Cancer remains one of the most pressing health challenges of the 21st century, with rising global incidence underscoring the need for innovative therapeutic strategies. Despite significant advances in biotechnology, curative outcomes remain limited, prompting interest in integrative approaches. Ayurveda, the traditional Indian system of medicine, suggests a holistic therapeutic framework that is now gaining molecular validation in oncology. In this review, the literature was systematically collected and analyzed from major databases, including PubMed, Scopus, and Web of Science, encompassing studies across ethnopharmacology, biochemistry, and cancer biology. The analysis focused on Ayurvedic phytochemicals that modulate ADP-ribosylation (ADPr), a dynamic post-translational modification central to DNA repair, chromatin organization, and cellular stress responses, with particular emphasis on poly (ADP-ribose) polymerase (PARP)-mediated pathways and their oncological relevance. We have also explored the role of p53, a key stress-response regulator intricately linked to ADPr dynamics, which acts as a downstream effector integrating these molecular events with cell fate decisions. Evidence indicates that several Ayurvedic compounds, including curcumin, resveratrol, and withaferin A, influence PARP–p53 signaling networks, thereby modulating DNA repair fidelity, apoptosis, and tumor adaptation. The review further addresses challenges related to the poor solubility of these phytochemicals and highlights recent advances in Phyto-nanomedicine-based delivery systems that enhance their stability and therapeutic efficacy. Overall, the synthesis of Ayurvedic pharmacology with molecular oncology reveals mechanistic insights that may inform the rational development of novel, mechanism-driven cancer therapeutics.

Despite advances in medicine, cancer remains one of the foremost global health concerns. Conventional treatments like surgery, radiotherapy, and chemotherapy have advanced with the emergence of targeted and immunotherapy approaches. However, therapeutic resistance and relapse remain major barriers to long-term success in cancer treatment, often driven by cancer stem cells (CSCs). These rare, resilient cells can survive therapy and drive tumour regrowth, urging deeper investigation into the mechanisms underlying their persistence. CSCs express ion channels typical of excitable tissues, which, beyond electrophysiology, critically regulate CSC fate. However, the underlying regulatory mechanisms of these channels in CSCs remain largely unexplored and poorly understood. Nevertheless, the therapeutic potential of targeting CSC ion channels is immense, as it offers a powerful strategy to disrupt vital signalling pathways involved in numerous pathological conditions. In this review, we explore the diverse repertoire of ion channels expressed in CSCs and highlight recent mechanistic insights into how these channels modulate CSC behaviours, dynamics, and functions. We present a concise overview of ion channel-mediated CSC regulation, emphasizing their potential as novel diagnostic markers and therapeutic targets, and identifying key areas for future research.

Mitochondrial signalling plays a fundamental role in orchestrating essential intracellular functions, including cellular respiration, proliferation, nucleic acid synthesis, and oxidative stress management. The activation of calpain, a group of Ca2+-dependent cysteine proteases, by ROS-induced oxidative stress is linked to cancer progression. Calpain can be activated by ROS either through intracellular Ca2+ elevation or via oxidative modifications of the protease, altering protein susceptibility to calpain cleavage. In tumour cell biology, ROS-activated calpains influence cell survival, migration, proliferation, apoptosis, and invasiveness. Several studies report unusual calpain expression in cancer cells. Various anticancer drugs induce cytotoxicity by activating calpain, significantly impacting cancer treatment strategies. This unique review explores the perspective of ROS-induced calpain activation and its pivotal role in cancer progression and therapeutics.

PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways. (Figure presented.) Mono-ADP-ribosylation has emerged as a crucial factor in innate immune responses, but is understudied due to the lack of sensitive detection methods. This study visualizes endogenous interferon-induced ADP-ribosylation and shows that PARP14 is a major enzyme responsible for this signalling event. Immunity responses induce PARP14-dependent ADP-ribosylation. SARS2-CoV2 Mac1 can remove PARP14-dependent ADP-ribosylation. PARP14, PARP9 and DTX3L regulate the formation of ubiquitin and ADPr foci in the cytoplasm. PARP14 activity is regulated by PARP9/DTX3L, through (1) the hydrolytic activity of PARP9 and (2) PARP14 interaction with DTX3L.

