Tim Vervliet

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP3R) via its BH4 domain, thereby suppressing IP3R Ca2+-flux properties and protecting against Ca2+-dependent apoptosis. Here, we directly compared IP3R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP3R nor inhibited IP3-induced Ca2+ 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 IP3R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP3R binding and inhibition. This difference in IP3R 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 IP3Rs. In agreement with the IP3R-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 IP3R, 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 IP3Rs and Ca2+-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca2+ signaling and apoptosis.

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival.

Cell-death and -survival decisions are critically controlled by intracellular Ca(2+) homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in these processes by mediating Ca(2+) flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca(2+) signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. In this review, we will focus on how the different IP3R isoforms (IP3R1, IP3R2 and IP3R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP3Rs by cellular factors like IP3, Ca(2+), Ca(2+)-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP3R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca(2+) store in cell death and survival and how IP3Rs and pro-survival/pro-death proteins can modulate the basal ER Ca(2+) leak. Third, we will review the regulation of the Ca(2+)-flux properties of the IP3R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP3R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Bcl-2 proteins and calcium signaling: complexity beneath the surface

Antiapoptotic Bcl-2-family members are well known for their ‘mitochondrial’ functions as critical neutralizers of proapoptotic Bcl-2-family members, including the executioner multidomain proteins Bax and Bak and the BH3-only proteins. It has been clear for more than 20 years that Bcl-2 proteins can impact intracellular Ca2+ homeostasis and dynamics. Moreover, altered Ca2+ signaling is increasingly linked to oncogenic behavior. Specifically targeting the Ca2+-signaling machinery may thus prove to be a valuable strategy for cancer treatment. Over 10 years ago a major controversy was recognized concerning whether or not Bcl-2 proteins exerted their antiapoptotic functions via Ca2+ signaling through lowering the filling state of the endoplasmic reticulum (ER) Ca2+ stores or by suppressing Ca2+ release from the ER without affecting the filling state of this Ca2+ store. Further research from different laboratories indicated a wide variety of mechanisms by which Bcl-2-family members can impact Ca2+ signaling. In this review, we propose that antiapoptotic Bcl-2-family members are multimodal regulators of intracellular Ca2+-signaling events in cell survival and cell death. We will discuss how different Bcl-2-family members impact cell survival and cell death by regulating Ca2+ transport systems at the ER, mitochondria and plasma membrane and by impacting the organization of organelles and how these insights can be exploited for causing cell death in cancer cells. Finally, we propose that the existing controversy reflects the diversity of links between Bcl-2 proteins and Ca2+ signaling, as certainly not all targets or mechanisms will be operative in every cell type and every condition.

IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDPS)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca2+ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDPS 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-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS to promote IP3R-mediated Ca2+ release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDPS-induced Ca2+ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDPS-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca2+ 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 IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca2+ signaling and cell death.

Regulation of inositol 1,4,5-trisphosphate receptors during endoplasmic reticulum stress.

The endoplasmic reticulum (ER) performs multiple functions in the cell: it is the major site of protein and lipid synthesis as well as the most important intracellular Ca(2+) reservoir. Adverse conditions, including a decrease in the ER Ca(2+) level or an increase in oxidative stress, impair the formation of new proteins, resulting in ER stress. The subsequent unfolded protein response (UPR) is a cellular attempt to lower the burden on the ER and to restore ER homeostasis by imposing a general arrest in protein synthesis, upregulating chaperone proteins and degrading misfolded proteins. This response can also lead to autophagy and, if the stress can not be alleviated, to apoptosis. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and IP3-induced Ca(2+) signaling are important players in these processes. Not only is the IP3R activity modulated in a dual way during ER stress, but also other key proteins involved in Ca(2+) signaling are modulated. Changes also occur at the structural level with a strengthening of the contacts between the ER and the mitochondria, which are important determinants of mitochondrial Ca(2+) uptake. The resulting cytoplasmic and mitochondrial Ca(2+) signals will control cellular decisions that either promote cell survival or cause their elimination via apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Bax Inhibitor-1 is a novel IP3 receptor-interacting and -sensitizing protein

