Publications by authors named "Geert Bultynck"

196 Publications

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca signalling through IP receptors.

Biochim Biophys Acta Mol Cell Res 2022 Jan 11;1869(4):119206. Epub 2022 Jan 11.

Division of Hematology/Oncology, Dept. Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Dept of Medicine, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address:

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IPR)-interacting protein by mass spectrometry. The PKM2:IPR interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IPR isoforms. Moreover, fluorescence microscopy indicated that both IPR and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IPR at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IPR isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IPR interaction and potentiated IPR-mediated Ca release both in intact cells (TAT-D5SD peptide) and in a unidirectional Ca flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IPR-mediated Ca signalling in intact cells without altering the ER Ca content. These data identify PKM2 as an IPR-interacting protein that inhibits intracellular Ca signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119206DOI Listing
January 2022

Killing in the name of: Reversing epigenetic silencing of ITPR3 to succumb cancer cells' resistance to chemotherapeutics.

Cell Calcium 2022 Jan 4;102:102526. Epub 2022 Jan 4.

KU Leuven, Laboratory of Molecular & Cellular Signaling, Department of Cellular & Molecular Medicine, Campus Gasthuisberg O/N-1, Herestraat 49 box 802, 3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.ceca.2022.102526DOI Listing
January 2022

Introducing the Special Issue on "Death mechanisms in cellular homeostasis" in honor of Dr. Peter Ruvolo.

Authors:
Geert Bultynck

Biochim Biophys Acta Mol Cell Res 2022 Jan 6;1869(4):119213. Epub 2022 Jan 6.

KU Leuven, Laboratory of Molecular & Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, BE-3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.bbamcr.2022.119213DOI Listing
January 2022

Keeping an eye on Ca signalling to tackle dry eye diseases.

EBioMedicine 2021 Dec 10;74:103741. Epub 2021 Dec 10.

KU Leuven and Leuven Kanker Institute, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, BE, 3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2021.103741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8671085PMC
December 2021

Preface to the Special Issue of the European Calcium Society in honor of Professor Sir Michael J. Berridge.

Biochim Biophys Acta Mol Cell Res 2022 Feb 11;1869(2):119172. Epub 2021 Nov 11.

KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, BE-3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.bbamcr.2021.119172DOI Listing
February 2022

Bcl-xL acts as an inhibitor of IPR channels, thereby antagonizing Ca-driven apoptosis.

Cell Death Differ 2021 Nov 8. Epub 2021 Nov 8.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 Box 802, Herestraat 49, 3000, Leuven, Belgium.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR 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 IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL's inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL's anti-apoptotic function.
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http://dx.doi.org/10.1038/s41418-021-00894-wDOI Listing
November 2021

Uniting the divergent Wolfram syndrome-linked proteins WFS1 and CISD2 as modulators of Ca signaling.

Sci Signal 2021 09 28;14(702):eabc6165. Epub 2021 Sep 28.

KU Leuven, Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, BE-3000 Leuven, Belgium.

[Figure: see text].
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http://dx.doi.org/10.1126/scisignal.abc6165DOI Listing
September 2021

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

Cell Mol Life Sci 2021 Oct 27;78(19-20):6541-6556. Epub 2021 Aug 27.

Lab for Molecular and Cellular Signalling, Department for Cellular and Molecular Medicine, Leuven Kanker Instituut, KU Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1007/s00018-021-03920-9DOI Listing
October 2021

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

Biochim Biophys Acta Mol Cell Res 2021 11 14;1868(12):119121. Epub 2021 Aug 14.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, 3000 Leuven, Belgium. Electronic address:

Recently, a functional IPR ortholog (CO.IPR-A) capable of IP-induced Ca release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IPRs, CO.IPR-A is not modulated by Ca, ATP or PKA. Protein-sequence analysis revealed that CO.IPR-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.IPR-A channels and modulate their Ca-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IPR isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IPR-A, thereby suppressing Ca flux through CO.IPR-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IPR-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IPRs, abrogated Bcl-2's ability to bind and inhibit CO.IPR-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IPR that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IPR function by exploiting this pre-existing site, thereby counteracting Ca-dependent apoptosis.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119121DOI Listing
November 2021

Getting old without type 2 IP3 receptors.

Cell Calcium 2021 09 24;98:102437. Epub 2021 Jun 24.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.ceca.2021.102437DOI Listing
September 2021

Bok joining the "Ca club".

