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1
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.
2
Theopold, U., M. Pintér, S. Daffre, Y. Tryselius, P. Friedrich, D. R. Nässel, and D. Hultmark. "CalpA, a Drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells." Molecular and Cellular Biology 15, no. 2 (February 1995): 824–34. http://dx.doi.org/10.1128/mcb.15.2.824.
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Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.
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Fontenele, Marcio, Bomyi Lim, Danielle Oliveira, Márcio Buffolo, David H. Perlman, Trudi Schupbach, and Helena Araujo. "Calpain A modulates Toll responses by limited Cactus/IκB proteolysis." Molecular Biology of the Cell 24, no. 18 (September 15, 2013): 2966–80. http://dx.doi.org/10.1091/mbc.e13-02-0113.
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Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IκB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFκB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IκB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFκB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFκB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system.
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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
5
Ilian, M. A., and N. E. Forsberg. "Gene expression of calpains and their specific endogenous inhibitor, calpastatin, in skeletal muscle of fed and fasted rabbits." Biochemical Journal 287, no. 1 (October 1, 1992): 163–71. http://dx.doi.org/10.1042/bj2870163.
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To investigate the role of calpains in myofibrillar protein degradation in skeletal muscle and the regulation of their activity in vivo, we studied the effects of fasting on gene expression of calpains and calpastatin in the skeletal muscle of rabbits. In response to fasting, myofibrillar protein degradation increased 2-fold and mRNA levels of calpain I, calpain II and calpastatin were also increased. However, calpain and calpastatin activities remained unchanged. To investigate this discrepancy, we analysed polysomal calpain mRNA. Results indicated that fasting caused a 2-fold increase in the loading of calpain I and II mRNAs on ribosomes. Thus transcription of genes encoding calpain may be increased during fasting to ensure adequate synthesis of the proteinases needed to mobilize muscle protein reserves. The effect of fasting on calpain and calpastatin mRNA expression is shared by cathepsin D and proteasome C2 but not by beta-actin, implying that fasting invokes control of several proteolytic systems in skeletal muscle and underscores the possibility that each proteolytic system plays a role in the adaptation of skeletal muscle to the fasted state.
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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.
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Miller, John A., Domenica E. Drouet, Leonid M. Yermakov, Mahmoud S. Elbasiouny, Fatima Z. Bensabeur, Michael Bottomley, and Keiichiro Susuki. "Distinct Changes in Calpain and Calpastatin during PNS Myelination and Demyelination in Rodent Models." International Journal of Molecular Sciences 23, no. 23 (December 6, 2022): 15443. http://dx.doi.org/10.3390/ijms232315443.
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Myelin forming around axons provides electrical insulation and ensures rapid and efficient transmission of electrical impulses. Disruptions to myelinated nerves often result in nerve conduction failure along with neurological symptoms and long-term disability. In the central nervous system, calpains, a family of calcium dependent cysteine proteases, have been shown to have a role in developmental myelination and in demyelinating diseases. The roles of calpains in myelination and demyelination in the peripheral nervous system remain unclear. Here, we show a transient increase of activated CAPN1, a major calpain isoform, in postnatal rat sciatic nerves when myelin is actively formed. Expression of the endogenous calpain inhibitor, calpastatin, showed a steady decrease throughout the period of peripheral nerve development. In the sciatic nerves of Trembler-J mice characterized by dysmyelination, expression levels of CAPN1 and calpastatin and calpain activity were significantly increased. In lysolecithin-induced acute demyelination in adult rat sciatic nerves, we show an increase of CAPN1 and decrease of calpastatin expression. These changes in the calpain-calpastatin system are distinct from those during central nervous system development or in acute axonal degeneration in peripheral nerves. Our results suggest that the calpain-calpastatin system has putative roles in myelination and demyelinating diseases of peripheral nerves.
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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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Mellgren, Ronald L., and Xinhua Huang. "Fetuin A Stabilizes m-Calpain and Facilitates Plasma Membrane Repair." Journal of Biological Chemistry 282, no. 49 (October 17, 2007): 35868–77. http://dx.doi.org/10.1074/jbc.m706929200.