Dear Editor, Bax Inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum (ER)-located protein that protects against ER stress-induced apoptosis.1 This function has been closely related to its ability to permeate Ca2+ from the ER2 and to lower the steady-state [Ca2+]ER.3 BI-1 may function as an H+/Ca2+-antiporter2 or Ca2+ channel.4 Recently, BI-1 was proposed as a negative regulator of autophagy through IRE1α.5 However, recent findings indicate that BI-1 may promote autophagy.6 The latter required the presence of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observations were explained through BI-1-enhanced IP3R activity, which lowered steady-state [Ca2+]ER, reducing ER-mitochondrial Ca2+ transfer and decreasing mitochondrial bio-energetics.7 However, direct evidence that BI-1 binds to IP3Rs and sensitizes IP3-induced Ca2+ release (IICR) is lacking. Therefore, we studied the regulation of IP3R function by BI-1 (see Supplementary Information for Methods). We constructed a 5xMyc-BI-1-expression plasmid, allowing the detection and purification of ectopically expressed BI-1 from transfected HeLa cells using anti-Myc-agarose beads (Figure 1a). Using isoform-specific IP3R antibodies, we demonstrated the co-immunoprecipitation of IP3R1 and IP3R3 with 5xMyc-BI-1 from HeLa cell lysates. Next, we screened for the subdomain of BI-1 responsible for IP3R interaction. We found that a synthetic Flag-tagged peptide containing BI-1s Ca2+-channel pore domain (CTP1; amino acids 198–217 of human BI-1) interacted with IP3R1 (Figure 1b). Lysates not exposed to Flag-CTP1 served as negative control. Moreover, proteolytic fragments of the IP3R containing its C terminus (indicated as IP3R1-Cterm in Figure 1b) were immunoprecipitated with Flag-CTP1. These C-terminal fragments were recognized by our antibody (Rbt03) that has its epitope in the last 15 C-terminal amino acids of the IP3R1.8 These fragments include the Ca2+-channel pore of the IP3R1, indicating that the Ca2+-channel pore domain of BI-1 interacted with the Ca2+-channel pore domain of IP3R1. Next, we examined the effect of BI-1 on IP3R function. Therefore, we used BI-1−/− mouse embryonic fibroblasts (MEF) and stably and ectopically overexpressed either empty vector (RFP-only), wild-type BI-1 or BI-1D213R with a bi-cistronic C-terminal IRES-RFP reporter. BI-1D213R is a mutant, in which the Asp213 critical for BI-1-mediated Ca2+ flux is altered into an Arg and which fails to lower [Ca2+]ER.4 BI-1-mRNA expression was detected using specific primers, and similar expression levels were found for wild-type BI-1 and BI-1D213R, while no signal was observed in vector-expressing BI-1−/− MEF cells (inset Figure 1c). Wild-type BI-1, but not BI-1D213R, overexpression significantly improved cell survival after thapsigargin exposure, an irreversible SERCA inhibitor, which kills cells through ER stress (empty vector: 33.65±4.48% wild-type BI-1: 44.39±5.31%* BI-1D213R: 34.14±4.19% surviving cells after 48 h, 20 nM thapsigargin normalized to vehicle-treated cells expressing empty vector. Mean±S.E.M. of four pooled experiments done in triplicates is shown, *P<0.05 Students t-test). These data indicate that BI-1s Ca2+-flux properties are essential for BI-1s anti-apoptotic function. Next, we analyzed the direct effect of ectopically expressed BI-1 on IP3R function in the absence of endogenous BI-1 (Figure 1c). We used a unidirectional 45Ca2+-flux assay in saponin-permeabilized BI-1−/− MEF cells, allowing direct ER access and an accurate analysis of IP3R function in the absence of plasmalemmal Ca2+ fluxes, SERCA activity or mitochondrial Ca2+ uptake.8 Cells ectopically overexpressing BI-1 displayed a sensitized IICR and concomitant decrease in EC50 from 3.57 μM to 2.25 μM IP3. To exclude that Ca2+ flux mediated by BI-1 indirectly sensitized IP3Rs through Ca2+-induced Ca2+ release, we examined the effect of BI-1D213R overexpression on IP3R function. BI-1D213R also sensitized IICR and concomitantly decreased the EC50 from 3.57 μM to 1.98 μM IP3. This correlates with the ability of BI-1D213R to co-immunoprecipitate with IP3Rs (Figure 1a). Collectively, these data indicate a direct sensitizing effect of BI-1 on IP3Rs, which may contribute to a decrease in steady-state [Ca2+]ER and mitochondrial bioenergetics and subsequent induction of basal autophagy. Figure 1 (a) Interaction of 5xMyc-BI-1 and 5xMyc-BI-1D213R with IP3R channels. BI-1 and BI-1D213R were expressed as 5xMyc-tagged fusion proteins. The empty 5xMyc vector was used as negative control. The vectors were transfected into HeLa cells for 2 days allowing ...