(Figure presented.)

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dualfunction enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+nanodomain, which forms when Ca2+channels open. Ca2+nanodomains arising from store-operated Orai1 Ca2+channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here,we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca2+channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.

A rise in cytosolic Ca2+ concentration is a universal signalling mechanism that regulates myriad physiological responses including exocytosis, contraction, cell growth and cell death. Cytosolic Ca2+ signals often occur in the form of repetitive Ca2+ transients, or oscillations, particularly following activation of G-protein-coupled receptors that engage the phospholipase C pathway. Ca2+ oscillations have also been seen within organelles including mitochondria and the nucleus. It is well established that signalling information is contained within the amplitude and frequency of the Ca2+ oscillations but recent studies reveal that the spatial profile of the Ca2+ transients is also used to selectively activate downstream responses.

It has now been well established that different mitochondrial compartments contain varieties of calpains. The expression levels of these calpains are tissue and cell type specific. Although, mitochondrial compartments contain different types of calpains, the precise location within mitochondria and their functions remain imprecise. The aim of the present review is to confer information concerning the localization of calpains in different mitochondrial compartments affiliated with their function, particularly in pathophysiological conditions. For instance, mitochondrial μ-calpain is located within the inner membrane, intermembrane space, and mitochondrial matrix depending on cell types. μ-Calpain activity facilitates cleavage of apoptosis-inducing factor (AIF) within inner membrane and intermembrane space, while the activated μ-calpain within matrix is associated with cleavage of complex I subunits and metabolic enzymes. Understandably, inhibition of the μ-calpain could be a potential strategy to ameliorate ischemia- reperfusion-associated injuries.

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

Protein isoforms are widely expressed in biological systems. How isoforms that co-exist within the same sub-cellular domain are differentially activated remains unclear. Here, we compare the regulatory mechanism of two closely related transcription factor isoforms, NFAT1 and NFAT4, that migrate from the cytoplasm to the nucleus following the increase in intracellular Ca2+ that accompanies the opening of store-operated Orai1/CRAC channels. We demonstrate that NFAT1 has a private line of communication with Orai1, activating in response to Ca2+ microdomains near the open channels. By contrast, NFAT4 stimulation requires both local Ca2+ entry and a nuclear Ca2+ rise. We mapped differences in nuclear location to amino acids within the SP-3 motif of the NFAT regulatory domain. The different Ca2+ dependencies enable agonists to recruit different isoform combinations as stimulus strength increases. Our study uncovers a mechanism whereby co-existing cytoplasmic transcription factor isoforms are differentially activated by distinct sub-cellular Ca2+ signals.

In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca2+ release-activated Ca2+ (CRAC) channels confined to PM microdomains with a similar number of dispersed CRAC channels to activate transcription factors, which subsequently increase nuclear gene expression. For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.

NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle [1]. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca2+-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus [2, 3]. In some cell types, NFAT can be activated by Ca2+ nanodomains near open store-operated Orai1 and voltage-gated Ca2+ channels in the plasma membrane [4, 5]. How local Ca2+ near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca2+ nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca2+ entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca 2+ nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1. © 2014 The Authors.

Calpains, a family of Ca2+-dependent cysteine proteases, can modulate their substrates structure and function through limited proteolytic activity. Calpain mediated proteolysis of intracellular proteins is a key step in various cellular processes such as cytoskeleton modulation, cell migration, cell cycle progression and apoptosis. Calpain activity is controlled in vivo by calpastatin, a multiheaded endogenous polypeptide encoded by the calpastatin gene that specifically inhibits calpain. Calpains have previously been considered as the cytoplasmic enzymes; however, recent research have demonstrated that m-calpain and calpastatin are present in endoplasmic reticulum and play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. This review summarizes function and regulation of the endoplasmic reticulum calpain system, focusing on the relevance of its roles in several cellular and biochemical events under normal and some pathophysiological conditions.