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Yeast two-hybrid experiments identified α2-Heremans-Schmid glycoprotein (human fetuin A) as a binding partner for calpain domain III (DIII). The tandem DIIIs of calpain-10 interacted under the most selective culture conditions, but DIIIs of m-calpain, calpain-3, and calpain-5 also interacted under less stringent selection. DIIIs of μ-calpain, calpain-6, and the tandem DIII-like domains of the Dictyostelium Cpl protein did not interact with α2-Heremans-Schmid glycoprotein in the yeast two-hybrid system. Bovine fetuin A stabilized proteolytic activity of purified m-calpain incubated in the presence of mm calcium chloride and prevented calcium-dependent m-calpain aggregation. Consistent with the yeast two-hybrid studies, fetuin A neither stabilized μ-calpain nor prevented its aggregation. Confocal immunofluorescence microscopy of scratch-damaged L6 myotubes demonstrated accumulation of m-calpain at the wound site in association with the membrane repair protein, dysferlin. m-Calpain also co-localized with fluorescein-labeled fetuin A at the wound site. The effect of fetuin A on calpain-mediated plasma membrane resealing was investigated using fibroblasts from Capns1-/- and Capns1+/+ mouse embryos. Capns1 encodes the small noncatalytic subunit that is required for the proteolytic function of m- and μ-calpains. Thus, Capns1-/- fibroblasts do not express these calpains in active form. Fetuin A increased resealing of scrape-damaged wild-type fibroblasts but not Capns1-/- fibroblasts. These studies identify fetuin A as a potential extracellular regulator of m-calpain at nascent sites of plasma membrane wounding.
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Bevers, Matthew B., and Robert W. Neumar. "Mechanistic Role of Calpains in Postischemic Neurodegeneration." Journal of Cerebral Blood Flow & Metabolism 28, no. 4 (December 12, 2007): 655–73. http://dx.doi.org/10.1038/sj.jcbfm.9600595.
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The calpain family of proteases is causally linked to postischemic neurodegeneration. However, the precise mechanisms by which calpains contribute to postischemic neuronal death have not been fully elucidated. This review outlines the key features of the calpain system, and the evidence for its causal role in postischemic neuronal pathology. Furthermore, the consequences of specific calpain substrate cleavage at various subcellular locations are explored. Calpain substrates within synapses, plasma membrane, endoplasmic reticulum, lysosomes, mitochondria, and the nucleus, as well as the overall effect of postischemic calpain activity on calcium regulation and cell death signaling are considered. Finally, potential pathways for calpain-mediated neurodegeneration are outlined in an effort to guide future studies aimed at understanding the downstream pathology of postischemic calpain activity and identifying optimal therapeutic strategies.
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Coria, María Sumampa, Dario Pighin, Gabriela Grigioni, and Gustavo Adolfo Palma. "Feeding strategies and ageing time alter calpain system proteins activities and meat quality of Braford steers." Animal Bioscience 35, no. 2 (February 1, 2022): 272–80. http://dx.doi.org/10.5713/ab.21.0227.
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Objective: The aim of this study was to evaluate the effect of ageing and feeding strategies on the calpain protease system and meat quality traits in Braford steers.Methods: Thirty Braford steers were employed; 15 animals were supplemented with corn silage during finishing and 15 were kept only on pasture. Meat quality traits and calpain system protein activity were evaluated in longissimus thoracis et lumborum (LTL) steaks aged for 2, 7, 14, and 21 days.Results: Aged meat showed higher pH and calcium content, while Warner Bratzler shear force (WBSF) decreased to day 21. No interaction between ageing and diet was seen for quality traits. Steers finished with corn silage showed higher values of water holding capacity, WBSF and free calcium, and lower values of pH and cooking loss. Calpain and calpastatin activities decreased with ageing. Finishing steers on pasture produced higher values of calpains and lower values of calpastatin activities. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group.Conclusion: These results suggest a diet by ageing interaction in calpains and calpastatin and this interaction impact in Warner Bratzler Shear Force in Braford LTL muscle.
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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.
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Pintér, M., and P. Friedrich. "The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster." Biochemical Journal 253, no. 2 (July 15, 1988): 467–73. http://dx.doi.org/10.1042/bj2530467.
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Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system.
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Kuchay, Shafi, Rafael Nunez, Amelia M. Bartholomew, and Athar H. Chishti. "Calpain I Null Mice Display Lymphoid Hyperplasia." Blood 104, no. 11 (November 16, 2004): 1268. http://dx.doi.org/10.1182/blood.v104.11.1268.1268.