Bcl-2 binds to and inhibits ryanodine receptors.

ABSTRACT The anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein not only counteracts apoptosis at the mitochondria by scaffolding pro-apoptotic Bcl-2-family members, but also acts at the endoplasmic reticulum, thereby controlling intracellular Ca2+ dynamics. Bcl-2 inhibits Ca2+ release by targeting the inositol 1,4,5-trisphosphate receptor (IP3R). Sequence analysis has revealed that the Bcl-2-binding site on the IP3R displays strong similarity with a conserved sequence present in all three ryanodine receptor (RyR) isoforms. We now report that Bcl-2 co-immunoprecipitated with RyRs in ectopic expression systems and in native rat hippocampi, indicating that endogenous RyR–Bcl-2 complexes exist. Purified RyR domains containing the putative Bcl-2-binding site bound full-length Bcl-2 in pulldown experiments and interacted with the BH4 domain of Bcl-2 in surface plasmon resonance (SPR) experiments, suggesting a direct interaction. Exogenous expression of full-length Bcl-2 or electroporation loading of the BH4 domain of Bcl-2 dampened RyR-mediated Ca2+ release in HEK293 cell models. Finally, introducing the BH4-domain peptide into hippocampal neurons through a patch pipette decreased RyR-mediated Ca2+ release. In conclusion, this study identifies Bcl-2 as a new inhibitor of RyR-based intracellular Ca2+-release channels.

The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond.

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca2+ signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca2+ transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca2+ oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2’s determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca2+-channel tail and might be more crucial for Bcl-XL’s function. Furthermore, one amino acid critically different in the sequence of Bcl-2’s and Bcl-XL’s BH4 domains underpins their selective effect on Ca2+ signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain’s binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2’s proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.

Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca2+-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca2+ release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XLK87D mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca2+ release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction.

Modulation of Ca2+ Signaling by Anti-apoptotic B-Cell Lymphoma 2 Proteins at the Endoplasmic Reticulum–Mitochondrial Interface

Mitochondria are important regulators of cell death and cell survival. Mitochondrial Ca2+ levels are critically involved in both of these processes. On the one hand, excessive mitochondrial Ca2+ leads to Ca2+-induced mitochondrial outer membrane permeabilization and thus apoptosis. On the other hand, mitochondria need Ca2+ in order to efficiently fuel the tricarboxylic acid cycle and maintain adequate mitochondrial bioenergetics. For obtaining this Ca2+, the mitochondria are largely dependent on close contact sites with the endoplasmic reticulum (ER), the so-called mitochondria-associated ER membranes. There, the inositol 1,4,5-trisphosphate receptors are responsible for the Ca2+ release from the ER. It comes as no surprise that this Ca2+ release from the ER and the subsequent Ca2+ uptake at the mitochondria are finely regulated. Cancer cells often modulate ER-Ca2+ transfer to the mitochondria in order to promote cell survival and to inhibit cell death. Important regulators of these Ca2+ signals and the onset of cancer are the B-cell lymphoma 2 (Bcl-2) family of proteins. An increasing number of reports highlight the ability of these Bcl-2-protein family members to finely regulate Ca2+ transfer from ER to mitochondria both in healthy cells and in cancer. In this review, we focus on recent insights into the dynamic regulation of ER-mitochondrial Ca2+ fluxes by Bcl-2-family members and how this impacts cell survival, cell death and mitochondrial energy production.