REVIEWS Drug Discovery Today Volume 24, Number 5 May 2019 Teaser This review provides an up-to-date overview of the different peptides that have 2+ emerged as modulators of ER–mitochondrial Ca fluxes with translational and therapeutic potential. Reviews KEYNOTE REVIEW Therapeutic implications of novel peptides targeting ER–mitochondria 2+ Ca -flux systems § Martijn Kerkhofs started Martijn Kerkhofs, Geert Bultynck, Tim Vervliet and his PhD in the LMCS research ,§ group, KU Leuven (Belgium), Giovanni Monaco in 2016. The main aim of his research work is to exploit KU Leuven, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Laboratory of Molecular the Bcl-2˘IP3R interaction in diffuse large B cell lymphomas and Cellular Signaling, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, 3000 Leuven, Belgium using IP3R-derived peptide tools to drive cell death in 2+ Bcl-2-dependent cancer cells and elucidating the Intracellular Ca -flux systems located at the ER–mitochondrial axis 2+ underlying molecular and cellular mechanisms. govern mitochondrial Ca balance and cell fate. Multiple yet incurable Geert Bultynck is a 2+ pathologies are characterized by insufficient or excessive Ca fluxes professor in the Laboratory of Molecular and Cellular toward the mitochondria, in turn leading to aberrant cell life or death Signaling (LMCS) in the Department of Cellular & dynamics. The discovery and ongoing molecular characterization of the 2+ Molecular Medicine, KU main interorganellar Ca gateways have resulted in a novel class of Leuven (Belgium), since 2008 onwards. His research aspires peptide tools able to regulate relevant protein–protein interactions (PPIs) to explore and to exploit the 2+ role of intracellular Ca -release channels and their underlying this signaling scenario. Here, we review peptides, molecularly 2+ modulation accessory proteins such as Bcl-2 proteins derived from Ca -flux systems or their accessory proteins. We discuss how in cell death and survival in health, disease and therapy. 2+ The aim is to develop novel therapeutic strategies to they alter Ca -signaling protein complexes and modulate cell survival in tackle diseases linked with altered activity of IP3 and light of their forthcoming therapeutic applications. ryanodine receptors. Tim Vervliet obtained his PhD in biomedical sciences in 2015. He is a post-doctoral Introduction researcher at KU Leuven 2+ 2+ (Belgium) in the LMCS In vertebrates, Ca outside bones and teeth amounts only to <1% of the total Ca . However, research group. His research 2+ this Ca is used intracellularly to drive various physiological processes, from fertilization, gene focuses on elucidating how endoplasmic reticulum- transcription, exocytosis and cell cycle progression to cell death [1–4]. The spatiotemporal located ryanodine receptor 2+ 2+ 2+ profile of these Ca signals and their decoding by Ca -binding proteins are key to this whole Ca -release channels are regulated by Bcl-2 proteins 2+ in health and disease. The aim is to apply these insights spectrum of functions. As such, the free cytosolic Ca concentrations are kept very low at 2+ 2+ and identify peptide tools to target neurodegenerative 100 nM. This is achieved by several Ca -transport systems that extrude Ca from the cytosol, diseases, myopathies and acute pancreatitis. + 2+ 2+ including Na /Ca exchangers, plasma membrane Ca ATPases, sarco/endoplasmic reticulum Giovanni Monaco is a senior 2+ 2+ 2+ (ER) Ca ATPases (SERCA) and Ca -binding proteins. Inside the cell, the majority of the Ca is post-doctoral researcher in the 2+ 2+ LMCS Lab, who received his m stored in the ER [(Ca )ER 500 M], although other compartments also store Ca , such as the PhD in biomedical sciences, KU 2+ Golgi and the lysosomes, whereas the mitochondria can transiently accumulate Ca . Leuven (Belgium), in 2013. His 2+ research interests range from Mitochondrial Ca accumulation is crucial for several mitochondrial functions, and by the cellular study of ER˘ extension cell survival and cell death (Fig. 1 provides a simplified overview). The Krebs cycle, 2+ mitochondria Ca -crosstalk 2+ for example, is dependent on Ca by virtue of its binding to and activation of enzymes such as and apoptosis signaling (e.g., Bcl-2 family member function) to the rational design of pyruvate, citrate and a-ketoglutarate dehydrogenase, driving ATP output. In the absence of such targeted therapies and peptide tools, exploiting the 2+ Ca fluxes, ATP output diminishes, resulting in the activation of AMP-activated kinases and molecular and metabolic vulnerabilities of leukemia and related hematological diseases. Corresponding authors: Bultynck, G. ([email protected]), Monaco, G. ([email protected]) § Shared authorship. 1359-6446/ã 2019 Elsevier Ltd. All rights reserved. 