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Abstract Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, homeostasis, cell proliferation, cell motility, and apoptosis. The conventional calpains include mu-calpain (calpain I) that requires micromolar calcium for half maximal activation and m-calpain (calpain II) that functions at millimolar calcium concentration in vitro. To evaluate the physiological function(s) of mu-calpain, we utilized genetically altered mu-calpain null mice as the model system. Spleen biopsy of mu-calpain null adult mice showed consistent expansion of white pulp (lymphoid hyperplasia) due to an increase in number and size of follicles and PALS (periarteriolar lymphatic sheets), relative to matched wild type mice. Also, cervical lymph nodes of mu-calpain null mice showed lymphoid hyperplasia and marked plasmacytosis. The majority of mu-calpain null mice displayed multi-organ lymphoid infiltration. Using immunofluorescence labeling and multiparameter flow cytometry, the cellular composition of the hematopoietic compartment was quantified in both bone marrow and spleen of mu-calpain null mice. The mu-calpain null mice exhibited an overall increase in the number of B-cell lineage; showing ~40% increase of B220+ cells in the bone marrow and ~19% increase of B220+ cells in the spleen. Interestingly, T-cell compartment (CD3+, CD4, CD8+) and NK lineage (NK1.1+) cells were significantly reduced. Myeloid specific GR1+, CD11b+ granulocytic cell lineage, and megakaryocytic CD41+ cells were also noticeably increased in the bone marrow of mu-calpain null mice. Together, these results reveal a central role of mu-calpain in the maintenance of lymphoid homeostasis, contributing to the regulation of B-cell lineage. Ongoing experiments involve investigating quantitative effects of mu-calpain deletion on the survival and apoptotic cell death of B220+ B-cell lineage in response to various insults such as Fas ligand, cycloheximide, irradiation, 9-AC, and staurosporine. The outcome of these studies will elucidate the precise biochemical function(s) of mu-calpain by identifying key substrates that regulate apoptosis in a physiologically relevant environment.
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Ennes-Vidal, Vítor, Marta Helena Branquinha, André Luis Souza dos Santos, and Claudia Masini d’Avila-Levy. "The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?" Cells 10, no. 2 (February 1, 2021): 299. http://dx.doi.org/10.3390/cells10020299.
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Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites?
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Kalbe, L., A. Leunda, T. Sparre, C. Meulemans, M. T. Ahn, T. Orntoft, M. Kruhoffer, B. Reusens, J. Nerup, and C. Remacle. "Nutritional regulation of proteases involved in fetal rat insulin secretion and islet cell proliferation." British Journal of Nutrition 93, no. 3 (March 2005): 309–16. http://dx.doi.org/10.1079/bjn20041313.
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Epidemiological studies have indicated that malnutrition during early life may programme chronic degenerative disease in adulthood. In an animal model of fetal malnutrition, rats received an isoenergetic, low-protein (LP) diet during gestation. This reduced fetal β-cell proliferation and insulin secretion. Supplementation during gestation with taurine prevented these alterations. Since proteases are involved in secretion and proliferation, we investigated which proteases were associated with these alterations and their restoration in fetal LP islets. Insulin secretion and proliferation of fetal control and LP islets exposed to different protease modulators were measured. Lactacystin and calpain inhibitor I, but not isovaleryl-l-carnitine, raised insulin secretion in control islets, indicating that proteasome and cysteinyl cathepsin(s), but not μ-calpain, are involved in fetal insulin secretion. Insulin secretion from LP islets responded normally to lactacystin but was insensitive to calpain inhibitor I, indicating a loss of cysteinyl cathepsin activity. Taurine supplementation prevented this by restoring the response to calpain inhibitor I. Control islet cell proliferation was reduced by calpain inhibitor I and raised by isovaleryl-l-carnitine, indicating an involvement of calpain. Calpain activity appeared to be lost in LP islets and not restored by taurine. Most modifications in the mRNA expression of cysteinyl cathepsins, calpains and calpastatin due to maternal protein restriction were consistent with reduced protease activity and were restored by taurine. Thus, maternal protein restriction affected cysteinyl cathepsins and the calpain–calpastatin system. Taurine normalised fetal LP insulin secretion by protecting cysteinyl cathepsin(s), but the restoration of LP islet cell proliferation by taurine did not implicate calpains.
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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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Kemp, C. M., T. Parr, R. G. Bardsley, and P. J. Buttery. "Comparison of the relative expression of caspase isoforms across muscle types." Proceedings of the British Society of Animal Science 2005 (2005): 107. http://dx.doi.org/10.1017/s1752756200010188.
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Toughness is a determinant of meat quality and a common cause of unacceptability in meat products. Calpain proteases are believed to be involved in meat tenderisation by post mortem degradation of myofibrillar proteins (Sensky et al., 2001). However other proteases are likely to contribute to the proteolysis involved in meat-conditioning (Sentandreu et al., 2002). Caspases are proteases involved in protein degradation in apoptosis. Like calpains caspases are activated early in pathological events associated with hypoxia/ischaemia, which is not dissimilar to the hypoxic conditions in muscle after slaughter. Caspases specifically cleave a number of proteins that are also targeted by calpains during post mortem proteolysis and also degrade the calpain-specific inhibitor calpastatin. The caspase system has a hierarchy of initiating isoforms (such as caspases 8 and 12) which activate effector caspases (such as 3 and 7) that cleave specific substrates. Caspases are regulated by inhibitors such as apoptosis repressor with caspase recruitment domain (ARC). Our hypothesis is that caspase activity may contribute to early post mortem proteolysis and tenderisation, similar to the calpain system. The aim of this study was to characterise the caspase system in various porcine skeletal muscles.