Cell Calcium 2021 09 2;98:102438. Epub 2021 Jul 2.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.ceca.2021.102438DOI Listing
September 2021

IP Receptor Biology and Endoplasmic Reticulum Calcium Dynamics in Cancer.

Prog Mol Subcell Biol 2021 ;59:215-237

Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Cancer Institute, KU Leuven, Leuven, Belgium.

Intracellular Ca signaling regulates a plethora of cellular functions. A central role in these processes is reserved for the inositol 1,4,5-trisphosphate receptor (IPR), a ubiquitously expressed Ca-release channel, mainly located in the endoplasmic reticulum (ER). Three IPR isoforms (IPR1, IPR2 and IPR3) 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 IPRs in cell function, their dysregulation is linked to multiple pathologies. In this review, we highlight the complex role of the IPR in cancer, as it participates in most of the so-called "hallmarks of cancer". In particular, the IPR directly controls cell death and cell survival decisions via regulation of autophagy and apoptosis. Moreover, the IPR impacts cellular proliferation, migration and invasion. Typical examples of the role of the IPRs in these various processes are discussed. The relative levels of the IPR 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 IPR by tumor suppressors and oncogenes can be exploited to develop novel therapeutic approaches to fight cancer.
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http://dx.doi.org/10.1007/978-3-030-67696-4_11DOI Listing
August 2021

I scream for ice cream - TRPC5 as cold sensor in teeth.

Cell Calcium 2021 May 11;97:102419. Epub 2021 May 11.

KU Leuven, Laboratory of Molecular & Cellular Signaling, Department of Cellular & Molecular Medicine, Campus Gasthuisberg O&N1 bus 802, 3000 Leuven, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.ceca.2021.102419DOI Listing
May 2021

SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System.

Stem Cell Rev Rep 2021 10 12;17(5):1855-1873. Epub 2021 May 12.

Stem Cell Institute, Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research.
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http://dx.doi.org/10.1007/s12015-021-10179-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8553725PMC
October 2021

Corrigendum to "Superoxide Anion Production and Bioenergetic Profile in Young and Elderly Human Primary Myoblasts".

Oxid Med Cell Longev 2021 2;2021:9764701. Epub 2021 Apr 2.

Department of Neuroscience Imaging and Clinical Sciences, University "G. d'Annunzio" Chieti-Pescara, Via dei Vestini 29, 66100 Chieti, Italy.

[This corrects the article DOI: 10.1155/2018/2615372.].
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http://dx.doi.org/10.1155/2021/9764701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046552PMC
April 2021

A comprehensive overview of the complex world of the endo- and sarcoplasmic reticulum Ca-leak channels.

Biochim Biophys Acta Mol Cell Res 2021 06 30;1868(7):119020. Epub 2021 Mar 30.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, B-3000 Leuven, Belgium. Electronic address:

Inside cells, the endoplasmic reticulum (ER) forms the largest Ca store. Ca is actively pumped by the SERCA pumps in the ER, where intraluminal Ca-binding proteins enable the accumulation of large amount of Ca. IP receptors and the ryanodine receptors mediate the release of Ca in a controlled way, thereby evoking complex spatio-temporal signals in the cell. The steady state Ca concentration in the ER of about 500 μM results from the balance between SERCA-mediated Ca uptake and the passive leakage of Ca. The passive Ca leak from the ER is often ignored, but can play an important physiological role, depending on the cellular context. Moreover, excessive Ca leakage significantly lowers the amount of Ca stored in the ER compared to normal conditions, thereby limiting the possibility to evoke Ca signals and/or causing ER stress, leading to pathological consequences. The so-called Ca-leak channels responsible for Ca leakage from the ER are however still not well understood, despite over 20 different proteins have been proposed to contribute to it. This review has the aim to critically evaluate the available evidence about the various channels potentially involved and to draw conclusions about their relative importance.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119020DOI Listing
June 2021

The role of Bcl-2 proteins in modulating neuronal Ca signaling in health and in Alzheimer's disease.

Biochim Biophys Acta Mol Cell Res 2021 05 9;1868(6):118997. Epub 2021 Mar 9.

KU Leuven, Laboratory of Molecular & Cellular Signaling, Department of Cellular & Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, BE-3000 Leuven, Belgium.

