Journal articles on the topic 'Calpain 1'
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1
Upla, Paula, Varpu Marjomäki, Liisa Nissinen, Camilla Nylund, Matti Waris, Timo Hyypiä, and Jyrki Heino. "Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1." Journal of Virology 82, no. 3 (November 21, 2007): 1581–90. http://dx.doi.org/10.1128/jvi.01375-07.
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ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.
2
Weber, Jonasz J., Eva Haas, Yacine Maringer, Stefan Hauser, Nicolas L. P. Casadei, Athar H. Chishti, Olaf Riess, and Jeannette Hübener-Schmid. "Calpain-1 ablation partially rescues disease-associated hallmarks in models of Machado-Joseph disease." Human Molecular Genetics 29, no. 6 (January 21, 2020): 892–906. http://dx.doi.org/10.1093/hmg/ddaa010.
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Abstract Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado–Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases.
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Ben-Aharon, Irit, Paula R. Brown, Nir Etkovitz, Edward M. Eddy, and Ruth Shalgi. "The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse." Reproduction 129, no. 4 (April 2005): 435–42. http://dx.doi.org/10.1530/rep.1.00255.
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There is some evidence suggesting that Ca2+is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+takes part in its regulation. However, the precise roles of Ca2+in spermatogenesis remain to be elucidated. Calpains are a family of Ca2+-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca2+-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain’s ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.
4
Baudry, Michel. "Calpain-1 and Calpain-2 in the Brain: Dr. Jekill and Mr Hyde?" Current Neuropharmacology 17, no. 9 (August 22, 2019): 823–29. http://dx.doi.org/10.2174/1570159x17666190228112451.
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While the calpain system has now been discovered for over 50 years, there is still a paucity of information regarding the organization and functions of the signaling pathways regulated by these proteases, although calpains play critical roles in many cell functions. Moreover, calpain overactivation has been shown to be involved in numerous diseases. Among the 15 calpain isoforms identified, calpain-1 (aka µ-calpain) and calpain-2 (aka m-calpain) are ubiquitously distributed in most tissues and organs, including the brain. We have recently proposed that calpain-1 and calpain- 2 play opposite functions in the brain, with calpain-1 activation being required for triggering synaptic plasticity and neuroprotection (Dr. Jekill), and calpain-2 limiting the extent of plasticity and being neurodegenerative (Mr. Hyde). Calpain-mediated cleavage has been observed in cytoskeleton proteins, membrane-associated proteins, receptors/channels, scaffolding/anchoring proteins, and protein kinases and phosphatases. This review will focus on the signaling pathways related to local protein synthesis, cytoskeleton regulation and neuronal survival/death regulated by calpain-1 and calpain-2, in an attempt to explain the origin of the opposite functions of these 2 calpain isoforms. This will be followed by a discussion of the potential therapeutic applications of selective regulators of these 2 calpain isoforms.
5
Murphy, Robyn M., Rodney J. Snow, and Graham D. Lamb. "μ-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans." American Journal of Physiology-Cell Physiology 290, no. 1 (January 2006): C116—C122. http://dx.doi.org/10.1152/ajpcell.00291.2005.
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μ-calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that μ-calpain becomes proteolytically active in the presence of 2–200 μM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; ∼1 μM) than the levels at which μ-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 μM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for μ-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with ∼15% autolysis observed after 1-min exposure to 2.5 μM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze μ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s “all-out” cycling, n = 8; and 70% V̇o2 peak until fatigue, n = 3) on the amount of autolyzed μ-calpain or calpain-3 in human muscle. No significant autolysis of μ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.
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McCartney, Christian-Scott E., Qilu Ye, Robert L. Campbell, and Peter L. Davies. "Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide." Journal of Biological Chemistry 293, no. 46 (September 25, 2018): 17716–30. http://dx.doi.org/10.1074/jbc.ra118.004803.
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Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit–containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.
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Covington, Marisa D., David D. Arrington, and Rick G. Schnellmann. "Calpain 10 is required for cell viability and is decreased in the aging kidney." American Journal of Physiology-Renal Physiology 296, no. 3 (March 2009): F478—F486. http://dx.doi.org/10.1152/ajprenal.90477.2008.
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Aging is associated with abnormalities in kidney function, but the exact mechanisms are unknown. We examined calpains 1, 2, and 10 protein levels in kidneys from rats, mice, and humans of various ages and determined whether calpain 10 is required for cell viability. Calpain 10 protein expression decreased in the kidney, but not in the liver, of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. There was no change in calpains 1 or 2 levels in the kidney or liver in control and caloric-restricted aging rats. Aging mice also exhibited decreased calpain 10 protein levels. Calpain 10 protein and mRNA levels decreased linearly in human kidney samples with age in the absence of changes in calpains 1 or 2. Our laboratory previously found calpain 10 to be expressed in both the cytosol and mitochondria of rabbit renal proximal tubular cells (RPTC). Adenoviral-delivered shRNA to rabbit RPTC decreased mitochondrial calpain 10 expression below detectable levels by 3 days while cytosolic calpain 10 levels remained unchanged at 3 days and decreased to ∼20% of control by 5 days. Knockdown of mitochondrial calpain 10 resulted in nuclear condensation and cleaved procaspase 3, markers of apoptosis. In summary, mitochondrial calpain 10 is required for cell viability and calpain 10 levels specifically decrease in aging rat, mice, and human kidney tissues when renal function decreases, suggesting that calpain 10 is required for renal function and is a biomarker of the aging kidney.
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Piper, Ann-Katrin, Reece A. Sophocleous, Samuel E. Ross, Frances J. Evesson, Omar Saleh, Adam Bournazos, Joe Yasa, et al. "Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy." American Journal of Physiology-Cell Physiology 318, no. 6 (June 1, 2020): C1226—C1237. http://dx.doi.org/10.1152/ajpcell.00408.2019.
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The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 ( CAPNS1−/−) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 ( CAPN1−/−) or -2 ( CAPN2−/−) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 ( CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.
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Pánico, Pablo, Marcia Hiriart, Patricia Ostrosky-Wegman, and Ana María Salazar. "TUG is a calpain-10 substrate involved in the translocation of GLUT4 in adipocytes." Journal of Molecular Endocrinology 65, no. 3 (October 2020): 45–57. http://dx.doi.org/10.1530/jme-19-0253.
