Identification and Optimization of a Novel Inhibitor of Mitochondrial Calpain 10 (original) (raw)

Mitochondrial calpain system: An overview

Calpain system is generally known to be comprised of three molecules: two Ca 2+ -dependent proteases: land m-calpains, and their endogenous inhibitor, calpastatin. While calpains have previously been considered as the cytoplasmic enzymes, research in the recent past demonstrated that l-calpain, m-calpain and calpain 10 are present in mitochondria, which play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. Although a number of original research articles on mitochondrial calpain system are available, yet to the best of our knowledge, a precise review article on mitochondrial calpain system has, however, not been available. This review outlines the key features of the mitochondrial calpain system, and its roles in several cellular and biochemical events under normal and some pathophysiological conditions.

N terminus of calpain 1 is a mitochondrial targeting sequence

Journal of Biological …, 2008

The ubiquitous m-and -calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, -calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of -calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form -calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic ␣-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of -calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.

Calpains, mitochondria, and apoptosis

Cardiovascular Research, 2012

Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca 2+-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca 2+ overload causes mitochondrial calpain 1 cleavage of the Na + /Ca 2+ exchanger leading to mitochondrial Ca 2+ accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-a-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca 2+ overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.

Targeted inhibition of calpain in mitochondria alleviates oxidative stress-induced myocardial injury

Acta Pharmacologica Sinica, 2020

The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.

Calcineurin-independent inhibition of mitochondrial Ca 2+ uptake by cyclosporin A

British Journal of Pharmacology, 2004

Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.British Journal of Pharmacology (2004) 141, 263–268. doi:10.1038/sj.bjp.0705609

Modulators of calpain activity: inhibitors and activators as potential drugs

Expert Opinion on Drug Discovery, 2020

Introduction Calpains are intracellular Ca 2+-dependent cysteine proteases with 15 known members in the enzyme family. They act as regulatory enzymes, their cleavage modifies the function of their substrates. As their substrates have important roles in many physiological processes, adequate function of calpains is mandatory for normal cellular functions. Adverse operation of them is often related with diseases (e.g. neurodegenerative disorders, cancer, type 2 diabetes mellitus or limb-girdle muscular dystrophy type 2A). Areas covered Herein, the authors give an overview about calpains, their structure, physiological and pathological functions. The challenges in the drug discovery of calpain inhibitors and activators are enlightened by summarising examples that eventuated good candidates. Going through these examples new and applicable strategies are discussed. Expert opinion Calpain enzymes are attractive targets to design inhibitors or activators for drug development. This research area has high potential, although it has many challenges. The selective and targeted inhibition or activation of calpains is needed. Thus the studies focused on the improvement of these properties of drug candidates. Keywordscalpain, cysteine proteases, calpastatin, calpain inhibitor, calpain activator, structure-activity-relationship (SAR) Article highlights • Calpains are intracellular cysteine proteases with very diverse physiological roles. • Aberrant activity of them; over-or under activation; is often involved in pathological functions and thus in disease. • Specific and effective inhibitors may be potent candidate to treat these diseases. • Peptide and peptidomimetic inhibitors were developed to increase the specificity and potency using different strategies. 4 calpain 1 and 2 [4]. These proteases, called conventional calpains, are ubiquitous and wellstudied members. Their activation needs micro-and millimolar concentration of Ca 2+ ion in vitro respectively [5]. They were called μand m-calpain based on this sensitivity for Ca 2+ ion. Both enzymes form heterodimers with a small regulatory subunit (calpain 4), and expressed in all tissues, while many of the later described members have tissue specific distribution (calpain 3, 6, 8, 9 11, 12, 15 and 16) [6] (Table 1). There are 15 calpain genes in the human genome (CAPN1 to CAPN16, no CAPN4). Calpains are classified as classical or non-classical calpains based on their domain structure; on what kind of conserved protein domains are attached to the well-conserved cysteine protease domain (CysPc) (Fig. 1A) [7]. The CysPc domain in calpain is composed of protease core 1 (PC1) and PC2 domains. Amino acids that form the catalytic triad were in distinct domains; the Cys is in the PC1 while the His and Asn are in the PC2 domain. The calpain 6 is the only member that does not have a catalytic triad; there is Lys instead of Cys. In the absence of Ca 2+ ions these two domains are far from each other and thus the catalytic triad is not assembled [8]. Both domains can bind Ca 2+ ion and that results in structural changes opening of the active site cleft and assembling the catalytic triad [9] (the relationship between the structure and activity is well summarised in Ref [10]). Although the whole 3D structure of only calpain 2 is known (Fig. 2A) [9,11], using this for 3D modelling of other calpains is an accepted strategy [12,13]. 2.2 Physiological role of Calpains 2.2.1 Regulation of enzyme activity Calpains are expressed in different organisms, for example in human, invertebrates, yeast, other fungi and bacteria. These intracellular enzymes cleave their substrate proteins in welldetermined manner. This limited proteolysis activates or deactivates the substrate proteins, thus calpains have regulatory or signalling function, rather than protein digestion in cells like proteasomal or lysosomal enzymes do. Because of this, their activity should be under strong

Mitochondrial localization of [mu]-calpain

Biochemical and Biophysical Research Communications, 2005

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80 kDa μ-calpain large subunit and 28 kDa small subunit. In rat cortex, antibodies against domains II and III of the large μ-calpain subunit also detected a 40 kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte μ-calpain with Ca2+. Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of μ-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca2+ released from mitochondrial stores.

Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Archives of Biochemistry and Biophysics, 2012

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37° C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca 2+ addition, Ca 2+ chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tertbutyl hydroperoxide (TBHP) or H 2 O 2 increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H 2 O 2 oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.

Heart mitochondria and calpain 1: Location, Function, and Targets

Biochimica et biophysica acta, 2015

Calpain 1 is a ubiquitous Ca(2+)-dependent cysteine protease. Although calpain 1 has been found in cardiac mitochondria, the exact location within mitochondrial compartments and its function remains unclear. The aims of the current review are to discuss the localization of calpain 1 in different mitochondrial compartment in relationship to its function, especially in pathophysiological conditions. Briefly, mitochondrial calpain 1 (mit-CPN1) is located within the intermembrane space and mitochondrial matrix. Activation of the mit-CPN1 within intermembrane space cleaves apoptosis inducing factor (AIF), whereas the activated mit-CPN1 within matrix cleaves complex I subunits and metabolic enzymes. Inhibition of the mit-CPN1 could be a potential strategy to decrease cardiac injury during ischemia-reperfusion.