The family of B-cell lymphoma-2 (Bcl-2) proteins exerts key functions in cellular health. Bcl-2 primarily acts in mitochondria where it controls the initiation of apoptosis. However, during the last decades, it has become clear that this family of proteins is also involved in controlling intracellular Ca signaling, a critical process for the function of most cell types, including neurons. Several anti- and pro-apoptotic Bcl-2 family members are expressed in neurons and impact neuronal function. Importantly, expression levels of neuronal Bcl-2 proteins are affected by age. In this review, we focus on the emerging roles of Bcl-2 proteins in neuronal cells. Specifically, we discuss how their dysregulation contributes to the onset, development, and progression of neurodegeneration in the context of Alzheimer's disease (AD). Aberrant Ca signaling plays an important role in the pathogenesis of AD, and we propose that dysregulation of the Bcl-2-Ca signaling axis may contribute to the progression of AD and that herein, Bcl-2 may constitute a potential therapeutic target for the treatment of AD.
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http://dx.doi.org/10.1016/j.bbamcr.2021.118997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041352PMC
May 2021

Balancing ER-Mitochondrial Ca Fluxes in Health and Disease.

Trends Cell Biol 2021 07 4;31(7):598-612. Epub 2021 Mar 4.

Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven Kanker Instituut, KU Leuven, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, BE-3000 Leuven, Belgium. Electronic address:

Organelles cooperate with each other to control cellular homeostasis and cell functions by forming close connections through membrane contact sites. Important contacts are present between the endoplasmic reticulum (ER), the main intracellular Ca-storage organelle, and the mitochondria, the organelle responsible not only for the majority of cellular ATP production but also for switching on cell death processes. Several Ca-transport systems focalize at these contact sites, thereby enabling the efficient transmission of Ca signals from the ER toward mitochondria. This provides tight control of mitochondrial functions at the microdomain level. Here, we discuss how ER-mitochondrial Ca transfers support cell function and how their dysregulation underlies, drives, or contributes to pathogenesis and pathophysiology, with a major focus on cancer and neurodegeneration but also with attention to other diseases such as diabetes and rare genetic diseases.
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http://dx.doi.org/10.1016/j.tcb.2021.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195822PMC
July 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability.

J Clin Invest 2021 04;131(7)

Physiology Group, Department of Basic and Applied Medical Sciences, Ghent University, Ghent, Belgium.

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc, resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.
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http://dx.doi.org/10.1172/JCI137752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011902PMC
April 2021

Cancer cell death strategies by targeting Bcl-2's BH4 domain.

Biochim Biophys Acta Mol Cell Res 2021 04 5;1868(5):118983. Epub 2021 Feb 5.

KU Leuven, Lab. Molecular & Cellular Signaling, Dep. Cellular & Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, BE-3000 Leuven, Belgium. Electronic address:

The Bcl-2-family proteins have long been known for their role as key regulators of apoptosis. Overexpression of various members of the family is associated with oncogenesis. Its founding member, anti-apoptotic Bcl-2 regulates cell death at different levels, whereby Bcl-2 emerged as a major drug target to eradicate cancers through cell death. This resulted in the development of venetoclax, a Bcl-2 antagonist that acts as a BH3 mimetic. Venetoclax already entered the clinic to treat relapse chronic lymphocytic leukemia patients. Here, we discuss the role of Bcl-2 as a decision-maker in cell death with focus on the recent advances in anti-cancer therapeutics that target the BH4 domain of Bcl-2, thereby interfering with non-canonical functions of Bcl-2 in Ca-signaling modulation. In particular, we critically discuss previously developed tools, including the peptide BIRD-2 (Bcl-2/IPR-disrupter-2) and the small molecule BDA-366. In addition, we present a preliminary analysis of two recently identified molecules that emerged from a molecular modeling approach to target Bcl-2's BH4 domain, which however failed to induce cell death in two Bcl-2-dependent diffuse large B-cell lymphoma cell models. Overall, antagonizing the non-canonical functions of Bcl-2 by interfering with its BH4-domain biology holds promise to elicit cell death in cancer, though improved tools and on-target antagonizing small molecules remain necessary and ought to be designed.
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http://dx.doi.org/10.1016/j.bbamcr.2021.118983DOI Listing
April 2021

BIRD-2, a BH4-domain-targeting peptide of Bcl-2, provokes Bax/Bak-independent cell death in B-cell cancers through mitochondrial Ca-dependent mPTP opening.