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The calpain-10 (CAPN10) protease is implicated in the translocation of the glucose transporter 4 (GLUT4), which is retained in the Golgi matrix via the Tether containing a UBX domain for GLUT4 (TUG) protein. Insulin stimulation induces the proteolytic processing of TUG, which leads to the translocation of GLUT4 to the cell membrane. We tested whether TUG is a CAPN10 substrate. Proteolysis of TUG by calpains was assessed using a cell-free system containing calpain-1 and TUG. In situ proteolysis of TUG by calpains was demonstrated in 3T3-L1 adipocytes in the presence of insulin or calpain inhibitors to modulate calpain activity. Proteolysis of TUG by CAPN10 was confirmed using transient or stable silencing of CAPN10 in 3T3-L1 adipocytes. Calpains proteolyzed the C-terminus of TUG in vitro. In adipocytes, insulin-induced cleavage of TUG was correlated with the activation of calpains. Treatment with calpain inhibitors reduced TUG cleavage, resulting in impaired GLUT4 translocation without altering Akt phosphorylation. Furthermore, CAPN10 but not calpain-1 or calpain-2 colocalized with GLUT4 in the absence of insulin, and their colocalization was reduced after stimulation with insulin. Finally, we demonstrated that CAPN10 knockdown reduced the proteolysis of TUG without altering the phosphorylation of Akt or the expression of the Usp25m protease. Thus, our results provide evidence that the TUG protein is cleaved by CAPN10 to regulate GLUT4 translocation.
10
Ou, B. R., and N. E. Forsberg. "Determination of skeletal muscle calpain and calpastatin activities during maturation." American Journal of Physiology-Endocrinology and Metabolism 261, no. 6 (December 1, 1991): E677—E683. http://dx.doi.org/10.1152/ajpendo.1991.261.6.e677.
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Our objectives were to characterize events underlying changes in skeletal muscle calpain and calpastatin activities, using maturation as a model. Muscle samples were taken from rabbits of four ages (newborn and 1, 2, and 5 mo old). Concentrations of RNA and protein and activities of calpains I and II and calpastatin were determined. Steady-state concentrations of mRNAs encoding calpain I, calpain II, calpastatin, alpha- and beta-tubulin, and beta-actin were determined using Northern blot analysis. Calpain and calpastatin activities declined markedly between birth and 1 mo of age and remained unchanged thereafter. Several factors accounted for the neonatal losses of calpains and calpastatin. First, muscle protein concentration increased between birth and 1 mo of age and diluted calpain and calpastatin specific activities. Second, there was a marked reduction of muscle RNA concentration between birth and 1 mo of age, which indicates that protein synthetic capacity declined with age. Finally, calpastatin mRNA concentration declined between birth and 1 mo of age and further contributed to developmental losses of calpastatin activity. Calpain I mRNA concentration was unaffected by age, and although calpain II mRNA concentration declined with age, losses were not detected between birth and 1 mo; hence age-related changes in calpain I and II activities are not mediated at the mRNA level. The age-related reductions in calpain II and calpastatin mRNA concentrations resembled age-related changes in alpha- and beta-tubulin and beta-actin mRNA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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Wang, Yubin, Yan Liu, Xiaoning Bi, and Michel Baudry. "Calpain-1 and Calpain-2 in the Brain: New Evidence for a Critical Role of Calpain-2 in Neuronal Death." Cells 9, no. 12 (December 16, 2020): 2698. http://dx.doi.org/10.3390/cells9122698.
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Calpains are a family of soluble calcium-dependent proteases that are involved in multiple regulatory pathways. Our laboratory has focused on the understanding of the functions of two ubiquitous calpain isoforms, calpain-1 and calpain-2, in the brain. Results obtained over the last 30 years led to the remarkable conclusion that these two calpain isoforms exhibit opposite functions in the brain. Calpain-1 activation is required for certain forms of synaptic plasticity and corresponding types of learning and memory, while calpain-2 activation limits the extent of plasticity and learning. Calpain-1 is neuroprotective both during postnatal development and in adulthood, while calpain-2 is neurodegenerative. Several key protein targets participating in these opposite functions have been identified and linked to known pathways involved in synaptic plasticity and neuroprotection/neurodegeneration. We have proposed the hypothesis that the existence of different PDZ (PSD-95, DLG and ZO-1) binding domains in the C-terminal of calpain-1 and calpain-2 is responsible for their association with different signaling pathways and thereby their different functions. Results with calpain-2 knock-out mice or with mice treated with a selective calpain-2 inhibitor indicate that calpain-2 is a potential therapeutic target in various forms of neurodegeneration, including traumatic brain injury and repeated concussions.
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Arora, A. S., P. de Groen, Y. Emori, and G. J. Gores. "A cascade of degradative hydrolase activity contributes to hepatocyte necrosis during anoxia." American Journal of Physiology-Gastrointestinal and Liver Physiology 270, no. 2 (February 1, 1996): G238—G245. http://dx.doi.org/10.1152/ajpgi.1996.270.2.g238.
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Calpain proteases contribute to hepatocyte necrosis during anoxia. Our aim was to ascertain the mechanism causing calpain activation during anoxia. In rat hepatocytes, a twofold increase in calpain activity occurred despite the lack of an increase in cytosolic Ca2+ concentration ([Ca2+]i). The increase in calpain activity was not associated with an increase in calpain mRNA or a decrease in calpastatin mRNA expression. Because phospholipid degradation products generated by phospholipases can activate calpains at physiological [Ca2+]i, we determined the effect of phospholipase inhibitors and activators on calpain activity. Pretreatment of hepatocytes with fluphenazine, a phospholipase inhibitor, decreased calpain activation and improved cell survival. Melittin, a phospholipase A2 activator, increased calpain activity and potentiated cell killing. Finally, phospholipid degradation preceded the increase in calpain activity. Thus the enhanced calpain activity occurring in hepatocytes during anoxia 1) is regulated at the posttranslational level and 2) appears to be dependent on phospholipase activity. These data suggest a novel cascade for degradative hydrolase activity during hepatocyte necrosis by anoxia with phospholipase-mediated activation of calpains.
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Kuchay, Shafi M., William P. Fay, and Athar H. Chishti. "Double Knockouts Reveal That Protein Tyrosine Phosphatase 1B Is a Physiological Substrate of Calpain-1 in Platelets." Blood 108, no. 11 (November 16, 2006): 396. http://dx.doi.org/10.1182/blood.v108.11.396.396.
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Abstract Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b3 subunit of αIibβ3 integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b3 subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.
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Moshal, Karni S., Mahavir Singh, Utpal Sen, Dorothea Susanne E. Rosenberger, Brooke Henderson, Neetu Tyagi, Hong Zhang, and Suresh C. Tyagi. "Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC." American Journal of Physiology-Heart and Circulatory Physiology 291, no. 6 (December 2006): H2825—H2835. http://dx.doi.org/10.1152/ajpheart.00377.2006.
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Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative tress.
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Vermaelen, Marianne, Pascal Sirvent, Fabrice Raynaud, Catherine Astier, Jacques Mercier, Alain Lacampagne, and Olivier Cazorla. "Differential localization of autolyzed calpains 1 and 2 in slow and fast skeletal muscles in the early phase of atrophy." American Journal of Physiology-Cell Physiology 292, no. 5 (May 2007): C1723—C1731. http://dx.doi.org/10.1152/ajpcell.00398.2006.
