Pore-forming proteins as drivers of membrane permeabilization in cell death pathways.

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

Sustainability in a broad sense: An essential aspect of scientific conferences.

This article reflects on sustainability in the context of scientific conferences with emphasis on environmental, diversity, inclusivity, and intellectual aspects. We argue that it is imperative to embrace sustainability as a broad concept during conference organization. In-person conferences have an obvious environmental impact but mitigating strategies can be implemented, such as incentivizing low-emission travel, offering fellowships to support sustainable traveling, and promoting use of public transport or car-pooling. Utilizing eco-conscious venues, catering, and accommodations, along with minimizing resource wastage, further reduces environmental impact. Additional considerations include facilitating hybrid format conferences that allow both in-person and online attendance. Hybrid conferences enhance global participation whilst reducing resource consumption and environmental impact. Often-overlooked benefits can arise from the simple recording of talks to enable asynchronous viewing for people unable to attend in person, in addition to providing a legacy of knowledge that, for example, could support the training of early career researchers (ECRs) or newcomers in the field. The longevity of a research field, intellectual sustainability, requires an inclusive conference atmosphere, offering optimal opportunities for ECRs, minority groups, and researchers from emerging countries. Diversity and inclusivity not only enrich conference experiences but also enhances creativity and innovation.

HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Carbohydrate Loading to Combat Acute Pancreatitis.

Acute pancreatitis is characterized by ATP deficiency and sustained Ca2+ overload in pancreatic acinar cells, leading to premature zymogen activation, auto-digestion of the pancreas, and necrosis. Recent research reveals a rational approach to ameliorate disease through galactose feeding, bypassing hexokinases to restore ATP levels and Ca2+ homeostasis, thereby reducing disease markers.

Dynamic control of mitochondria-associated membranes by kinases and phosphatases in health and disease.

Membrane-contact sites are getting more and more credit for their indispensable role in maintenance of cell function and homeostasis. In the last decades, the ER-mitochondrial contact sites in particular received a lot of attention. While our knowledge of ER-mitochondrial contact sites increases steadily, the focus often lies on a static exploration of their functions. However, it is increasingly clear that these contact sites are very dynamic. In this review, we highlight the dynamic nature of ER-mitochondrial contact sites and the role of kinases and phosphatases therein with a focus on recent findings. Phosphorylation events allow for rapid integration of information on the protein level, impacting protein function, localization and interaction at ER-mitochondrial contact sites. To illustrate the importance of these events and to put them in a broader perspective, we connect them to pathologies like diabetes type II, Parkinson's disease and cancer.

IP3 Receptor Biology and Endoplasmic Reticulum Calcium Dynamics in Cancer.

Intracellular Ca2+ signaling regulates a plethora of cellular functions. A central role in these processes is reserved for the inositol 1,4,5-trisphosphate receptor (IP3R), a ubiquitously expressed Ca2+-release channel, mainly located in the endoplasmic reticulum (ER). Three IP3R isoforms (IP3R1, IP3R2 and IP3R3) exist, encoded respectively by ITPR1, ITPR2 and ITPR3. The proteins encoded by these genes are each about 2700 amino acids long and assemble into large tetrameric channels, which form the target of many regulatory proteins, including several tumor suppressors and oncogenes. Due to the important role of the IP3Rs in cell function, their dysregulation is linked to multiple pathologies. In this review, we highlight the complex role of the IP3R in cancer, as it participates in most of the so-called "hallmarks of cancer". In particular, the IP3R directly controls cell death and cell survival decisions via regulation of autophagy and apoptosis. Moreover, the IP3R impacts cellular proliferation, migration and invasion. Typical examples of the role of the IP3Rs in these various processes are discussed. The relative levels of the IP3R isoforms expressed and their subcellular localization, e.g. at the ER-mitochondrial interface, is hereby important. Finally, evidence is provided about how the knowledge of the regulation of the IP3R by tumor suppressors and oncogenes can be exploited to develop novel therapeutic approaches to fight cancer.

Sensory neuropathy-causing mutations in ATL3 affect ER-mitochondria contact sites and impair axonal mitochondrial distribution.

Axonopathies are neurodegenerative disorders caused by axonal degeneration, affecting predominantly the longest neurons. Several of these axonopathies are caused by genetic defects in proteins involved in the shaping and dynamics of the endoplasmic reticulum (ER); however, it is unclear how these defects impinge on neuronal survival. Given its central and widespread position within a cell, the ER is a pivotal player in inter-organelle communication. Here, we demonstrate that defects in the ER fusion protein ATL3, which were identified in patients suffering from hereditary sensory and autonomic neuropathy, result in an increased number of ER-mitochondria contact sites both in HeLa cells and in patient-derived fibroblasts. This increased contact is reflected in higher phospholipid metabolism, upregulated autophagy and augmented Ca2+ crosstalk between both organelles. Moreover, the mitochondria in these cells display lowered motility, and the number of axonal mitochondria in neurons expressing disease-causing mutations in ATL3 is strongly decreased. These results underscore the functional interdependence of subcellular organelles in health and disease and show that disorders caused by ER-shaping defects are more complex than previously assumed.

Onco-IP3Rs Feed Cancerous Cravings for Mitochondrial Ca(2.).