Cell Calcium 2021 03 12;94:102333. Epub 2021 Jan 12.

Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven Cancer Institute (LKI), KU Leuven, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000, Leuven, Belgium. Electronic address:

Anti-apoptotic Bcl-2 critically controls cell death by neutralizing pro-apoptotic Bcl-2-family members at the mitochondria. Bcl-2 proteins also act at the endoplasmic reticulum, the main intracellular Ca-storage organelle, where they inhibit IP receptors (IPR) and prevent pro-apoptotic Ca-signaling events. IPR channels are targeted by the BH4 domain of Bcl-2. Some cancer types rely on the IPR-Bcl-2 interaction for survival. We previously developed a cell-permeable, BH4-domain-targeting peptide that can abrogate Bcl-2's inhibitory action on IPRs, named Bcl-2 IP receptor disrupter-2 (BIRD-2). This peptide kills several Bcl-2-dependent cancer cell types, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukaemia (CLL) cells, by eliciting intracellular Ca signalling. However, the exact mechanisms by which these excessive Ca signals triggered by BIRD-2 provoke cancer cell death remain elusive. Here, we demonstrate in DLBCL that although BIRD-2 activates caspase 3/7 and provokes cell death in a caspase-dependent manner, the cell death is independent of pro-apoptotic Bcl-2-family members, Bim, Bax and Bak. Instead, BIRD-2 provokes mitochondrial Ca overload that is rapidly followed by opening of the mitochondrial permeability transition pore (mPTP). Inhibiting mitochondrial Ca overload using Ru265, an inhibitor of the mitochondrial Ca uniporter complex counteracts BIRD-2-induced cancer cell death. Finally, we validated our findings in primary CLL patient samples where BIRD-2 provoked mitochondrial Ca overload and Ru265 counteracted BIRD-2-induced cell death. Overall, this work reveals the mechanisms by which BIRD-2 provokes cell death, which occurs via mitochondrial Ca overload but acts independently of pro-apoptotic Bcl-2-family members.
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http://dx.doi.org/10.1016/j.ceca.2020.102333DOI Listing
March 2021

EPIC3, a novel Ca indicator located at the cell cortex and in microridges, detects high Ca subdomains during Ca influx and phagocytosis.

Cell Calcium 2020 12 9;92:102291. Epub 2020 Oct 9.

Neutrophil Signalling Group, Cardiff University Medical School, Cardiff, CF14 4XN, UK. Electronic address:

The construction of a low affinity Ca-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca influx on intra-wrinkle Ca in the macrophage cell line, RAW 264.7. During experimentally-induced Cainflux, EPIC3 reported Ca concentrations at the cell cortex in the region of 30-50 μM, with peak locations towards the tips of wrinkles reaching 80 μM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca activated protease calpain-1) and released the C-terminal fluors. The cortical Ca levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50-80 μM) during phagocytosis. As phagocytosis was completed, hotspots of Ca near the phagosome were also observed.
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http://dx.doi.org/10.1016/j.ceca.2020.102291DOI Listing
December 2020

Transmembrane BAX Inhibitor-1 Motif Containing Protein 5 (TMBIM5) Sustains Mitochondrial Structure, Shape, and Function by Impacting the Mitochondrial Protein Synthesis Machinery.

Cells 2020 09 23;9(10). Epub 2020 Sep 23.

Institute for Molecular Medicine, Johannes Gutenberg University Medical Center Mainz, D-55131 Mainz, Germany.

The Transmembrane Bax Inhibitor-1 motif (TMBIM)-containing protein family is evolutionarily conserved and has been implicated in cell death susceptibility. The only member with a mitochondrial localization is TMBIM5 (also known as GHITM or MICS1), which affects cristae organization and associates with the Parkinson's disease-associated protein CHCHD2 in the inner mitochondrial membrane. We here used CRISPR-Cas9-mediated knockout HAP1 cells to shed further light on the function of TMBIM5 in physiology and cell death susceptibility. We found that compared to wild type, -knockout cells were smaller and had a slower proliferation rate. In these cells, mitochondria were more fragmented with a vacuolar cristae structure. In addition, the mitochondrial membrane potential was reduced and respiration was attenuated, leading to a reduced mitochondrial ATP generation. TMBIM5 did not associate with Mic10 and Mic60, which are proteins of the mitochondrial contact site and cristae organizing system (MICOS), nor did knockout affect their expression levels. -knockout cells were more sensitive to apoptosis elicited by staurosporine and BH3 mimetic inhibitors of Bcl-2 and Bcl-XL. An unbiased proteomic comparison identified a dramatic downregulation of proteins involved in the mitochondrial protein synthesis machinery in TMBIM5-knockout cells. We conclude that TMBIM5 is important to maintain the mitochondrial structure and function possibly through the control of mitochondrial biogenesis.
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http://dx.doi.org/10.3390/cells9102147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598220PMC
September 2020

Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease.

Ann Clin Transl Neurol 2020 10 19;7(10):1962-1972. Epub 2020 Sep 19.

Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Objective: ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene.

Methods: Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca imaging.

Results: Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca -transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function.

Interpretation: Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease-causing gene for CMT and indicates altered Ca homeostasis in disease pathogenesis.
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http://dx.doi.org/10.1002/acn3.51190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545616PMC
October 2020

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models.

Cell Death Dis 2020 09 17;11(9):769. Epub 2020 Sep 17.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut (LKI), Leuven, Belgium.

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.
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http://dx.doi.org/10.1038/s41419-020-02944-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498462PMC
September 2020

MICAL2 is essential for myogenic lineage commitment.

Cell Death Dis 2020 08 18;11(8):654. Epub 2020 Aug 18.

Translational Cardiomyology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.
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http://dx.doi.org/10.1038/s41419-020-02886-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434877PMC
August 2020

Necroptosis in Immuno-Oncology and Cancer Immunotherapy.

Cells 2020 08 1;9(8). Epub 2020 Aug 1.

Department of Cellular and Molecular Medicine, Laboratory of Cell Stress & Immunity (CSI), KU Leuven, 3000 Leuven, Belgium.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.
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http://dx.doi.org/10.3390/cells9081823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464343PMC
August 2020

DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition.

Biomolecules 2020 07 21;10(7). Epub 2020 Jul 21.

Lab. Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg ON-I, KU Leuven, 3000 Leuven, Belgium.

Anti-apoptotic Bcl-2-family members are frequently dysregulated in both blood and solid cancers, contributing to their survival despite ongoing oncogenic stress. Yet, such cancer cells often are highly dependent on Bcl-2 for their survival, a feature that is exploited by so-called BH3-mimetic drugs. Venetoclax (ABT-199) is a selective BH3-mimetic Bcl-2 antagonist that is currently used in the clinic for treatment of chronic lymphocytic leukemia patients. Unfortunately, venetoclax resistance has already emerged in patients, limiting the therapeutic success. Here, we examined strategies to overcome venetoclax resistance. Therefore, we used two diffuse large B-cell lymphoma (DLBCL) cell lines, Riva WT and venetoclax-resistant Riva (VR). The latter was obtained by prolonged culturing in the presence of venetoclax. We report that Riva VR cells did not become more sensitive to BIRD-2, a peptide targeting the Bcl-2 BH4 domain, and established cross-resistance towards BDA-366, a putative BH4-domain antagonist of Bcl-2. However, we found that Bcl-XL, another Bcl-2-family protein, is upregulated in Riva VR, while Mcl-1 expression levels are not different in comparison with Riva WT, hinting towards an increased dependence of Riva VR cells to Bcl-XL. Indeed, Riva VR cells could be resensitized to venetoclax by A-1155463, a selective BH3 mimetic Bcl-XL inhibitor. This is underpinned by siRNA experiments, demonstrating that lowering Bcl-XL-expression levels also augmented the sensitivity of Riva VR cells to venetoclax. Overall, this work demonstrates that Bcl-XL upregulation contributes to acquired resistance of DLBCL cancer cells towards venetoclax and that antagonizing Bcl-XL can resensitize such cells towards venetoclax.
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http://dx.doi.org/10.3390/biom10071081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408247PMC
July 2020

Small molecule 3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3).

FEBS Lett 2020 09 20;594(18):3067-3075. Epub 2020 Jul 20.

Laboratory of Physiopharmacology, University of Antwerp, Belgium.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway.
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http://dx.doi.org/10.1002/1873-3468.13878DOI Listing
September 2020
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