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Calpains have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. However, limited data are available about the specific involvement of each calpain in the early stages of muscle atrophy. The aims of this study were to determine whether calpains 1 and 2 are autolyzed after a short period of muscle disuse, and, if so, where in the myofibers the autolyzed products are localized. In the rat soleus muscle, 5 days of immobilization increased autolyzed calpain 1 in the particulate and not the soluble fraction. Conversely, autolyzed calpain 2 was not found in the particulate fraction, whereas it was increased in the soluble fraction after immobilization. In the less atrophied plantaris muscle, no difference was noted between the control and immobilized groups whatever the fraction or calpain. Other proteolytic pathways were also investigated. The ubiquitin-proteasome pathway was activated in both skeletal muscles, and caspase 3 was activated only in the soleus muscle. Taken together, our data suggest that calpains 1 and 2 are involved in atrophy development in slow type muscle exclusively and that they have different regulation and protein targets. Moreover, the activation of proteolytic pathways appears to differ in slow and fast muscles, and the proteolytic mechanisms involved in fast-type muscle atrophy remain unclear.
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Liu, Xiuli, Juanita J. Rainey, Jay F. Harriman, and Rick G. Schnellmann. "Calpains mediate acute renal cell death: role of autolysis and translocation." American Journal of Physiology-Renal Physiology 281, no. 4 (October 1, 2001): F728—F738. http://dx.doi.org/10.1152/ajprenal.2001.281.4.f728.
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The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only μ- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT μ- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either μ- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either μ- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.
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Elagib, Kamaleldin E., Lorrie L. Delehanty, Ivailo Mihaylov, and Adam Goldfarb. "Calpain Regulation of Megakaryopoiesis through a Positive Regulatory Loop Involving P-TEFb, GATA-1, and RUNX1." Blood 114, no. 22 (November 20, 2009): 566. http://dx.doi.org/10.1182/blood.v114.22.566.566.
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Abstract Abstract 566 GATA-1 and RUNX1 cooperate in programming megakaryocytic development through the critical intermediation of the active P-TEFb kinase complex (Cdk9/cyclin T1). RUNX1 on its own traps P-TEFb in inactive chromatin loops and causes RNA pol II (RNAP II) stalling. GATA-1 by contrast remodels chromatin loops and promotes RNAP II elongation. Thus, P-TEFb most likely integrates and resolves conflicting signals from RUNX1 and GATA-1 to coordinate orderly activation of megakaryocytic target genes during development. P-TEFb activity is tightly regulated by a large network of interacting factors including Cdk9, cyclin T1, HEXIM1 and 7SK snRNA, RNA processing factors, and transcriptional regulators. During megakaryocytic differentiation, global activation of P-TEFb involves dissociation of HEXIM1 and recruitment of GATA-1, in a manner dependent on Cdk9 activity. The current studies address factors that regulate this dramatic reconfiguration of the P-TEFb complex during initiation of megakaryocytic differentiation. Candidate factors were identified based on two criteria: participation in the P-TEFb complex and specific upregulation in megakaryocytic differentiation. Notably, analysis of a P-TEFb interactome database identified the protease calpain 2 and its cofactor calpain S1 as participants in this complex (Jeronimo CD. Et. al. Mol Cell 2007). We confirmed a physical interaction by coimmunoprecipitation of endogenous calpain 2 and cyclin T1. Analysis of gene expression databases revealed three striking features of calpain 2: 1) strong upregulation early in megakaryocytic differentiation, 2) defective upregulation in GATA-1-deficient megakaryocytes, and 3) defective upregulation in megakaryocytes expressing GATA-1s, a mutant form associated with Down syndrome-associated megakaryocytic disorders (DS-TMD and DS-AMKL). The role of calpain in megakaryocytic differentiation of primary human CD34+ progenitors was assessed by shRNA knockdown (kd) of calpain S1, a required cofactor for calpains 1 and 2, as well as by treatment of cells with the calpain inhibitors calpeptin and Calpain Inhibitor III. All three approaches blocked cellular enlargement, CD41 upregulation, and polyploidization, indicating a critical role for calpain activity in early steps of megakaryocytic differentiation. We next addressed the hypothesis that calpain contributed to megakaryocytic differentiation through positive regulation of P-TEFb activity. In support of this hypothesis, calpain inhibition prevented the P-TEFb-driven processes of RNAP II hyperphosphorylation and HEXIM1 upregulation, both normally seen in megakaryocytic differentiation. In addition, calpain inhibition blocked the transcriptional cooperation of RUNX1 and GATA-1, which we have previously shown to be dependent on P-TEFb activity. How calpain activity regulates P-TEFb remains unclear, but in vitro and in vivo assays identified cyclin T1 and RNAP II as highly sensitive targets of calpain 2/S1 protease activity. Because P-TEFb remodeling in megakaryopoiesis requires Cdk9 kinase activity, we examined the possibility that calpain itself might be regulated by Cdk9, a notion supported by multiple experiments. In particular, Cdk9 inhibition by shRNA kd or flavopiridol treatment prevented the calpain-dependent cleavage of cyclin T1 and RNAP II normally seen in megakaryocytic differentiation. Furthermore, using purified, recombinant factors, in vitro calpain 2 cleavage of the RNAP II carboxy terminal domain (CTD) showed an absolute requirement for active P-TEFb complex. We thus postulate the existence of a novel regulatory circuit in which P-TEFb and calpain control the activity of one another during megakaryocytic differentiation. The participation of RUNX1 and GATA-1 in this circuit is suggested by the requirements for P-TEFb and calpain activity for their transcriptional cooperation. In addition, a murine strain with megakaryocytic GATA-1 deficiency, the GATA-1Lo strain, showed drops in platelet counts when treated with the calpain inhibitor E64, in contrast to to wild type counterparts, which responded with increased platelet counts. This novel regulatory circuit most likely has clinical relevance for at least two reasons: 1) P-TEFb inhibition in GATA-1Lo mice has been shown to elicit a disorder resembling the DS-TMD, and 2) megakaryocytes expressing GATA-1s display defective upregulation of calpains 2 and S1. Disclosures: No relevant conflicts of interest to declare.
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Larsen, Anna K., René Lametsch, John S. Elce, Jørgen K. Larsen, Bo Thomsen, Martin R. Larsen, Moira A. Lawson, Peter A. Greer, and Per Ertbjerg. "Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin." Biochemical Journal 411, no. 3 (April 14, 2008): 657–66. http://dx.doi.org/10.1042/bj20070522.
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Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs.
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Muniappan, Latha, Michihiro Okuyama, Aida Javidan, Devi Thiagarajan, Weihua Jiang, Jessica J. Moorleghen, Lihua Yang, et al. "Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice." Arteriosclerosis, Thrombosis, and Vascular Biology 41, no. 5 (May 5, 2021): 1694–709. http://dx.doi.org/10.1161/atvbaha.120.315546.