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Hata, Shoji, Naoko Doi, Fujiko Kitamura, and Hiroyuki Sorimachi. "Stomach-specific Calpain, nCL-2/Calpain 8, Is Active without Calpain Regulatory Subunit and Oligomerizes through C2-like Domains." Journal of Biological Chemistry 282, no. 38 (July 23, 2007): 27847–56. http://dx.doi.org/10.1074/jbc.m703168200.
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Calpains constitute a family of intracellular Ca2+-regulated cysteine proteases that are indispensable in the regulation of a wide variety of cellular functions. The improper activation of calpain causes lethality or various disorders, such as muscular dystrophies and tumor formation. nCL-2/calpain 8 is predominantly expressed in the stomach, where it appears to be involved in membrane trafficking in the gastric surface mucus cells (pit cells). Although the primary structure of nCL-2 is quite similar to that of the ubiquitous m-calpain large subunit, the enzymatic properties of nCL-2 have never been reported. Here, to characterize nCL-2, the recombinant protein was prepared using an Escherichia coli expression system and purified to homogeneity. nCL-2 was stably produced as a soluble and active enzyme without the conventional calpain regulatory subunit (30K). Purified nCL-2 showed Ca2+-dependent activity, with half-maximal activity at about 0.3 mm Ca2+, similar to that of m-calpain, whereas its optimal pH and temperature were comparatively low. Immunoprecipitation analysis revealed that nCL-2 exists in both monomeric and homo-oligomeric forms, but not as a heterodimer with 30K or 30K-2, and that the oligomerization occurs through domains other than the 5EF-hand domain IV, most probably through domain III, suggesting a novel regulatory system for nCL-2.
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Yamaura, Ikuya, Eiichi Tani, Takaomi C. Saido, Koichi Suzuki, Nobutaka Minami, and Yukio Maeda. "Calpain-calpastatin system of canine basilar artery in vasospasm." Journal of Neurosurgery 79, no. 4 (October 1993): 537–43. http://dx.doi.org/10.3171/jns.1993.79.4.0537.
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✓ Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, while contraction was induced in the normal canine basilar artery by a local application of KCl or serotonin after transclival exposure. The control animals were injected with saline instead of fresh blood. The activation of μ-calpain, a Ca++-dependent neutral protease, in the basilar artery was studied by evaluating the conversion from its inactivated into its activated form on immunoblots. In addition, the activity of calpastatin, an intrinsic inhibitor of calpain, in the basilar artery was determined by assay. The majority of the μ-calpain was inactivated in the control group. In the spastic group, μ-calpain was generally activated markedly in the early stage of vasospasm and moderately thereafter. The contraction induced by KCl or serotonin application was classified into the early phasic and the later tonic stages; μ-calpain was usually activated in the phasic stage and inactivated in the tonic stage. Calpastatin activity was significantly decreased during vasospasm, whereas it was not significantly changed in KCl- or serotonin-induced contraction. The final activity of μ-calpain results from the balance of μ-calpain and calpastatin. This suggests that μ-calpain activity was enhanced continuously in the spastic group and transiently in the KCl or serotonin group, and that the continuous activation of μ-calpain during vasospasm probably induced more proteolytic changes compared to those in the KCl or serotonin group.
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Alexa, Anita, Zoltán Bozóky, Attila Farkas, Peter Tompa, and Peter Friedrich. "Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+Ions." Journal of Biological Chemistry 279, no. 19 (February 19, 2004): 20118–26. http://dx.doi.org/10.1074/jbc.m311969200.
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The effect of Ca2+in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+binding and Ca2+sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+relay system. Wild-typeDrosophilaCalpain B domain III binds two to three Ca2+ions with aKdof 3400 μm. Phospholipids lower this value to 220 μm. Ca2+binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+need of both enzymes, lowering half-maximal Ca2+concentration from 8.6 to 1.3 mmfor Calpain B and from 250 to 7 μmfor m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this “extended transducer” mechanism is presented.
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Yajima, Yukiko, Mayumi Sato, Hiroyuki Sorimachi, Mitsushi Inomata, Masatoshi Maki, and Seiichi Kawashima. "Calpain System Regulates the Differentiation of Adult Primitive Mesenchymal ST-13 Adipocytes." Endocrinology 147, no. 10 (October 1, 2006): 4811–19. http://dx.doi.org/10.1210/en.2005-1647.
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The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-l-leucyl-l-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-α (C/EBPα), peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor α, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca2+-dependent degradation of PPARγ and C/EBPα but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARγ and C/EBPα. Under both conditions, calpain inhibitors almost completely prevented C/EBPα cleavage but partially blocked the decrease of PPARγ. Two ubiquitous forms of calpain, μ- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of μ- but not m-calpain was found in the nucleus. Finally, stable dominant-negative μ-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARγ and C/EBPα during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.