Calpains, a family of Ca2+ -dependent cysteine proteases, can modulate their substrates structure and function through limited proteolytic activity. Calpain mediated proteolysis of intracellular proteins is a key step in various cellular processes such as cytoskeleton modulation, cell migration, cell cycle progression and apoptosis. Calpain activity is controlled in vivo by calpastatin, a multiheaded endogenous polypeptide encoded by the calpastatin gene that speciflcally inhibits calpain. Calpains have previously been considered as the cytoplasmic enzymes; however, recent research have demonstrated that m-calpain and calpastatin are present in endoplasmic reticulum and play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. This reviewsummarizes function and regulation of the endoplasmic reticulum calpain system, focusing on the relevance of its roles in several cellular and biochemical events under normal and some pathophysiological conditions.

Cytoplasmic Ca2+ is an universal intracellular messenger that activates cellular responses over a broad temporal range, from neurotransmitter release to cell growth and proliferation.1,2 Inherent to the use of the multifarious Ca2+ signal is the question of specificity: how can some Ca2+-dependent responses be activated in a cell and not others? A rise in cytoplasmic Ca2+ can evoke a response either by binding directly to the target (as occurs with certain Ca2+-activated K + and Cl- channels, for example) or through recruitment of intermediary proteins, such as calmodulin and troponin C. A substantial body of evidence has now established that Ca2+-binding proteins differ both in their affinities for Ca2+ and in their on- and off-rates for Ca2+ binding/unbinding. Furthermore, different Ca2+- binding proteins often occupy distinct locations within the cell. Therefore, the size, kinetics and spatial profile of a cytoplasmic Ca2+ signal are all important in determining which Ca2+-dependent response will be activated, when and for how long.3 © 2013 Landes Bioscience.

Ca 2+-dependent gene expression is critical for cell growth, proliferation, plasticity, and adaptation [1-3]. Because a common mechanism in vertebrates linking cytoplasmic Ca 2+ signals with activation of protein synthesis involves the nuclear factor of activated T cells (NFAT) family of transcription factors [4, 5], we have quantified protein expression in single cells following physiological Ca 2+ signals by using NFAT-driven expression of a genetically encoded fluorescent protein. We find that gene expression following CRAC channel activation is an all-or-nothing event over a range of stimulus intensities. Increasing agonist concentration recruits more cells but each responding cell does so in an essentially digital manner. Furthermore, Ca 2+-dependent gene expression shows both short-term memory and strong synergy, where two pulses of agonist, which are ineffectual individually, robustly activate gene expression provided that the time interval between them is short. Such temporal filtering imparts coincidence detection to Ca 2+-dependent gene activation. The underlying molecular basis mapped to time-dependent, nonlinear accumulation of nuclear NFAT. Local Ca 2+ near CRAC channels has to rise above a threshold level to drive gene expression, providing analog control to the digital activation process and a means to filter out fluctuations in background noise from activating transcription while ensuring robustness and high fidelity in the excitation-transcription coupling mechanism. © 2012 Elsevier Ltd All rights reserved.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca2+ oscillations. Although oscillations arise from regenerative Ca2+ release, they are sustained by store-operated Ca2+entry through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca2+ oscillations, CRAC channel activity, and downstream Ca2+-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca 2+ sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca2+ oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca2+signals and downstream responses.