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Objective: Cytoskeletal structural proteins maintain cell structural integrity by bridging extracellular matrix with contractile filaments. During abdominal aortic aneurysm (AAA) development, (1) aortic medial degeneration is associated with loss of smooth muscle cell integrity and (2) fibrogenic mesenchymal cells mediate extracellular matrix remodeling. Calpains cleave cytoskeletal proteins that maintain cell structural integrity. Pharmacological inhibition of calpains exert beneficial effects on Ang II (angiotensin II)–induced AAAs in LDLR −/− (low-density receptor deficient) mice. Here, we evaluated the functional contribution of fibrogenic mesenchymal cells-derived calpain-2 on (1) cytoskeletal structural protein and extracellular matrix alterations and (2) AAA progression. Approach and Results: Calpain-2 protein and cytoskeletal protein (filamin and talin) fragmentation are significantly elevated in human and Ang II–induced AAAs in mice. To examine the relative contribution of calpain-2 in AAA development, calpain-2 floxed mice in an LDLR −/− background were bred to mice with a tamoxifen-inducible form of Cre under control of either the ubiquitous promoter, chicken β-actin, or fibrogenic mesenchymal cell-specific promoter, Col1α2 (collagen type 1 alpha 2). Ubiquitous or fibrogenic mesenchymal cell-specific depletion of calpain-2 in mice suppressed Ang II–induced AAAs, filamin/talin fragmentation, while promoting extracellular matrix protein, collagen in the aortas. Calpain-2 silencing in aortic smooth muscle cells or fibroblasts reduced Ang II–induced filamin fragmentation. In addition, silencing of filamin in aortic SMCs significantly reduced collagen protein. Furthermore, calpain-2 deficiency suppressed rupture of established Ang II–induced AAAs in mice. Conclusions: Our studies implicate that calpain-2 deficiency prevents (1) Ang II–induced cytoskeletal structural protein fragmentation and AAA development and (2) stabilize and suppress rupture of established AAAs in mice.
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Wieschhaus, Adam, Anwar Khan, Asma Zaidi, Henry Rogalin, Toshihiko Hanada, Fei Liu, Lucia De Franceschi, Carlo Brugnara, Alicia Rivera, and Athar H. Chishti. "Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology." Biochemical Journal 448, no. 1 (October 18, 2012): 141–52. http://dx.doi.org/10.1042/bj20121008.
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Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+–Cl− co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.
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Gafni, Juliette, Evan Hermel, Jessica E. Young, Cheryl L. Wellington, Michael R. Hayden, and Lisa M. Ellerby. "Inhibition of Calpain Cleavage of Huntingtin Reduces Toxicity." Journal of Biological Chemistry 279, no. 19 (February 23, 2004): 20211–20. http://dx.doi.org/10.1074/jbc.m401267200.
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Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in anin vitrocell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.
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Kuchay, Shafi M., Nayoung Kim, Elizabeth A. Grunz, William P. Fay, and Athar H. Chishti. "Double Knockouts Reveal that Protein Tyrosine Phosphatase 1B Is a Physiological Target of Calpain-1 in Platelets." Molecular and Cellular Biology 27, no. 17 (June 18, 2007): 6038–52. http://dx.doi.org/10.1128/mcb.00522-07.
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ABSTRACT Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1 −/−) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1 −/− platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1 −/− mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.
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Zhao, Ming, Yang Yang, Xueyuan Bi, Xiaojiang Yu, Hanghuan Jia, Huanle Fang, and Weijin Zang. "Acetylcholine Attenuated TNF-α-Induced Apoptosis in H9c2 Cells: Role of Calpain and the p38-MAPK Pathway." Cellular Physiology and Biochemistry 36, no. 5 (2015): 1877–89. http://dx.doi.org/10.1159/000430157.
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Background: Previous studies have shown that inflammation is associated with excessive activation of calpains. Acetylcholine (ACh) has been reported to inhibit pro-inflammatory cytokine release and protect against cardiomyocyte injury. However, there is no direct evidence regarding whether ACh can regulate calpains to exert cardioprotection. To this end, we investigated the effect of ACh on tumour necrosis factor alpha (TNF-α)-induced cardiomyocyte injury and further explored the underlying mechanism. Methods: Flow cytometry and transmission electron microscopy were performed to evaluate apoptosis and cellular ultrastructure. Western blotting was performed to assess changes in protein expression. siRNA was employed to silence specific proteins. Results: TNF-α treatment increased the expression of cleaved caspase-3, calpain-1 and p38-mitogen-activated protein kinase (p38-MAPK). The calpain inhibitor PD150606 and the p38-MAPK inhibitor SB203580 inhibited apoptosis induced by TNF-α. Moreover, SB203580 decreased the expression and activity of calpain-1, possibly related to the up-regulation of calpastatin. ACh significantly inhibited TNF-α-induced cell apoptosis, as evidenced by decreases in caspase-3 cleavage, p38-MAPK phosphorylation, and calpain-1 expression and activity as well as increases in calpastatin expression. These beneficial effects of ACh were abolished by atropine or M2AChR siRNA. Conclusion: Our results suggest that ACh ameliorated TNF-α-induced calpain activation by decreasing p38-MAPK phosphorylation and enhancing calpastatin expression, indicating that calpain may be an important link between inflammatory factors and myocardial cell apoptosis.
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McClung, J. M., A. R. Judge, E. E. Talbert, and S. K. Powers. "Calpain-1 is required for hydrogen peroxide-induced myotube atrophy." American Journal of Physiology-Cell Physiology 296, no. 2 (February 2009): C363—C371. http://dx.doi.org/10.1152/ajpcell.00497.2008.
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Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 μM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (−81%), α-actinin (−40%), desmin (−79%), talin (−37%), and troponin I (−80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate αII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 μM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.
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Douillard, Aymeric, Olivier Galbes, Bernadette Rossano, Barbara Vernus, Anne Bonnieu, Robin Candau, and Guillaume Py. "Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment." Canadian Journal of Physiology and Pharmacology 89, no. 2 (February 2011): 117–25. http://dx.doi.org/10.1139/y10-114.
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Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg–1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.
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GOLL, DARREL E., VALERY F. THOMPSON, HONGQI LI, WEI WEI, and JINYANG CONG. "The Calpain System." Physiological Reviews 83, no. 3 (July 2003): 731–801. http://dx.doi.org/10.1152/physrev.00029.2002.
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Goll, Darrel E., Valery F. Thompson, Hongqi Li, Wei Wei, and Jinyang Cong. The Calpain System. Physiol Rev 83: 731–801, 2003; 10.1152/physrev.00029.2002.—The calpain system originally comprised three molecules: two Ca2+-dependent proteases, μ-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both μ- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55–65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six “domains” in the 80-kDa subunit: 1) a 19-amino acid NH2-terminal sequence; 2) and 3) two domains that constitute the active site, IIa and IIb; 4) domain III; 5) an 18-amino acid extended sequence linking domain III to domain IV; and 6) domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of μ- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.