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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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Miyazaki, Takuro, and Akira Miyazaki. "Impact of Dysfunctional Protein Catabolism on Macrophage Cholesterol Handling." Current Medicinal Chemistry 26, no. 9 (May 21, 2019): 1631–43. http://dx.doi.org/10.2174/0929867325666180326165234.
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Protein catabolism in macrophages, which is accomplished mainly through autophagy- lysosomal degradation, ubiquitin-proteasome system, and calpains, is disturbed in atheroprone vessels. Moreover, growing evidence suggests that defects in protein catabolism interfere with cholesterol handling in macrophages. Indeed, decreases in autophagy facilitate the deposition of cholesterol in atheroprone macrophages and the subsequent development of vulnerable atherosclerotic plaques due to impaired catabolism of lipid droplets and limited efferocytic clearance of dead cells. The proteasome is responsible for the degradation of ATP-binding cassette transporters, which leads to impaired cholesterol efflux from macrophages. Overactivation of conventional calpains contributes to excessive processing of functional proteins, thereby accelerating receptor-mediated uptake of oxidized low-density lipoproteins (LDLs) and slowing cholesterol efflux. Furthermore, calpain-6, an unconventional nonproteolytic calpain in macrophages, potentiates pinocytotic uptake of native LDL and attenuates the efferocytic clearance of dead cells. Herein, we focus on recent progress in understanding how defective protein catabolism is associated with macrophage cholesterol handling and subsequent atherogenesis.
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Sensky, P. L., T. Parr, S. N. Brown, R. G. Bardsley, P. J. Buttery, and J. D. Wood. "Relationship between pigmeat toughness and the calpain proteolytic system." Proceedings of the British Society of Animal Science 1996 (March 1996): 156. http://dx.doi.org/10.1017/s1752756200593521.
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Variability in the rate of postmortem meat tenderisation occurs in the carcasses of pigs, even those of similar genotype raised under common conditions (Koohmaraie et al, 1995). Recent work has shown that administration of synthetic (β-agonists enhances muscle growth, reduces tenderisation and increases the activity of calpastatin, the natural inhibitor of the proteolytic enzymes μ-calpain and m-calpain, after slaughter (Kretchmar et al., 1990, Parr et al., 1992). Therefore, it is possible that part of the above variability may be related to levels of natural β-agonists, i.e. adrenaline. In this study the relationship between pigmeat toughness and the calpain proteolytic system has been investigated, using adrenaline infusion in an attempt to perturb the calpain system and create a wide range of meat qualities.
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Sensky, P. L., T. Parr, S. N. Brown, R. G. Bardsley, P. J. Buttery, and J. D. Wood. "Relationship between pigmeat toughness and the calpain proteolytic system." Proceedings of the British Society of Animal Science 1996 (March 1996): 156. http://dx.doi.org/10.1017/s0308229600031238.
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Variability in the rate of postmortem meat tenderisation occurs in the carcasses of pigs, even those of similar genotype raised under common conditions (Koohmaraie et al, 1995). Recent work has shown that administration of synthetic (β-agonists enhances muscle growth, reduces tenderisation and increases the activity of calpastatin, the natural inhibitor of the proteolytic enzymes μ-calpain and m-calpain, after slaughter (Kretchmar et al., 1990, Parr et al., 1992). Therefore, it is possible that part of the above variability may be related to levels of natural β-agonists, i.e. adrenaline. In this study the relationship between pigmeat toughness and the calpain proteolytic system has been investigated, using adrenaline infusion in an attempt to perturb the calpain system and create a wide range of meat qualities.
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Weber, Heike, Ludwig Jonas, Saskia Hühns, and Peter Schuff-Werner. "Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat." American Journal of Physiology-Gastrointestinal and Liver Physiology 286, no. 6 (June 2004): G932—G941. http://dx.doi.org/10.1152/ajpgi.00406.2003.
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Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed μ-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg ip), pancreatitis was induced by cerulein injections (10 μg/kg ip; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both μ-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity ( P < 0.01), pancreatic edema formation ( P < 0.05), histological score for eight parameters ( P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.
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Haim, K., I. Ben-Aharon, and R. Shalgi. "Expression and immunolocalization of the calpain–calpastatin system during parthenogenetic activation and fertilization in the rat egg." Reproduction 131, no. 1 (January 2006): 35–43. http://dx.doi.org/10.1530/rep.1.00697.
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Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca2+-dependent thiol protease. The calpain–calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain–calpastatin interplay throughout the process.Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110kDa calpastatin and β-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static.We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.
