A single-center study of bone mineral density in adult patients with severe hemophilia A in correlation with markers of bone metabolism.

INTRODUCTION: Osteopenia and osteoporosis are well-known hemophilia A comorbidities. The pathogenesis of bone turnover alteration resulting in reduced bone mass includes impaired osteoblastic differentiation and disinhibition of RANKL-induced osteoclastogenesis as a result of a low FVIII level. AIM: To evaluate the bone mineral density (BMD) in adult patients with severe hemophilia A and assess a possible correlation with the bone remodeling biomarkers OPG/RANKL, CTX-1, osteocalcin, and Vit D. MATERIALS AND METHODS: 28 male subjects with severe hemophilia A and 33 age-matched controls were recruited. The biomarkers were tested with the ELISA assay and BMD with DEXA of the lumbar spine (LS) and total hip (TH). RESULTS: The patients had lower LS-BMD (-0.955±0.145 vs. 1.118±0.079, p=0.05) and TH-BMD (-0.840±0.147 vs. 0.951±0.075, p=0.05) than those of the controls. The TH T-scores were -1.41±0.91 vs. 0.4±0.49 (p=0.05) and the LS T-scores -1.16±1.046 vs. 0.14±0.72 (p=0.05). 66.6% of patients under 50 years had osteopenia and 8.3% had osteoporosis. Fifty percent of those over 50 years old had osteopenia and 20% had osteoporosis. We found significantly higher OPG levels (123.69±107.05 vs. 41.98±18.95, p=0.05) than that in controls and lower sRANKL levels (23.49±29.39 vs. 131.32±201.27, p=0.05) and sRANKL/OPG ratio (0.27±0.35 vs. 5.28±10.01, p=0.05) than those in controls. A positive correlation was found between sRANKL and the BMD T-score of lumbar spine (p=0.001) in the patient group. CONCLUSIONS: sRANKL level and ratio can be used as predictors of low BMD.

Allosteric cross-talk between the hydrophobic cleft and the BH4 domain of Bcl-2 in control of inositol 1,4,5-trisphosphate receptor activity.

Aim: Inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous calcium (Ca2+) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP3R-independent action at the mitochondria (Oncotarget. 2016;7:55704-20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IP3R:Bcl-2 interaction. Methods: Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IP3R:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction. Results: It was shown that occlusion of Bcl-2's hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability (Po) regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2. Conclusions: Bcl-2 is an important regulator of IP3R activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IP3R activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL's inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL's anti-apoptotic function.

Tracing the evolutionary history of Ca2+-signaling modulation by human Bcl-2: Insights from the Capsaspora owczarzaki IP3 receptor ortholog.

Recently, a functional IP3R ortholog (CO.IP3R-A) capable of IP3-induced Ca2+ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP3Rs, CO.IP3R-A is not modulated by Ca2+, ATP or PKA. Protein-sequence analysis revealed that CO.IP3R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP3R-A channels and modulate their Ca2+-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP3R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP3R-A, thereby suppressing Ca2+ flux through CO.IP3R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP3R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP3Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP3R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP3R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP3R function by exploiting this pre-existing site, thereby counteracting Ca2+-dependent apoptosis.

Bcl-2-Protein Family as Modulators of IP3 Receptors and Other Organellar Ca2+ Channels.

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 Ca2+ homeostasis and dynamics, showing proapoptotic and antiapoptotic functions. Bcl-2 family proteins specifically target several intracellular Ca2+-transport systems, including organellar Ca2+ channels: inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), Ca2+-release channels mediating Ca2+ flux from the endoplasmic reticulum, as well as voltage-dependent anion channels (VDACs), which mediate Ca2+ 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 IP3Rs. Distinct interaction sites for different Bcl-2 family members were identified in the primary structure of IP3Rs. The unique molecular profiles of these Bcl-2 proteins may account for their distinct functional outcomes when bound to IP3Rs. Furthermore, Bcl-2 inhibitors used in cancer therapy may affect IP3R function as part of their proapoptotic effect and/or as an adverse effect in healthy cells.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Pathophysiological consequences of isoform-specific IP3 receptor mutations.

Ca2+ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca2+-signaling network is the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a tetrameric channel that mediates Ca2+ release from the endoplasmic reticulum (ER) in response to IP3. IP3R function is controlled by a myriad of factors, such as Ca2+, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP3R-mediated Ca2+ signaling is the result of the existence of three main isoforms (IP3R1, IP3R2 and IP3R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP3R1 is highly expressed in neurons, IP3R2 in cardiomyocytes and hepatocytes and IP3R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP3R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP3R1 to dysfunctional exocrine tissues and autoimmune diseases for IP3R2 and -3. Over the past years, several IP3R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP3R isoform and the subsequent molecular mechanisms underlying the etiology of the disease.

Mutations Associated with Imatinib Mesylate Resistance - Review.