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Alvarez-Arce, Alejandro, Irene Lee-Rivera, Edith López, Arturo Hernández-Cruz, and Ana María López-Colomé. "Thrombin-Induced Calpain Activation Promotes Protease-Activated Receptor 1 Internalization." International Journal of Cell Biology 2017 (2017): 1–14. http://dx.doi.org/10.1155/2017/1908310.
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The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1–4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2concentration ([Ca+2]i) mediated byGq11αcoupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation ofμ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation ofα-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novelμ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.
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NATH, Rathna, Kadee J. RASER, Daniel STAFFORD, Iradj HAJIMOHAMMADREZA, Avigail POSNER, Hamish ALLEN, Robert V. TALANIAN, Po-wai YUEN, Richard B. GILBERTSEN, and Kevin K. W. WANG. "Non-erythroid α-spectrin breakdown by calpain and interleukin 1 β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis." Biochemical Journal 319, no. 3 (November 1, 1996): 683–90. http://dx.doi.org/10.1042/bj3190683.
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The cytoskeletal protein non-erythroid α-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), α-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1β-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. α-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(β)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.
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Arnandis, Teresa, Ivan Ferrer-Vicens, Luis Torres, Concha García, Elena R. Garcia-Trevijano, Rosa Zaragoza, and Juan R. Viña. "Differential functions of calpain 1 during epithelial cell death and adipocyte differentiation in mammary gland involution." Biochemical Journal 459, no. 2 (March 28, 2014): 355–68. http://dx.doi.org/10.1042/bj20130847.
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The results of the present study unveil novel and important nuclear functions of calpains in tissue remodelling. Although inducing nuclear permeability during programmed cell death, histone H3 is identified as a new target of calpain-1 upon adipocyte differentiation.
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Macqueen, Daniel J., and Alexander H. Wilcox. "Characterization of the definitive classical calpain family of vertebrates using phylogenetic, evolutionary and expression analyses." Open Biology 4, no. 4 (April 2014): 130219. http://dx.doi.org/10.1098/rsob.130219.
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The calpains are a superfamily of proteases with extensive relevance to human health and welfare. Vast research attention is given to the vertebrate ‘classical’ subfamily, making it surprising that the evolutionary origins, distribution and relationships of these genes is poorly characterized. Consequently, there exists uncertainty about the conservation of gene family structure, function and expression that has been principally defined from work with mammals. Here, more than 200 vertebrate classical calpains were incorporated in phylogenetic analyses spanning an unprecedented range of taxa, including jawless and cartilaginous fish. We demonstrate that the common vertebrate ancestor had at least six classical calpains, including a single gene that gave rise to CAPN11 , 1 , 2 and 8 in the early jawed fish lineage, plus CAPN3 , 9 , 12 , 13 and a novel calpain gene, hereafter named CAPN17 . We reveal that while all vertebrate classical calpains have been subject to persistent purifying selection during evolution, the degree and nature of selective pressure has often been lineage-dependent. The tissue expression of the complete classic calpain family was assessed in representative teleost fish, amphibians, reptiles and mammals. This highlighted systematic divergence in expression across vertebrate taxa, with most classic calpain genes from fish and amphibians having more extensive tissue distribution than in amniotes. Our data suggest that classical calpain functions have frequently diverged during vertebrate evolution and challenge the ongoing value of the established system of classifying calpains by expression.
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Laajala, Mira, Minna M. Hankaniemi, Juha A. E. Määttä, Vesa P. Hytönen, Olli H. Laitinen, and Varpu Marjomäki. "Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein." Viruses 11, no. 12 (November 28, 2019): 1106. http://dx.doi.org/10.3390/v11121106.
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Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.
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Suzuki, K., K. Shimizu, T. Hamamoto, Y. Nakagawa, T. Murachi, and T. Yamamuro. "Characterization of proteoglycan degradation by calpain." Biochemical Journal 285, no. 3 (August 1, 1992): 857–62. http://dx.doi.org/10.1042/bj2850857.
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Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.
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Rintanen, Nina, Mikko Karjalainen, Jonna Alanko, Lassi Paavolainen, Anita Mäki, Liisa Nissinen, Moona Lehkonen, et al. "Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway." Molecular Biology of the Cell 23, no. 3 (February 2012): 448–63. http://dx.doi.org/10.1091/mbc.e11-06-0548.
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Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.
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Meier, Markus, Harald H. Klein, Jan Kramer, Maren Drenckhan, and Morten Schütt. "Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling." Journal of Endocrinology 193, no. 1 (April 2007): 45–51. http://dx.doi.org/10.1677/joe.1.07087.
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Calpains are a family of non-lysosomal cytoplasmatic cysteine proteases. Since calpain 10 (CAPN10), a member of the calpain family of proteases, has been found to represent a putative diabetes susceptibility gene, it was argued that calpains may be involved in the development of type 2 diabetes. The functional role of calpains in insulin signaling and/or insulin action is, however, not clear. We investigated the effects of the calpains 1 and 2 inhibitor PD151746 on insulin signaling and insulin action in human hepatoma G2 cells (HepG2). HepG2 cells were incubated without (−PD) or with (+PD) 5.33 μmol/l PD151746 for different times and then stimulated with 100 nmol/l insulin for 0 (t0), 5 (t5), 15 (t15), 30 (t30), 45 (t45), and 60 (t60) min. After solubilization of the cells, insulin receptor kinase activity, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol-3 kinase (PI3-kinase), PI3-kinase activity, Thr308 phosphorlyation of Akt, amount of protein tyrosine phosphatase-ε (PTPε), and glycogen synthase activity were determined. Incubation with PD151746 resulted in a significant reduction of insulin-stimulated glycogen synthesis compared with cells not pre-incubated with the calpain inhibitor (−PD: t0, 4.90 ± 1.20%; t5, 5.90 ± 1.02%; t15, 5.29 ± 0.95%; t30, 5.60 ± 1.10%; t45, 5.52 ± 0.90%; t60, 5.67 ± 0.97%;+PD: t0, 4.56 ± 1.10%; t5, 6.16 ± 1.05%; t15, 7.52 ± 1.09%; t30, 7.68 ± 1.10%; t45, 8.28 ± 0.89%; t60, 7.69 ± 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPε in the cells after 12 h (−PD: t1, 0.85 ± 0.18 RU (Relative unit); t8, 0.87 ± 0.18 RU; t12, 0.9 ± 0.13 RU; +PD: t1, 0.92 ± 0.21 RU; t8, 1.1 ± 0.15 RU; t12, 1.34 ± 0.16 RU; P < 0.05). Calpain inhibition with PD151746 had no effect on the insulin stimulation of the investigated insulin signaling parameters. These results in HepG2 cells suggest that calpains play a role in the hepatic regulation of insulin-stimulated glycogen synthesis independent of the PI3-kinase/Akt signaling pathway.
