Tomas Luyten

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.

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.

mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

Autophagy is a lysosomal degradation pathway important for cellular homeostasis and survival. Inhibition of the mammalian target of rapamycin (mTOR) is the best known trigger for autophagy stimulation. In addition, intracellular Ca2+ regulates autophagy, but its exact role remains ambiguous. Here, we report that the mTOR inhibitor rapamycin, while enhancing autophagy, also remodeled the intracellular Ca2+-signaling machinery. These alterations include a) an increase in the endoplasmic-reticulum (ER) Ca2+-store content, b) a decrease in the ER Ca2+-leak rate, and c) an increased Ca2+ release through the inositol 1,4,5-trisphosphate receptors (IP3Rs), the main ER-resident Ca2+-release channels. Importantly, buffering cytosolic Ca2+ with BAPTA impeded rapamycin-induced autophagy. These results reveal intracellular Ca2+ signaling as a crucial component in the canonical mTOR-dependent autophagy pathway.

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.

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 ...

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.

Alzheimer's disease-associated peptide Aβ42 mobilizes ER Ca2+ via InsP3R-dependent and -independent mechanisms

Dysregulation of Ca2+ homeostasis is considered to contribute to the toxic action of the Alzheimers disease (AD)-associated amyloid-β-peptide (Aβ). Ca2+ fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca2+ fluxes induced by Aβ42. Here, we investigated the contribution of Ca2+ release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca2+ homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 induced an elevation in intracellular Ca2+. The Aβ42-stimulated Ca2+ signals persisted in the absence of extracellular Ca2+ indicating a significant contribution of Ca2+ release from the ER Ca2+ store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca2+ release. The Ca2+ mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors, revealing an additional direct effect of Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42.

Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca2+-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2 +☆

Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3Rs role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

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.