1092 www.drugdiscoverytoday.com https://doi.org/10.1016/j.drudis.2019.03.020 Drug Discovery Today Volume 24, Number 5 May 2019 REVIEWS Energy status ER-mitochondria Mitochondrial Ca2+ and cell fate Ca2+ fluxes and integrity Bioenergetic crisis Suppressed ER Cell death Attenuated Low bioenergetics Ca2+ Autophagy Normal Physiological bioenergetics KEYNOTE REVIEW Cell survival Enhanced Reviews Enhanced bioenergetics Enhanced cell survival 2+ Excessive Ca Mitochondrial permeabilization Damaged mitochondria Mitophagy or cell death Drug discovery today FIGURE 1 2+ 2+ Endoplasmic reticulum (ER)–mitochondrial Ca exchange regulates cell death and survival, as well as the energy status of the cell. Different levels of Ca 2+ 2+ signaling impact the cell in a different way. Mitochondrial Ca uptake directly stimulates mitochondrial energy production. Hence, lack of mitochondrial Ca 2+ causes a bioenergetic crisis, possibly leading to cell death. If the cell experiences suboptimal Ca exchange between ER and mitochondria, autophagy can be activated to supplement the low bioenergetic state. This can lead to physiological bioenergetics and cell survival; however, if the autophagic response is not 2+ sufficient to rescue the cells, a bioenergetic crisis could yet ensue. By contrast, enhanced ER–mitochondrial Ca flux and subsequent enhanced bioenergetics 2+ make cells more resilient and thus enhance cell survival. Lastly, Ca overload in the mitochondria can underlie mitochondrial damage. This can cause cell death but in some cases it is a trigger for mitophagy, a mitochondria-specific form of autophagy, to clear defective mitochondria, allowing the cell to survive. 2+ subsequent energy re-supplementation via the cytosolic recycling To enable ER–mitochondrial Ca fluxes, the ER lumen needs to 2+ 2+ process of autophagy [5]. Conversely, an excess of Ca in the maintain sufficiently high Ca levels, which are sustained by 2+ mitochondria, often in combination with the production of SERCA activity and ER luminal Ca -binding proteins [14]. Fur- 2+ reactive oxygen species (ROS) opens the mitochondrial permeabil- thermore, two important channel families are responsible for Ca ity transition pore (mPTP), which renders the inner mitochondrial release from the ER: the inositol 1,4,5-trisphosphate (IP3) receptor membrane permeable for all kinds of solutes and hampers (IP3R) [15], which is expressed in most cell types, and the ryano- mitochondrial function [5,6]. Dysfunctional mitochondria are dine receptor (RyR) [16], which has a more distinct expression removed by mitophagy, the selective degradation of mitochondria pattern, for example cardiomyocytes, skeletal muscle fibers, neu- by autophagy, to prevent cell damage and death [6,7]. Mitochon- rons, liver and pancreas. The IP3R is gated by IP3, which is formed 2+ 2+ drial Ca accumulation happens through ‘quasi-synaptic’ Ca from phosphatidyl inositol 4,5-bisphosphate (PIP2) in response to fluxes between the ER and the mitochondria [8,9]. Given the activation of G-protein-coupled receptors (GPCRs) or tyrosine importance of mitochondria in the regulation of cell fate, an kinases [15]. RyRs are activated in response to a variety of physio- 2+ 2+ adequate functioning of the ER–mitochondrial Ca -signaling axis logical agonists, including Ca itself, cyclic-ADP ribose and is important for cell survival. Many parameters impact the extent NAADP or via coupling with dihydropyridine receptors (DHPRs) 2+ of ER–mitochondrial Ca fluxes. These factors include: expres- in skeletal muscles [16]. Furthermore, IP3Rs and RyRs are tetra- 2+ sion, properties and regulation of Ca -release channels and meric channels encoded by three distinct genes. They give rise to pumps at the ER [10,11], expression and regulation of mitochon- the expression of three different isoforms each (IP3R1, IP3R2 and 2+ drial Ca -uptake proteins [12,13] and the highly negative mito- IP3R3, and RyR1, RyR2 and RyR3, respectively), which are differ- chondrial membrane potential (DCm), approximately À180 mV entially activated and regulated [10,17]. Upon activation of these 2+ 2+ [5] (the main molecular players involved in this Ca communi- channels, Ca is released from the ER into the cytosol [15,16]. 2+ cation are illustrated in Fig. 2). Mitochondrial Ca uptake is mediated by two main channels: the www.drugdiscoverytoday.com 1093 REVIEWS Drug Discovery Today Volume 24, Number 5 May 2019 SR/ER Reviews KEYNOTE REVIEW Krebs Cycle ATP ADP Cx43 ~ –180 mv m Mitochondrion Ca2+ ΔΨ Drug discovery today FIGURE 2 2+ 2+ 2+ Overview of the ER–mitochondria Ca -signaling toolkit and its regulators. The ER harbors two main Ca -release channels: IP3R and the RyR. Both Ca -release channels are regulated by members of the Bcl-2 protein family, namely Bcl-2 and Nrh for the IP3R and Bcl-2 and Bcl-XL for the RyR. To retain its filling state, the ER also harbors the SERCA. SERCA activity is controlled by PLB, in itself subject to dephosphorylation by PP1. This dephosphorylation manages PLB activity, enabling 2+ increased inhibition of SERCA. On the mitochondrial side, Ca traverses the OMM via VDAC1. VDAC1 conductivity can be modified by interacting proteins such 2+ as HKI/II or Bcl-XL. After crossing the OMM, Ca is transported to the matrix via the MCU. MCU itself is regulated through phosphorylation, for example by CaMKII, but sensitization also occurs through an interaction with FHIT.