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Ono, Yasuko, Kazumi Kakinuma, Fukuyo Torii, Akihiro Irie, Kazuhiro Nakagawa, Siegfried Labeit, Keiko Abe, Koichi Suzuki, and Hiroyuki Sorimachi. "Possible Regulation of the Conventional Calpain System by Skeletal Muscle-specific Calpain, p94/Calpain 3." Journal of Biological Chemistry 279, no. 4 (November 1, 2003): 2761–71. http://dx.doi.org/10.1074/jbc.m308789200.
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Pandurangan, Muthuraman, Inho Hwang, Chinzorio Orhirbat, Yang Jieun, and Soo-Hyun Cho. "The calpain system and diabetes." Pathophysiology 21, no. 2 (June 2014): 161–67. http://dx.doi.org/10.1016/j.pathophys.2014.01.003.
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Storr, Sarah J., Neil O. Carragher, Margaret C. Frame, Tim Parr, and Stewart G. Martin. "The calpain system and cancer." Nature Reviews Cancer 11, no. 5 (April 21, 2011): 364–74. http://dx.doi.org/10.1038/nrc3050.
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Kar, Pulak, Krishna Samanta, Soni Shaikh, Animesh Chowdhury, Tapati Chakraborti, and Sajal Chakraborti. "Mitochondrial calpain system: An overview." Archives of Biochemistry and Biophysics 495, no. 1 (March 2010): 1–7. http://dx.doi.org/10.1016/j.abb.2009.12.020.
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Hopkins, D. L., and J. M. Thompson. "Factors contributing to proteolysis and disruption of myofibrillar proteins and the impact on tenderisation in beef and sheep meat." Australian Journal of Agricultural Research 53, no. 2 (2002): 149. http://dx.doi.org/10.1071/ar01079.
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This review seeks to examine the current theories of tenderisation with respect to red meat where tenderisation is defined as the reduction in toughness post-rigor. This examination is in the light of recent research on meat biochemistry, and from this, areas of research that may prove fruitful are highlighted. Based on available data, the major candidate to explain tenderisation post-rigor is the calpain protease system. Evidence that change in the binding of actomyosin (the complex of contractile proteins formed at rigor) or cleavage of myofibrillar proteins due to Ca2+ ions contributes to tenderisation is far from compelling. Equally it appears that the cathepsin proteases are unlikely to have a role in early post-mortem cleavage of proteins (proteolysis) and thus tenderisation. The mode of action of the calpains is not yet fully defined and questions remain as to the role of m-calpain given the in vitro requirement for a Ca2+ ion concentration exceeding that observed in post-mortem muscle. The existence of the calpains in living muscle and other tissues suggests a mode of action more subtle than currently thought. Additionally, the observation that the degradation of myofibrillar proteins occurs in the presence of effective synthetic and natural calpain inhibitors suggests that other enzymes may also have a role in tenderisation. Inevitably, the accumulated evidence points to a complex system likely to involve interacting proteases and ions, and only through open minded investigation with reliance on developments in the medical and biochemical fields will a more complete model of tenderisation be developed.
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Yang, Jie, Fei Xiang, Peng-Cheng Cai, Yu-Zhi Lu, Xiao-Xiao Xu, Fan Yu, Feng-Zhi Li, et al. "Activation of calpain by renin-angiotensin system in pleural mesothelial cells mediates tuberculous pleural fibrosis." American Journal of Physiology-Lung Cellular and Molecular Physiology 311, no. 1 (July 1, 2016): L145—L153. http://dx.doi.org/10.1152/ajplung.00348.2015.
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Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.
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Schneider, Martha, Kerstin Ackermann, Melissa Stuart, Claudia Wex, Ulrike Protzer, Hermann M. Schätzl, and Sabine Gilch. "Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain." Journal of Virology 86, no. 18 (July 11, 2012): 10112–22. http://dx.doi.org/10.1128/jvi.01001-12.
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The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5−/−mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.
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Benetti, Roberta, Tamara Copetti, Stefania Dell'Orso, Edon Melloni, Claudio Brancolini, Martin Monte, and Claudio Schneider. "The Calpain System Is Involved in the Constitutive Regulation of β-Catenin Signaling Functions." Journal of Biological Chemistry 280, no. 23 (April 7, 2005): 22070–80. http://dx.doi.org/10.1074/jbc.m501810200.
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β-Catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of β-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3β complex, which targets it for proteasomal degradation. It has been recently shown that Ca2+ release from internal stores results in nuclear export and calpain-mediated degradation of β-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of β-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous β-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of β-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on β-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3β and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated β-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.
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MEYER, Sheryl L., Donna BOZYCZKO-COYNE, Satish K. MALLYA, Chrysanthe M. SPAIS, Ron BIHOVSKY, John K. KAWOOYA, Diane M. LANG, Richard W. SCOTT, and Robert SIMAN. "Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system." Biochemical Journal 314, no. 2 (March 1, 1996): 511–19. http://dx.doi.org/10.1042/bj3140511.
