Santeri Kiviluoto

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2s inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. 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.

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.

STIM1 as a key regulator for Ca2+ homeostasis in skeletal-muscle development and function

Stromal interaction molecules (STIM) were identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry (SOCE) and Ca2+-release-activated Ca2+ (CRAC) channels in non-excitable cells. STIM proteins target Orai1-3, tetrameric Ca2+-permeable channels in the plasma membrane. Structure-function analysis revealed the molecular determinants and the key steps in the activation process of Orai by STIM. Recently, STIM1 was found to be expressed at high levels in skeletal muscle controlling muscle function and properties. Novel STIM targets besides Orai channels are emerging.Here, we will focus on the role of STIM1 in skeletal-muscle structure, development and function. The molecular mechanism underpinning skeletal-muscle physiology points toward an essential role for STIM1-controlled SOCE to drive Ca2+/calcineurin/nuclear factor of activated T cells (NFAT)-dependent morphogenetic remodeling programs and to support adequate sarcoplasmic-reticulum (SR) Ca2+-store filling. Also in our hands, STIM1 is transiently up-regulated during the initial phase of in vitro myogenesis of C2C12 cells. The molecular targets of STIM1 in these cells likely involve Orai channels and canonical transient receptor potential (TRPC) channels TRPC1 and TRPC3. The fast kinetics of SOCE activation in skeletal muscle seem to depend on the triad-junction formation, favoring a pre-localization and/or pre-formation of STIM1-protein complexes with the plasma-membrane Ca2+-influx channels. Moreover, Orai1-mediated Ca2+ influx seems to be essential for controlling the resting Ca2+ concentration and for proper SR Ca2+ filling. Hence, Ca2+ influx through STIM1-dependent activation of SOCE from the T-tubule system may recycle extracellular Ca2+ losses during muscle stimulation, thereby maintaining proper filling of the SR Ca2+ stores and muscle function. Importantly, mouse models for dystrophic pathologies, like Duchenne muscular dystrophy, point towards an enhanced Ca2+ influx through Orai1 and/or TRPC channels, leading to Ca2+-dependent apoptosis and muscle degeneration. In addition, human myopathies have been associated with dysfunctional SOCE. Immunodeficient patients harboring loss-of-function Orai1 mutations develop myopathies, while patients suffering from Duchenne muscular dystrophy display alterations in their Ca2+-handling proteins, including STIM proteins. In any case, the molecular determinants responsible for SOCE in human skeletal muscle and for dysregulated SOCE in patients of muscular dystrophy require further examination.

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.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

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 ...

The transmembrane Bax inhibitor motif (TMBIM) containing protein family: Tissue expression, intracellular localization and effects on the ER CA2+-filling state☆

Bax inhibitor-1 (BI-1) is an evolutionarily conserved pH-dependent Ca²⁺ leak channel in the endoplasmic reticulum and the founding member of a family of six highly hydrophobic mammalian proteins named transmembrane BAX inhibitor motif containing (TMBIM) 1-6 with BI-1 being TMBIM6. Here we compared the structure, subcellular localization, tissue expression and the effect on the cellular Ca²⁺ homeostasis of all family members side by side. We found that all TMBIM proteins possess the di-aspartyl pH sensor responsible for pH sensing identified in TMBIM6 and its bacterial homologue BsYetJ. TMBIM1-3 and TMBIM4-6 represent two phylogenetically distinct groups that are localized in the Golgi apparatus (TMBIM1-3), endoplasmic reticulum (TMBIM4-6) or mitochondria (TMBIM5) but share a common structure of at least seven transmembrane domains with the last domain being semi-hydrophobic. TMBIM1 is mainly expressed in muscle, TMBIM2 and 3 in the nervous system, TMBIM4 and 5 are ubiquitously expressed and TMBIM6 in skeletal muscle, kidney, liver and spleen. All TMBIM proteins reduce the Ca²⁺ content of the endoplasmic reticulum, and all but TMBIM5 also reduce the cytosolic resting Ca²⁺ concentration. These results suggest that the TMBIM family has comparable functions in the maintenance of intracellular Ca²⁺ homeostasis in a wide variety of tissues. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Bax Inhibitor-1-mediated Ca2+ leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1s Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca(2+)-channel pore-dead mutant BI-1 (BI-1(D213R)) was developed. We determined whether BI-1 behaves as a bona fide H+/Ca2+ antiporter or as an ER Ca(2+)-leak channel by investigating the effect of pH on unidirectional Ca(2+)-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1(-/-) cells increased the ER Ca(2+)-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1(D231R) expression in BI-1(-/-), despite its ER localization, did not increase the ER Ca(2+)-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca2+ leak was blocked. Finally, a peptide representing the Ca(2+)-channel pore of BI-1 promoting Ca2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca(2+)-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.

Bax Inhibitor-1 Is Likely a pH-Sensitive Calcium Leak Channel, Not a H+/Ca2+ Exchanger

The structure of a bacterial Bax inhibitor-1 homolog suggests that Bax inhibitor-1 is an ER-localized Ca2+ leak channel tightly controlled by the intracellular pH. The endoplasmic reticulum (ER) plays a key role in the synthesis, folding, and sorting of proteins, and disturbances of this delicate system can cause cell death. The ER also serves as the major intracellular calcium (Ca2+) store, and release of Ca2+ from this store controls diverse cellular functions. At the interface of both these functions of the ER is Bax inhibitor-1 (BI-1), an evolutionarily conserved multifunctional protein that mediates Ca2+ efflux from the ER and protects against ER stress. Several mechanisms have been proposed to explain how BI-1 might mediate Ca2+ efflux from the ER. Chang et al. present structural evidence that a bacterial homolog of BI-1, BsYetJ, is a pH-sensitive Ca2+ leak channel. This finding not only sheds a new light on ER Ca2+ efflux mediated by BI-1, but also provides a tentative function for other members of the BI-1 protein family.