NFATs are a family of Ca2+-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of numerous distinct cell types and organ systems. Here, we visualize NFAT1 movement in and out of the nucleus in response to transient activation of store-operated Ca2+ release-activated Ca2+ (CRAC) channels in nonexcitable cells. We show that NFAT migration is exquisitely sensitive to Ca2+ microdomains near open CRAC channels. Another Ca2+-permeable ion channel (TRPC3) was ineffective in driving NFAT1 to the nucleus. NFAT1 movement is temporally dissociated from the time course of the Ca2+ signal and remains within the nucleus for 10 times longer than the duration of the trigger Ca2+ signal. Kinetic analyses of each step linking CRAC channel activation to NFAT1 nuclear residency reveals that the rate-limiting step is transcription factor exit from the nucleus. The slow deactivation of NFAT provides a mechanism whereby Ca2+-dependent responses can be sustained despite the termination of the initial Ca 2+ signal and helps explain how gene expression in nonexcitable cells can continue after the primary stimulus has been removed. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Previously, we reported that bovine pulmonary smooth muscle endoplasmic reticulum (ER) membrane possesses associated m-calpain and calpastatin and ER lumen contains only m-calpain. Herein, we report characteristic properties of ER membrane m-calpain (MCp), calpastatins and lumen m-calpain (LCp) and a brief comparative study between MCp and LCp. MCp containing 80 kDa large and 28 kDa small subunit is non-phosphorylated, whereas LCp containing only 80 kDa large subunit is phosphorylated. Optimum pH, Ca2+ concentration and pI value of both MCp and LCp are 7.5, 5 mM and 4.5, respectively. MCp and LCp have similar kinetic parameters and circular dichroism (CD) spectra. Autolysis of MCp and LCp are different. Coimmunoprecipitation studies revealed that LCp is associated with ERp57 in the ER lumen, which suggests that the regulation of LCp differs from the regulation of MCp. In presence of Ca2+, the activated LCp cleaves inositol 1,4,5-trisphosphate receptor-1 (IP3R1) in the ER lumen, whereas the activated MCp cleaves Na+/Ca2+ exchanger-1 (NCX1) in the ER membrane. We have determined pI (4.6 and 4.7, respectively) and IC50 (0.52 and 0.8 nM, respectively) values of 110 and 70 kDa calpastatins. For first time, we have determined the characteristic properties, regulation and functional activity of LCp in the ER lumen. © The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na+/Ca2+ exchanger (NCX) in the ER. Treatment of the ER with Ca2+ (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 μM) or MDL28170 (10 μM), or Ca2+ chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca2+ overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Aims: We sought to identify, purify and partially characterize a protein inhibitor of Na+/K+-ATPase in cytosol of pulmonary artery smooth muscle. Main methods: (i) By spectrophotometric assay, we identified an inhibitor of Na+/K+-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na+/K+-ATPase Α2Β1 and Α1Β1 isozymes for determining some characteristics of the inhibitor. Key findings: We identified a novel endogenous protein inhibitor of Na+/K+-ATPase having an apparent mol mass of ~70kDa in the cytosolic fraction of the smooth muscle. The IC50 value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the ΑΒ2Α1 and Β121 isozymes of the Na+/K+-ATPase; (ii) it interacted reversibly to the E1 site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na+/K+-ATPase exists as (ΑΒ)2 diprotomer. Significance: The inhibitor binds to the Na+/K+-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where Α2 is more sensitive than α1. © 2010 Elsevier Inc.

Calpain system is generally known to be comprised of three molecules: two Ca2+-dependent proteases: μ- and m-calpains, and their endogenous inhibitor, calpastatin. While calpains have previously been considered as the cytoplasmic enzymes, research in the recent past demonstrated that μ-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. © 2009 Elsevier Inc.

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na +/Ca 2+ exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 μM) in presence of CaCl 2 (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca 2+ chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca 2+ overload, which could arise due to inhibition of Ca 2+ efflux by the forward-mode NCX and that could lead to sustained Ca 2+ overload in the smooth muscle leading to pulmonary hypertension. © 2010 Springer Science+Business Media, LLC.

We identified α2, α1, and β1 isoforms of Na+/K+-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α2 β1 isozyme of Na+/K+-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C12E8), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C12E8, whereas C12E8 was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α2 β1 isozyme of Na+/K+-ATPase elicited higher E1Na-E2 K transition compared with that of the C12E8- and Triton X-100-purified enzyme. The rate of Na+ efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C12E8-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α2β1 isozyme of Na+/K+-ATPase possessed more organized secondary structure compared to the C12E8- and Triton X-100-purified isozyme. © Springer Science+Business Media, LLC. 2008.