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Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.
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Cunha, Telma F., Jose B. N. Moreira, Nathalie A. Paixão, Juliane C. Campos, Alex W. A. Monteiro, Aline V. N. Bacurau, Carlos R. Bueno, Julio C. B. Ferreira, and Patricia C. Brum. "Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice." Journal of Applied Physiology 112, no. 11 (June 1, 2012): 1839–46. http://dx.doi.org/10.1152/japplphysiol.00346.2011.
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Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.
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Zaman, Vandana, Kelsey P. Drasites, Ali Myatich, Ramsha Shams, Donald C. Shields, Denise Matzelle, Azizul Haque, and Narendra L. Banik. "Inhibition of Calpain Attenuates Degeneration of Substantia Nigra Neurons in the Rotenone Rat Model of Parkinson’s Disease." International Journal of Molecular Sciences 23, no. 22 (November 10, 2022): 13849. http://dx.doi.org/10.3390/ijms232213849.
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In the central nervous system (CNS), calcium homeostasis is a critical determinant of neuronal survival. Calpain, a calcium-dependent neutral protease, is widely expressed in the brain, including substantia nigra (SN) dopaminergic (DA) neurons. Though calpain is implicated in human Parkinson’s disease (PD) and corresponding animal models, the roles of specific ubiquitous calpain isoforms in PD, calpain-1 and calpain-2, remain poorly understood. In this study, we found that both isoforms are activated in a nigrostriatal pathway with increased phosphorylated synuclein following the administration of rotenone in Lewis rats, but calpain isoforms played different roles in neuronal survival. Although increased expression of calpain-1 and calpain-2 were detected in the SN of rotenone-administered rats, calpain-1 expression was not altered significantly after treatment with calpain inhibitor (calpeptin); this correlated with neuronal survival. By contrast, increased calpain-2 expression in the SN of rotenone rats correlated with neuronal death, and calpeptin treatment significantly attenuated calpain-2 and neuronal death. Calpain inhibition by calpeptin prevented glial (astroglia/microglia) activation in rotenone-treated rats in vivo, promoted M2-type microglia, and protected neurons. These data suggest that enhanced expression of calpain-1 and calpain-2 in PD models differentially affects glial activation and neuronal survival; thus, the attenuation of calpain-2 may be important in reducing SN neuronal loss in PD.
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Nian, Hong, and Binyun Ma. "Calpain–calpastatin system and cancer progression." Biological Reviews 96, no. 3 (January 20, 2021): 961–75. http://dx.doi.org/10.1111/brv.12686.
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Thompson, Valery F., Sandra Saldaña, Jinyang Cong, Dennis M. Luedke, and Darrel E. Goll. "The calpain system in human placenta." Life Sciences 70, no. 21 (April 2002): 2493–508. http://dx.doi.org/10.1016/s0024-3205(02)01506-0.
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Sultan, Karim R., Bernd T. Dittrich, Elmi Leisner, Nina Paul, and Dirk Pette. "Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle." American Journal of Physiology-Cell Physiology 280, no. 2 (February 1, 2001): C239—C247. http://dx.doi.org/10.1152/ajpcell.2001.280.2.c239.
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The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (∼2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of μ-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in μ-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS.
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Carragher, N. O., M. A. Westhoff, D. Riley, D. A. Potter, P. Dutt, J. S. Elce, P. A. Greer, and M. C. Frame. "v-Src-Induced Modulation of the Calpain-Calpastatin Proteolytic System Regulates Transformation." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 257–69. http://dx.doi.org/10.1128/mcb.22.1.257-269.2002.
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ABSTRACT v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G1 stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.
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Smith, Ira J., Stewart H. Lecker, and Per-Olof Hasselgren. "Calpain activity and muscle wasting in sepsis." American Journal of Physiology-Endocrinology and Metabolism 295, no. 4 (October 2008): E762—E771. http://dx.doi.org/10.1152/ajpendo.90226.2008.
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Muscle wasting in sepsis reflects activation of multiple proteolytic mechanisms, including lyosomal and ubiquitin-proteasome-dependent protein breakdown. Recent studies suggest that activation of the calpain system also plays an important role in sepsis-induced muscle wasting. Perhaps the most important consequence of calpain activation in skeletal muscle during sepsis is disruption of the sarcomere, allowing for the release of myofilaments (including actin and myosin) that are subsequently ubiquitinated and degraded by the 26S proteasome. Other important consequences of calpain activation that may contribute to muscle wasting during sepsis include degradation of certain transcription factors and nuclear cofactors, activation of the 26S proteasome, and inhibition of Akt activity, allowing for downstream activation of Foxo transcription factors and GSK-3β. The role of calpain activation in sepsis-induced muscle wasting suggests that the calpain system may be a therapeutic target in the prevention and treatment of muscle wasting during sepsis. Furthermore, because calpain activation may also be involved in muscle wasting caused by other conditions, including different muscular dystrophies and cancer, calpain inhibitors may be beneficial not only in the treatment of sepsis-induced muscle wasting but in other conditions causing muscle atrophy as well.