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Bartus, Raymond T. "The Calpain Hypothesis of Neurodegeneration: Evidence for a Common Cytotoxic Pathway." Neuroscientist 3, no. 5 (September 1997): 314–27. http://dx.doi.org/10.1177/107385849700300513.
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Calpain's general function and pathogenic role in the CNS are reviewed. Collectively, the literature indicates that calpain proteolysis plays a common and important role in a variety of acute neurodegenerative conditions, including focal ischemia (stroke), global ischemia, traumatic brain injury, and spinal cord injury. This evidence indicates that 1) calpain is activated in an abnormally sustained fashion during cellular events commonly associated with neurodegeneration (e.g., excessive interstitial glutamate and cytosolic calcium); 2) many of calpain's preferred substrates are degraded as important components in these neurodegenerative conditions; 3) calpain activation occurs early in the pathogenic cascade of each, prior to onset of substantial cell death; and 4) calpain inhibitors can effectively reduce the severity of neuronal damage and loss of function normally associated with these acute neurodegenerative perturbations. Calpain proteolysis is also implicated in chronic neurodegenerative diseases, with the strength of current evidence varying among specific diseases. The evidence accumulated for a plausible role in Alzheimer's disease (AD) is currently the strongest. For example, empirical links have been established between abnormal calpain proteolysis and 1) the cellular formation of classic Alzheimer's pathology, such as β-amyloid plaques, neurofibrillary tangles, and Alz-50 immunoreactivity; 2) the brain regions with greatest concentrations of AD-related pathology; and 3) the degeneration of key brain pathways vulnerable in the early stages of the disease. Similar, though less extensive, evidence exists for a potential role of abnormal calpain proteolysis in Parkinson's disease. Finally, for several other chronic neurodegenerative conditions (e.g., Huntington's disease and amyotrophic lateral sclerosis), early evidence is emerging that calpain may also play some pathogenic role. Thus, these data support the possibility that uncontrolled calpain proteolysis may contribute to and/or accelerate the loss of neurons associated with a wide range of neurodegenerative conditions and may, therefore, represent an important, final common cytotoxic pathway for many diverse forms of neurodegeneration. NEUROSCIENTIST 3:314–327, 1997
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Goll, Darrel E., Valery F. Thompson, Richard G. Taylor, and Ahmed Ouali. "The calpain system and skeletal muscle growth." Canadian Journal of Animal Science 78, no. 4 (December 1, 1998): 503–12. http://dx.doi.org/10.4141/a98-081.
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The first protein of a group of proteins now identified as belonging to the calpain system was purified in 1976. The calpain system presently is known to be constituted of three well-characterized proteins; several lesser studied proteins that have been isolated from invertebrates; and 10 mRNAs, two each in Drosophila and C. elegans and six in vertebrates, that encode proteins, which, based on sequence homology, belong to the calpain family. The three well-characterized proteins in the calpain family include two Ca2+-dependent proteolytic enzymes, µ-calpain and m-calpain, and a protein, calpastatin, that has no known activity other than to inhibit the two calpains. A substantial amount of experimental evidence accumulated during the past 25 yr has shown that the calpain system has an important role both in rate of skeletal muscle growth and in rate and extent of postmortem tenderization. Calpastatin seems to be the variable component of the calpain system, and skeletal muscle calpastatin activity is highly related to rate of muscle protein turnover and rate of postmortem tenderization. The current paradigm is that high calpastatin activity: 1) decreases rate of muscle protein turnover and hence is associated with an increased rate of skeletal muscle growth; and 2) decreases calpain activity in postmortem muscle and hence is associated with a lower rate of postmortem tenderization. This article summarizes some of the known properties of the calpain system and discusses the potential importance of the calpain system to animal science. Key words: Calpain, calpastatin, postmortem tenderization, skeletal muscle growth
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Saatman, Kathryn E., Babak Abai, Ashley Grosvenor, Christian K. Vorwerk, Douglas H. Smith, and David F. Meaney. "Traumatic Axonal Injury Results in Biphasic Calpain Activation and Retrograde Transport Impairment in Mice." Journal of Cerebral Blood Flow & Metabolism 23, no. 1 (January 2003): 34–42. http://dx.doi.org/10.1097/01.wcb.0000035040.10031.b0.
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Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intra-axonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.
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Nwankwo, Jennifer O., Sha Huang, Jongyoon Han, and Athar H. Chishti. "Genetic Deletion of Calpain-1 Improves Reticulocyte Deformability in a Mouse Model of Severe Sickle Cell Disease." Blood 124, no. 21 (December 6, 2014): 2698. http://dx.doi.org/10.1182/blood.v124.21.2698.2698.
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Abstract Introduction: Sickle cell disease (SCD) is caused by the substitution of a valine residue for glutamic acid in the β-globin chain of hemoglobin. The resulting sickle hemoglobin rapidly polymerizes upon deoxygenation, forming sickle RBCs (sRBCs) with reduced deformability. Less deformable sRBCs have an impaired ability to navigate the microvasculature, thereby potentiating vaso-occlusive crises, the hallmark of SCD. Deoxygenation-mediated sickling also induces influx of calcium leading to calpain-1 mediated RBC dehydration via the Gardos channel. Calpains are calcium-activated cysteine proteases involved in numerous cellular processes. In mammals, calpain-1 and calpain-2 are the dominantly expressed isoforms, along with the endogenous inhibitor, calpastatin. Previously, we have shown that genetic deletion of calpain-1 improves RBC deformability in C57BL/6 mice (Wieschhaus et al, Biochem J 2012). Similarly, pharmacological inhibition of calpain-1 prevents dehydration of sRBCs in the SAD model of mild SCD (De Franceschi, et al, FASEB J 2013). Here, our objective is to determine whether genetic deletion of calpain-1 in the HbSS-Townes mice – a severe SCD mouse model, improves RBC deformability in both normoxic and hypoxic conditions. Methods: Transgenic SCD mice (HbSS-Townes) expressing >99% human HbS were backcrossed with calpain-1 knockout (CKO) mice to obtain sickle mice lacking calpain-1 (HbSS-Townes-CKO). Casein zymography was performed to confirm the complete loss of calpain-1 activity in the RBCs of the HbSS-Townes-CKO mice. Thiazole orange-based FACS revealed that HbSS-Townes and HbSS-Townes-CKO mice express ~60% reticulocytes. The elevated reticulocyte count prompted us to evaluate the effect of calpain-1 deficiency on reticulocyte deformability. We utilized a microfluidic-based flow cytometer to assess single reticulocyte deformability following thiazole orange staining upon treatment with calcium ionophore (A23187) and CaCl2. We further probed the molecular mechanisms of RBC deformability by assessing calcium-induced proteolysis of RBC cytoskeletal proteins. RBCs were harvested from HbSS-Townes and HbSS-Townes-CKO mice, treated with A23187 and CaCl2 to activate calpain-1, and subjected to RBC ghosts preparation and gel electrophoresis. Cytoskeletal