, several Bcl-2 family members have emerged as critical modulators of intracellular Ca 2+ homeostasis and dynamics, showing proapoptotic and antiapoptotic functions. Bcl-2 family proteins specifically target several intracellular Ca 2+-transport systems, including organellar Ca 2+ channels: inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs), Ca 2+-release channels mediating Ca 2+ flux from the endoplasmic reticulum, as well as voltage-dependent anion channels (VDACs), which mediate Ca 2+ flux across the mitochondrial outer membrane into the mitochondria. Although the formation of protein complexes between Bcl-2 proteins and these channels has been extensively studied, a major advance during recent years has been elucidating the complex interaction of Bcl-2 proteins with IP 3 Rs. Distinct interaction sites for different Bcl-2 family members were identified in the primary structure of IP 3 Rs. The unique molecular profiles of these Bcl-2 proteins may account for their distinct functional outcomes when bound to IP 3 Rs. Furthermore, Bcl-2 inhibitors used in cancer therapy may affect IP 3 R function as part of their proapoptotic effect and/or as an adverse effect in healthy cells. B-CELL LYMPHOMA-2 (Bcl-2) FAMILY OF PROTEINS T he Bcl-2 family of proteins consists of proand antiapoptotic members, which are characterized by the presence of at least one of the four highly conserved α-helical motifs, termed Bcl-2 homology (BH) domains (Adams and Cory 1998). The antiapoptotic family members, such as Bcl-2, Bcl-Xl, and Mcl-1, contain all four BH domains where the BH1, BH2, and BH3 domains form a hydrophobic cleft (Fig. 1A). The hydrophobic cleft is separated from the amino-terminal BH4 domain by an unstructured
2013
Disrupting inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP 3 R-derived peptide (TAT-IDP S )) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca 2 þ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP 3 R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP S in a more heterogeneous Bcl-2-dependent cancer model using a set of 'primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP S to promote IP 3 R-mediated Ca 2 þ release. Although total IP 3 R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP 3 R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP S -induced Ca 2 þ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP 3 R2-protein level, but not with IP 3 R1-, IP 3 R3-or total IP 3 R-expression levels. Inhibiting or knocking down IP 3 R2 activity in SU-DHL-4-reduced TAT-IDP S -induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP 3 R2 and to promote IP 3 -induced pro-apoptotic Ca 2 þ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP 3 R hyperactivity. In particular, cancer cells expressing high levels of IP 3 R2 are addicted to IP 3 R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca 2 þ signaling and cell death.
Cell Death and Differentiation, 2012
Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP 3 R) via its BH4 domain, thereby suppressing IP 3 R Ca 2 þ -flux properties and protecting against Ca 2 þ -dependent apoptosis. Here, we directly compared IP 3 R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP 3 R nor inhibited IP 3 -induced Ca 2 þ release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP 3 R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP 3 R binding and inhibition. This difference in IP 3 R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP 3 Rs. In agreement with the IP 3 R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP 3 R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP 3 Rs and Ca 2 þ -driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca 2 þ signaling and apoptosis.
The FEBS journal, 2018
B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the antiapoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2's antiapoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the center of Bcl-2's BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen proapoptotic Ca2+ -release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability, and function of full-length Bcl-2 as a regulator of Ca2+ signaling and cell death. Our results indicate that full-length Bcl-2 Ile14Gly/Val15Gly, in contrast to wild-type Bcl-2, (a) displayed severely reduced structural stabi...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
Bcl-2 is the founding member of a large family of apoptosis regulating proteins. Bcl-2 is a prime target for novel therapeutics because it is elevated in many forms of cancer and contributes to cancer progression and therapy resistance based on its ability to inhibit apoptosis. Bcl-2 interacts with proapoptotic members of the Bcl-2 family to inhibit apoptosis and small molecules that disrupt this interaction have already entered the cancer therapy arena. A separate function of Bcl-2 is to inhibit Ca 2+ signals that promote apoptosis. This function is mediated through interaction of the Bcl-2 BH4 domain with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca 2+ channel. A novel peptide inhibitor of this interaction enhances proapoptotic Ca 2+ signals. In preliminary experiments this peptide enhanced ABT-737 induced apoptosis in chronic lymphocytic leukemia cells. These findings draw attention to the BH4 domain as a potential therapeutic target. This review summarizes what is currently known about the BH4 domain of Bcl-2, its interaction with the IP3R and other proteins, and the part it plays in Bcl-2's anti-apoptotic function. In addition, we speculate on how the BH4 domain of Bcl-2 can be targeted therapeutically not only for diseases associated with apoptosis resistance, but also for diseases associated with accelerated cell death.
Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
Journal of Peptide Science, 2006
Solid phase synthesis of BH4, the 26 amino-acid domain (6 RTGYDNREIVMKYIHYKLSQRGYEWD 31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15 N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant β-sheeted structures in water that convert progressively into α-helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.
Peptide screening to knockdown Bcl-2's anti-apoptotic activity: Implications in cancer treatment
International Journal of Biological Macromolecules , 2013
Bcl-2 (B cell lymphoma-2) is an anti-apoptotic member of Bcl-2 family and its overexpression causes development of several types of cancer. The BH3 domain of pro-apoptotic and BH3-only proteins is capable of binding to Bcl-2 protein to induce apoptosis. This binding is the basis for the development of novel anticancer drug which would likely antagonize Bcl-2 overexpression. In this study we have identified BH3 domain of Bax (Bax BH3) as potentially the best Bcl-2 antagonist by performing docking of BH3 peptides (peptides representing BH3 domain of pro-apoptotic and BH3-only proteins) into the Bcl-2 hydrophobic groove formed by BH3, BH1 and BH2 domains (also referred as BH3 cleft). To predict the best small antagonist for Bcl-2, three groups of small peptides (pentapeptide, tetrapeptide and tripeptide) were designed and screened against Bcl-2 which revealed the structural importance of a set of residues playing a vital role in interaction with Bcl-2. The docking and scoring function identified KRIG and KRI as specific pep-tides among the screened small peptides responsible for Bcl-2 neutralization and would induce apoptosis. The applied pharmacokinetic and pharmacological filters to all small peptides signify that only IGD has drug-like properties and displayed good oral bioavailability. However, the obtained binding affinity of IGD to Bcl-2 was diminutive. Hence deprotonation, amidation, acetylation, benzoylation, benzylation, and addition of phenyl, deoxyglucose and glucose fragments were performed to increase the binding affinity and to prevent its rapid degradation. Benzoylated IGD tripeptide (IGD bzo) was observed to have increased binding affinity than IGD with acceptable pharmacokinetic filters. In addition, stability of Bcl-2/IGD bzo complex was validated by Molecular Dynamics (MD) simulations revealing improved binding energy, salt bridges and strong interaction energies. This study suggests a new molecule that inhibits Bcl-2 associated cancer/tumor regression.
Journal of Biological Chemistry, 2010
Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosisresistant nature of cancer cells.
Journal of Biological Chemistry, 2010
The anti-apoptotic proteins of the Bcl2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl2 with bilayerreconstituted purified VDAC was demonstrated, with Bcl2 decreasing channel conductance. Expression of Bcl2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration-and time-dependent manner, to immobilized Bcl2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl2 prevented Bcl2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl2-GFPmediated protection against apoptosis. These results point to Bcl2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.