Aims: We sought to determine the mechanisms of an increase in Ca2+ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na+/K+-ATPase inhibition by ouabain. Main methods: The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na+ and Ca2+ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings: We identified the α2β1 and α1β1 isozymes of Na+/K+-ATPase in caveolae vesicles, and only the α1β1 isozyme in noncaveolae fraction of the plasma membrane. The α2-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na+/H+-exchange inhibitor) and tetrodotoxin (voltage-gated Na+-channel inhibitor) pretreatment prevented ouabain induced increase in Na+ and Ca2+ levels. Ouabain induced increase in Ca2+ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na+/Ca2+-exchange inhibitor) and verapamil (L-type Ca2+-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca2+ level in the caveolae vesicles, indicating that apart from Na+/Ca+-exchanger and L-type Ca2+-channels, "slip-mode conductance" of Na+ channels could also be involved in this scenario. Significance: Inhibition of α2 isoform of Na+/K+-ATPase by ouabain plays a crucial role in modulating the Ca2+ influx regulatory components in the caveolae microdomain for marked increase in (Ca2+)i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension. © 2008 Elsevier Inc. All rights reserved.

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 μM) stimulates μ-calpain activity and subsequently cleaves Na+/Ca2+ exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca2+ chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca2+ level in the mitochondria, which subsequently dissociates μ-calpain-calpastatin association leading to the activation of μ-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110 kDa) resulting in the doublet of ∼54-56 kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified μ-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca2+ overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle. © 2008 Elsevier Inc. All rights reserved.

Treatment of bovine pulmonary smooth muscle cells with the TxA2 mimetic, U46619 stimulated [Ca2+]i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (KV channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca2+]i, indicating a role of KV-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the KV-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca2+]i in the cells. Calphostin C pretreatment also markedly reduced p47phox phosphorylation and translocation to the membrane and association with p22phox, a component of Cyt.b558 of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O2{radical dot}--mediated regulation of KV-LVOCC axis leading to an increase in [Ca2+]i by U46619 in the cells. © 2009 Elsevier Inc. All rights reserved.

Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that μ-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that μ-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that μ-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between μ-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria. © 2007 Elsevier Inc. All rights reserved.

Ca2+ is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca2+)i level in situ. The Ca2+ signal inducing contraction in cardiac muscle originates from two sources. Ca2+ enters the cell through voltage dependent Ca2+ channels. This Ca2+ binds to and activates Ca2+ release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca2+ induced Ca2+ release (CICR) process. Entry of Ca2+ with each contraction requires an equal amount of Ca2+ extrusion within a single heartbeat to maintain Ca2+ homeostasis and to ensure relaxation. Cardiac Ca2+ extrusion mechanisms are mainly contributed by Na+/Ca2+ exchanger and ATP dependent Ca2+ pump (Ca2+-ATPase). These transport systems are important determinants of (Ca2+)i level and cardiac contractility. Altered intracellular Ca2+ handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the β-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/ intracellular Ca2+ release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the β-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca2+ regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca2+ handling such as RyR, SERCA2a, and PLN. Controlling these molecular functions by different agents have been found to be beneficial in some experimental conditions. © Springer Science+Business Media, LLC 2006.

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na2CO3 and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale. © 2007 Elsevier B.V. All rights reserved.

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of μ-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of μ-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as μ-calpain and calpastatins. Immunoprecipitation study revealed molecular association between μ-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of μ-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis. © 2007 Elsevier Inc. All rights reserved.

The properties of Ca2+-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn- phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C 12E8) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca 2+-ATPase with a greater specific activity than solubilization with C12E8 or Triton X-100. DHPC was determined to be superior to C12E8; while that the C12E8 was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca2+-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C12E8 and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca 2+ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C12E8 and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca2+-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C12E8 and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca2+ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca2+-ATPase retained more organized and native secondary conformation compared to C12E8 and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C12E8 and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca 2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme. © 2005 Elsevier B.V. All rights reserved.