proteins were visualized and quantified. Guided by our previous findings that calpain-1 knockout (CKO) mice exhibit a clot retraction defect (Kuchay, et al, MCB 2007), we harvested un-coagulated blood from HbSS-Townes and HbSS-Townes-CKO mice, and assessed their clot formation phenotype in calcium-free RPMI medium at different time intervals. Results: Casein zymography revealed a complete loss of calpain-1 enzyme activity (indicated by lack of casein cleavage) in the HbSS-Townes-CKO mice. Furthermore, reticulocytes from HbSS-Townes-CKO mice are more deformable than their HbSS-Townes counterparts under steady state as well as upon treatment with calcium ionophore and CaCl2. An improvement in sickle reticulocyte deformability has significant potential to prevent vaso-occlusive crisis under situations of hypoxia-induced ischemia reperfusion injury – a hypothesis that is currently being tested. In addition, our preliminary results show that RBC ghosts from HbSS-Townes mice exhibit a transglutaminase-mediated cross-linking defect in the cytoskeleton that is restored in the HbSS-Townes-CKO mice. This finding has significant implications for an increased in vivo RBC life span of HbSS-Townes-CKO mice. Finally, we found that HbSS-Townes-CKO mice display a whole blood clot retraction defect, similar to our previous findings on platelets harvested from calpain-1 knockout (CKO) mice. Our findings showing improved sickle cell deformability and clot retraction phenotype are consistent with recently published findings demonstrating an interaction between RBCs and platelets in clot formation and in vivo thrombosis. To test this intriguing hypothesis, we are currently evaluating whether HbSS-Townes-CKO mice exhibit reduced thrombus formation upon laser-induced in vivo thrombosis. Together, our results report the first mouse model of severe sickle cell disease lacking calpain-1 activity, and unveil a new role for calpain-1 in the multi-step process regulating SCD pathogenesis. These findings might serve as a basis for exploring calpain-1 inhibition as a therapeutic target for SCD. Disclosures No relevant conflicts of interest to declare.
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Watson, P. H., S. T. Mortimer, K. K. W. Wang, D. E. Croall, and D. A. Hanley. "Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments." Journal of Molecular Endocrinology 15, no. 1 (August 1995): 61–71. http://dx.doi.org/10.1677/jme.0.0150061.
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ABSTRACT Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca2+-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca2+-regulated PTH secretion. The present study was undertaken to determine the presence of calciumactivated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71·5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47–84 and bPTH69–84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments.
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Liu, Ming Cheng, Veronica Akle, Wenrong Zheng, Jitendra R. Dave, Frank C. Tortella, Ronald L. Hayes, and Kevin K. W. Wang. "Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis." Biochemical Journal 394, no. 3 (February 24, 2006): 715–25. http://dx.doi.org/10.1042/bj20050905.
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A major theme of TBI (traumatic brain injury) pathology is the over-activation of multiple proteases. We have previously shown that calpain-1 and -2, and caspase-3 simultaneously produced αII-spectrin BDPs (breakdown products) following TBI. In the present study, we attempted to identify a comprehensive set of protease substrates (degradome) for calpains and caspase-3. We further hypothesized that the TBI differential proteome is likely to overlap significantly with the calpain- and caspase-3-degradomes. Using a novel HTPI (high throughput immunoblotting) approach and 1000 monoclonal antibodies (PowerBlot™), we compared rat hippocampal lysates from 4 treatment groups: (i) naïve, (ii) TBI (48 h after controlled cortical impact), (iii) in vitro calpain-2 digestion and (iv) in vitro caspase-3 digestion. In total, we identified 54 and 38 proteins that were vulnerable to calpain-2 and caspase-3 proteolysis respectively. In addition, the expression of 48 proteins was down-regulated following TBI, whereas that of only 9 was up-regulated. Among the proteins down-regulated in TBI, 42 of them overlapped with the calpain-2 and/or caspase-3 degradomes, suggesting that they might be proteolytic targets after TBI. We further confirmed several novel TBI-linked proteolytic substrates, including βII-spectrin, striatin, synaptotagmin-1, synaptojanin-1 and NSF (N-ethylmaleimide-sensitive fusion protein) by traditional immunoblotting. In summary, we demonstrated that HTPI is a novel and powerful method for studying proteolytic pathways in vivo and in vitro.
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Yeh, J.-Y., B.-R. Ou, and N. E. Forsberg. "Effects of dexamethasone on muscle protein homeostasis and on calpain and calpastatin activities and gene expression in rabbits." Journal of Endocrinology 141, no. 2 (May 1994): 209–17. http://dx.doi.org/10.1677/joe.0.1410209.
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Abstract The objectives were to investigate the mechanisms by which glucocorticoids control proteolysis in muscle cells and the relationship between the calpain:calpastatin system and proteolysis in muscle. Female rabbits were treated with 1 mg dexamethasone (Dex)/kg body weight per day for 0, 1, 2 or 4 days after which animals were killed and muscle samples taken for analyses. Dex reduced urinary Nτ-methylhistidine (NMH) 48% (day 4 versus day 1 of Dex treatment) and muscle NMH concentrations by 49% (day 1) to 40% (day 2) respectively, suggesting that protein degradation was reduced. To investigate whether the changes in apparent proteolysis were related to calpains, we examined the effects of Dex on muscle calpain and calpastatin activities. These were unaffected by Dex. This implies that Dex-dependent changes in degradation are not mediated by changes in muscle calpain or calpastatin activities. We studied the effects of Dex on calpain and calpastatin gene expression as a means of clarifying the relationships between proteinase gene expression and proteinase activities. μ-Calpain mRNA concentration was unaffected by Dex but m-calpain mRNA and calpastatin mRNA concentrations were reduced by 42–55% and 40% respectively. Dex had a similar effect on β-actin mRNA. Although calpain and calpastatin genes behaved as housekeeping genes, changes in their expression mimicked apparent changes in proteolysis. The observation that calpain and calpastatin activities were unchanged indicates that additional regulation of the calpain:calpastatin system exists at other sites in muscle cells. To determine whether Dex-dependent changes in proteolysis were mediated indirectly, we assayed the effects of Dex on plasma thyroid hormone concentrations. Dex reduced both plasma triiodothyronine (T3) and thyroxine (T4) concentrations by as much as 68% and 67% respectively, and reduced the T3/T4 ratio by 74% following 4 days of Dex treatment. Thyroid hormones regulate muscle proteolysis and their lower concentrations in Dex-treated rabbits may account for the reductions in muscle NMH concentration and urinary NMH excretion caused by Dex. Journal of Endocrinology (1994) 141, 209–217
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Lehti, Maarit, Riikka Kivelä, Paavo Komi, Jyrki Komulainen, Heikki Kainulainen, and Heikki Kyröläinen. "Effects of fatiguing jumping exercise on mRNA expression of titin-complex proteins and calpains." Journal of Applied Physiology 106, no. 4 (April 2009): 1419–24. http://dx.doi.org/10.1152/japplphysiol.90660.2008.