The uncontrolled proliferation of cancer cells requires functional mitochondrial metabolism, which uses Ca(2+) as a cofactor. IP3 receptors (IP3Rs) from endoplasmic reticulum (ER) Ca(2+) stores provide the supply of Ca(2+) to mitochondria. A new study by Cardenas et al. shows that, in contrast to normal cells, cancer cells critically depend on ER-mitochondrial Ca(2+) fluxes for their survival by sustaining the production of mitochondrial substrates used for nucleotide biosynthesis and proliferation.

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.

The SH3-binding domain of Cx43 participates in loop/tail interactions critical for Cx43-hemichannel activity.

Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca2+ concentration ([Ca2+]i) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca2+]i, which may be involved in pathological hemichannel opening.

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.

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 IP3R's 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.

Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment?

Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca2+ are postulated to contribute to RIBE. Ca2+ is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca2+ by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca2+ and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca2+ signaling as a key player in modulating the RIBE and the overall response to radiation.

The anti-metastatic micro-environment of the bone: Importance of osteocyte Cx43 hemichannels.

Bone metastases of tumor cells are a common and life-threatening feature of a variety of late-stage cancers, including breast cancers. However, until now, much less has been known about the intrinsic anti-metastatic properties of the bones and how these could be exploited to prevent or treat bone metastases. Very recently, native Cx43 hemichannels present in osteocytes have been identified as important anti-metastatic signaling complexes by establishing high local extracellular ATP levels. Moreover, bisphosphonate drugs, applied as adjuvant therapies in the treatment of breast cancer patients and bone diseases, are known to display anti-metastatic properties. Now, it became clear that these compounds exert their effects through osteocyte Cx43 hemichannels, thereby triggering their opening and promoting ATP release in the extracellular micro-environment. Hence, endogenous osteocyte Cx43 hemichannels emerge as important and promising therapeutic targets for the prevention of bone metastases and/or clinical treatment of bone-metastasized breast cancers.

ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca(2+) signaling.

Intracellular Ca(2+) signals that arise from the endoplasmic reticulum (ER), the major intracellular Ca(2+)-storage organelle, impact several mitochondrial functions and dictate cell survival and cell death processes. Furthermore, alterations in Ca(2+) signaling in cancer cells promote survival and establish a high tolerance towards cell stress and damage, so that the on-going oncogenic stress does not result in the activation of cell death. Over the last years, the mechanisms underlying these oncogenic alterations in Ca(2+) signaling have started to emerge. An important aspect of this is the identification of several major oncogenes, including Bcl-2, Bcl-XL, Mcl-1, PKB/Akt, and Ras, and tumor suppressors, such as p53, PTEN, PML, BRCA1, and Beclin 1, as direct and critical regulators of Ca(2+)-transport systems located at the ER membranes, including IP3 receptors and SERCA Ca(2+) pumps. In this way, these proteins execute part of their function by controlling the ER-mitochondrial Ca(2+) fluxes, favoring either survival (oncogenes) or cell death (tumor suppressors). Oncogenic mutations, gene deletions or amplifications alter the expression and/or function of these proteins, thereby changing the delicate balance between oncogenes and tumor suppressors, impacting oncogenesis and favoring malignant cell function and behavior. In this review, we provided an integrated overview of the impact of the major oncogenes and tumor suppressors, often altered in cancer cells, on Ca(2+) signaling from the ER Ca(2+) stores. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart.

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia-reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

IP3 Receptor Properties and Function at Membrane Contact Sites.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a ubiquitously expressed Ca2+-release channel localized in the endoplasmic reticulum (ER). The intracellular Ca2+ signals originating from the activation of the IP3R regulate multiple cellular processes including the control of cell death versus cell survival via their action on apoptosis and autophagy. The exact role of the IP3Rs in these two processes does not only depend on their activity, which is modulated by the cytosolic composition (Ca2+, ATP, redox status, …) and by various types of regulatory proteins, including kinases and phosphatases as well as by a number of oncogenes and tumor suppressors, but also on their intracellular localization, especially at the ER-mitochondrial and ER-lysosomal interfaces. At these interfaces, Ca2+ microdomains are formed, in which the Ca2+ concentration is finely regulated by the different ER, mitochondrial and lysosomal Ca2+-transport systems and also depends on the functional and structural interactions existing between them. In this review, we therefore discuss the most recent insights in the role of Ca2+ signaling in general, and of the IP3R in particular, in the control of basal mitochondrial bioenergetics, apoptosis, and autophagy at the level of inter-organellar contact sites.

Alterations in Ca2+ Signalling via ER-Mitochondria Contact Site Remodelling in Cancer.

Inter-organellar contact sites establish microdomains for localised Ca2+-signalling events. One of these microdomains is established between the ER and the mitochondria. Importantly, the so-called mitochondria-associated ER membranes (MAMs) contain, besides structural proteins and proteins involved in lipid exchange, several Ca2+-transport systems, mediating efficient Ca2+ transfer from the ER to the mitochondria. These Ca2+ signals critically control several mitochondrial functions, thereby impacting cell metabolism, cell death and survival, proliferation and migration. Hence, the MAMs have emerged as critical signalling hubs in physiology, while their dysregulation is an important factor that drives or at least contributes to oncogenesis and tumour progression. In this book chapter, we will provide an overview of the role of the MAMs in cell function and how alterations in the MAM composition contribute to oncogenic features and behaviours.