Hristina Ivanova

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.

The C Terminus of Bax Inhibitor-1 Forms a Ca2+-permeable Channel Pore

Background: Evolutionary conserved Bax inhibitor-1 (BI-1) protects against ER stress-mediated apoptosis. Results: We identified a Ca2+-permeable channel pore in the C terminus of BI-1. Critical pore properties are an α-helical structure and two aspartate residues conserved among animals, but not among plants and yeast. Conclusion: C-terminal domain of BI-1 harbors a Ca2+-permeable channel pore. Significance: BI-1 has Ca2+ channel properties likely relevant for its function in ER stress and apoptosis. 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 Ca2+ homeostasis by acting as a Ca2+-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca2+ pore responsible for its Ca2+-leak properties. We utilized a set of C-terminal peptides to screen for Ca2+ leak activity in unidirectional 45Ca2+-flux experiments and identified an α-helical 20-amino acid peptide causing Ca2+ leak from the ER. The Ca2+ leak was independent of endogenous ER Ca2+-release channels or other Ca2+-leak mechanisms, namely translocons and presenilins. The Ca2+-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca2+-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 Ca2+-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca2+-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca2+ leak. Thus, we elucidated residues critically important for BI-1-mediated Ca2+ 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 Ca2+-leak properties of BI-1 are an added function during evolution.

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-XL-binding sites on type 1 IP3 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-2s 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.

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.

The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP 3 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-2s 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-2β, 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-2s 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.

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 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.

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 (IP3R) 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 IP3R 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.