Chronic myeloid leukemia (CML) arises from the fusion of the BCR and the ABL1 genes. The BCR gene (chromosome 22q11.2) and the ABL1 gene (chromosome 9q34) fuse together due to reciprocal chromosome translocation forming the Philadelphia chromosome (Ph). This fusion gene codes tyrosine kinase which accelerates the cell division and reduces DNA repair. Imatinib mesylate is a selective inhibitor of this tyrosine kinase. It is the first-line treatment for CML-patients. However, it became clear that Philadelphia-positive (Ph+) cells could evolve to elude inhibition due to point mutations within the BCR-ABL kinase domain. To date more than 40 mutations have been identified and their early detection is important for clinical treatment. With the development of the new tyrosine kinase inhibitors (TKIs), associated with these mutations, the resistance problem seems to diminish, as some of the new drugs are less prone to resistance. The aim of this review is to focus on the diff erent mutations leading to resistance.

A double point mutation at residues Ile14 and Val15 of Bcl-2 uncovers a role for the BH4 domain in both protein stability and function.

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 stability and a shortened protein half-life; (b) failed to interact with Bcl-2-associated X protein (BAX), to inhibit the inositol 1,4,5-trisphosphate receptor (IP3 R) and to protect against Ca2+ -mediated apoptosis. We conclude that the hydrophobic face of Bcl-2's BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2's ability to modulate the function of its targets, like IP3 R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain.

Endoplasmic Reticulum-Mitochondrial Ca2+ Fluxes Underlying Cancer Cell Survival.

Calcium ions (Ca2+) are crucial, ubiquitous, intracellular second messengers required for functional mitochondrial metabolism during uncontrolled proliferation of cancer cells. The mitochondria and the endoplasmic reticulum (ER) are connected via "mitochondria-associated ER membranes" (MAMs) where ER-mitochondria Ca2+ transfer occurs, impacting the mitochondrial biology related to several aspects of cellular survival, autophagy, metabolism, cell death sensitivity, and metastasis, all cancer hallmarks. Cancer cells appear addicted to these constitutive ER-mitochondrial Ca2+ fluxes for their survival, since they drive the tricarboxylic acid cycle and the production of mitochondrial substrates needed for nucleoside synthesis and proper cell cycle progression. In addition to this, the mitochondrial Ca2+ uniporter and mitochondrial Ca2+ have been linked to hypoxia-inducible factor 1α signaling, enabling metastasis and invasion processes, but they can also contribute to cellular senescence induced by oncogenes and replication. Finally, proper ER-mitochondrial Ca2+ transfer seems to be a key event in the cell death response of cancer cells exposed to chemotherapeutics. In this review, we discuss the emerging role of ER-mitochondrial Ca2+ fluxes underlying these cancer-related features.

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.

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels.

Ca2+ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP3R), i.e. the main intracellular Ca2+ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP3Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP3R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP3R1 in GST-pull downs and their overall ability to inhibit IP3-induced Ca2+ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a "humanized" version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP3R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP3R channels.

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca2+ signaling.

Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca2+ homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca2+-transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1µM) did neither trigger Ca2+ release by itself nor affect agonist-induced Ca2+ release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca2+ signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca2+ overload, since Ca2+ buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca2+ homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca2+ overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition.

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2ß, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

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 Ca(2+)-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 Ca(2+) 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-XL(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca(2+) 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.

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.

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.

Profiling of the Bcl-2/Bcl-X(L)-binding sites on type 1 IP(3) receptor.

Several members of the anti-apoptotic Bcl-2-protein family, including Bcl-2, Bcl-X(L) and Mcl-1, directly bind and regulate the inositol 1,4,5-trisphosphate receptor (IP(3)R), one of the two main intracellular Ca(2+)-release channel types present in the endoplasmic reticulum. However, the molecular determinants underlying their binding to the IP(3)R remained a matter of debate. One interaction site for Bcl-2 was proposed in the central part of the modulatory domain [Y.P. Rong, A.S. Aromolaran, G. Bultynck, F. Zhong, X. Li, K. McColl, S. Matsuyama, S. Herlitze, H.L. Roderick, M.D. Bootman, G.A. Mignery, J.B. Parys, H. De Smedt, C.W. Distelhorst, Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals, Mol. Cell 31 (2008) 255-265] and another site in the C-terminal domain of the IP(3)R encompassing the sixth transmembrane domain, to which Bcl-2, Bcl-X(L) and Mcl-1 can bind [E.F. Eckenrode, J. Yang, G.V. Velmurugan, J.K. Foskett, C. White, Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca(2+) signaling, J. Biol. Chem. 285 (2010) 13678-13684]. Here, we investigated and compared the binding of Bcl-2 and Bcl-X(L) to both sites. Two different IP(3)R domains were used for the C-terminal site: one lacking and one containing the sixth transmembrane domain. Our results show that elements preceding the C-terminal cytosolic tail located at the sixth transmembrane domain of IP(3)R1 were critical for recruiting both Bcl-2 and Bcl-X(L) to the C-terminal part of the IP(3)R. Furthermore, consistent with our previous observations, Bcl-X(L) bound with higher efficiency to the C-terminal part of the IP(3)R and to a much lesser extent to the central, modulatory domain, while Bcl-2 targeted both sites with similar efficiencies. In conclusion, IP(3)R harbors two different binding sites for anti-apoptotic Bcl-2 proteins, one in the central, modulatory domain and one in the C-terminal domain near the Ca(2+)-channel pore.

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.