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Eccentric exercise induced by electrostimulation increases mRNA expression of titin-complex proteins in rodent skeletal muscle. In this study, mRNA expression of titin, muscle LIM protein (MLP), cardiac ankyrin repeat protein (CARP), ankyrin repeat domain protein 2 (Ankrd2), diabetes-related ankyrin repeat protein (DARP), and calcium-activated proteinases, calpains, were investigated in human skeletal muscle after fatiguing jumping exercise. Fatiguing jumping exercise did not change mRNA expression of titin, DARP, calpain 1, or calpain 3. MLP, Ankrd2 and calpain 2 mRNA levels were increased 2 days postexercise. CARP mRNA level was already elevated 30 min and remained elevated 2 days postexercise. Increased mRNA expression of MLP, CARP, and Ankrd2, observed for the first time in human skeletal muscle, may be part of the signaling activated by physical exercise. The rapid increase in the level of CARP mRNA nominates CARP as one of the first genes to respond to exercise. The increase in the mRNA level of calpain 2 suggests its involvement in myofiber remodeling after strenuous jumping exercise.
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Verheijden, Kim A. T., Ramon Sonneveld, Marinka Bakker-van Bebber, Jack F. M. Wetzels, Johan van der Vlag, and Tom Nijenhuis. "The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury." Journal of the American Society of Nephrology 29, no. 8 (June 28, 2018): 2099–109. http://dx.doi.org/10.1681/asn.2016111248.
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BackgroundThe hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive.MethodsWe tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes.ResultsCompared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects.ConclusionsWe elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.
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RAVULAPALLI, Ravikiran, Beatriz GARCIA DIAZ, Robert L. CAMPBELL, and Peter L. DAVIES. "Homodimerization of calpain 3 penta-EF-hand domain." Biochemical Journal 388, no. 2 (May 24, 2005): 585–91. http://dx.doi.org/10.1042/bj20041821.
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Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass Mr 80000) and small (Mr 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution. Molecular modelling suggests that there would be no barriers to the dimerization of the whole enzyme through the PEF domains. This orientation would place the catalytic centres at opposite ends of the dimer.
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Parnaud, Géraldine, Eva Hammar, Dominique G. Rouiller, and Domenico Bosco. "Inhibition of calpain blocks pancreatic β-cell spreading and insulin secretion." American Journal of Physiology-Endocrinology and Metabolism 289, no. 2 (August 2005): E313—E321. http://dx.doi.org/10.1152/ajpendo.00006.2005.
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In addition to promoting insulin secretion, an increase in cytosolic Ca2+ triggered by glucose has been shown to be crucial for spreading of β-cells attached on extracellular matrix (804G matrix). Calpains are Ca2+-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca2+-induced insulin secretion and spreading of rat pancreatic β-cells. The results indicate calpain dependency of β-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca2+]i. Finally, calpeptin was shown to affect the [Ca2+]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic β-cells. It is therefore suggested that calpain could be a mediator of Ca2+-induced-insulin secretion and β-cell spreading.
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Berkholz, Janine, Andreas Zakrzewicz, and Barbara Munz. "skNAC depletion stimulates myoblast migration and perturbs sarcomerogenesis by enhancing calpain 1 and 3 activity." Biochemical Journal 453, no. 2 (June 28, 2013): 303–10. http://dx.doi.org/10.1042/bj20130195.
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skNAC (skeletal and heart muscle specific variant of nascent polypeptide-associated complex α) is a skeletal and heart muscle-specific protein known to be involved in the regulation of sarcomerogenesis. The respective mechanism, however, is largely unknown. In the present paper, we demonstrate that skNAC regulates calpain activity. Specifically, we show that inhibition of skNAC gene expression leads to enhanced, and overexpression of the skNAC gene to repressed, activity of calpain 1 and, to a lesser extent, calpain 3 in myoblasts. In skNAC siRNA-treated cells, enhanced calpain activity is associated with increased migration rates, as well as with perturbed sarcomere architecture. Treatment of skNAC-knockdown cells with the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) reverts both the positive effect on myoblast migration and the negative effect on sarcomere architecture. Taken together, our data suggest that skNAC controls myoblast migration and sarcomere architecture in a calpain-dependent manner.
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Briz, Victor, and Michel Baudry. "Calpains: Master Regulators of Synaptic Plasticity." Neuroscientist 23, no. 3 (May 17, 2016): 221–31. http://dx.doi.org/10.1177/1073858416649178.
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Although calpain was proposed to participate in synaptic plasticity and learning and memory more than 30 years ago, the mechanisms underlying its activation and the roles of different substrates have remained elusive. Recent findings have provided evidence that the two major calpain isoforms in the brain, calpain-1 and calpain-2, play opposite functions in synaptic plasticity. In particular, while calpain-1 activation is the initial trigger for certain forms of synaptic plasticity, that is, long-term potentiation, calpain-2 activation restricts the extent of plasticity. Moreover, while calpain-1 rapidly cleaves regulatory and cytoskeletal proteins, calpain-2-mediated stimulation of local protein synthesis reestablishes protein homeostasis. These findings have important implications for our understanding of learning and memory and disorders associated with impairment in these processes.
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Yoshikawa, Yoshiro, Hiroji Hagihara, Yoshimi Ohga, Chikako Nakajima-Takenaka, Ken-ya Murata, Shigeki Taniguchi, and Miyako Takaki. "Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 4 (April 2005): H1690—H1698. http://dx.doi.org/10.1152/ajpheart.00666.2004.
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We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of α-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion ( n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly ( P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo2) intercepts (PVA-independent Vo2; Vo2 for the total Ca2+ handling in excitation-contraction coupling and basal metabolism) of Vo2-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly ( P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O2 costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of α-fodrin and the immunostaining of 150-kDa products of α-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of α-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca2+ handling by directly inhibiting the proteolysis of α-fodrin.
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Fareed, Moin U., Amy R. Evenson, Wei Wei, Michael Menconi, Vitaliy Poylin, Victoria Petkova, Bernadette Pignol, and Per-Olof Hasselgren. "Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 290, no. 6 (June 2006): R1589—R1597. http://dx.doi.org/10.1152/ajpregu.00668.2005.
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Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.
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SAVART, M., V. PALLET, P. LETRAD, C. BOSSUET, and A. DUCASTAING. "Calpain 1-protein kinase C complex: effect of calpain inhibitors after dissociation." Biochimie 73, no. 11 (November 1991): 1409–16. http://dx.doi.org/10.1016/0300-9084(91)90172-w.
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