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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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Anagli, J., J. Hagmann, and E. Shaw. "Investigation of the role of calpain as a stimulus-response mediator in human platelets using new synthetic inhibitors." Biochemical Journal 274, no. 2 (March 1, 1991): 497–502. http://dx.doi.org/10.1042/bj2740497.
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A series of peptidyl diazomethanes and monofluoromethane with structures specific for calpain have been synthesized and tested for their ability to inhibit calpain activity in vivo, using human platelets as a model system. Calpain activity in vivo was determined by observing proteolysis of actin-binding protein and talin, two known substrates of calpain. Very potent inhibitors, which emerged from this study, were used to investigate the role of calpain in some platelet response processes. Our results show that calpain-mediated proteolysis in platelets is not an obligatory event leading to change of cell shape, adhesion to glass and spreading, aggregation and 5-hydroxytryptamine release. Two of the inhibitors were iodinated with 125I and used to radiolabel the enzyme in vivo. To our knowledge, this work also represents the first report describing the affinity labelling of calpain in human platelets using irreversible radioactive inhibitors.
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Ma, Xiufeng, Hui Chang, Zhe Wang, Shenhui Xu, Xin Peng, Jie Zhang, Xia Yan, Tingyun Lei, Huiping Wang, and Yunfang Gao. "Differential activation of the calpain system involved in individualized adaptation of different fast-twitch muscles in hibernating Daurian ground squirrels." Journal of Applied Physiology 127, no. 2 (August 1, 2019): 328–41. http://dx.doi.org/10.1152/japplphysiol.00124.2019.
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We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels ( Spermophilus dauricus). Results from morphological indices showed various degrees of atrophy in the order LG > PL > EDL. Furthermore, all three muscles underwent fast-to-slow fiber-type conversion in hibernation. In regard to the calpain system in the LG muscle, cytosolic Ca2+ increased significantly in hibernation, followed by recovery in posthibernation. Furthermore, calpastatin expression significantly decreased, and calpain 1 and 2 expression significantly increased, which may be responsible for the increased degradation of desmin during hibernation compared with that during summer activity. In the EDL muscle, Ca2+ overload was observed during interbout arousal, and calpastatin showed an increase during hibernation and interbout arousal, which could explain the increased levels of troponin T during both periods compared with levels during summer activity. These findings suggest that cytosolic Ca2+ overload and subsequent calpain 1 and 2 activation may be an important mechanism of LG muscle atrophy during hibernation. Cytosolic Ca2+ homeostasis and high expression of calpain inhibitor calpastatin during hibernation may also be an important mechanism for the EDL muscle to maintain muscle mass. Thus, the differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy of different fast-twitch muscles during hibernation. NEW & NOTEWORTHY We found that the extent of both muscle atrophy and calpain system activation differed in fast-twitch lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) skeletal muscles in hibernating Daurian ground squirrels, but similar hierarchies in the order of LG > PL > EDL. The differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy in different fast-twitch muscles during hibernation.
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Weber, Jonasz Jeremiasz, Priscila Pereira Sena, Elisabeth Singer, and Huu Phuc Nguyen. "Killing Two Angry Birds with One Stone: Autophagy Activation by Inhibiting Calpains in Neurodegenerative Diseases and Beyond." BioMed Research International 2019 (February 14, 2019): 1–13. http://dx.doi.org/10.1155/2019/4741252.
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Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca2+-dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.
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Coria, María Sumampa, Pablo Sebastián Reineri, Dario Pighin, Maria Guadalupe Barrionuevo, Pedro Gabriel Carranza, Gabriela Grigioni, and Gustavo Adolfo Palma. "Feeding strategies alter gene expression of the calpain system and meat quality in the longissimus muscle of Braford steers." Asian-Australasian Journal of Animal Sciences 33, no. 5 (May 1, 2020): 753–62. http://dx.doi.org/10.5713/ajas.19.0163.
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Objective: The aim of the present study was to determine the effect of supplementing pasture-finished steers with corn silage on the expression level of the calpain system proteins and beef tenderization.Methods: Thirty Braford steers grazing on summer pasture were used for the study. For 120 days fifteen animals were supplemented with corn silage at 1% of body weight per head per day (Suppl) whereas the remaining 15 steers only received pasture (Contr). Carcass and meat traits were evaluated and compared between groups. Gene expression and activities of proteases (calpain 1 and calpain 2) and inhibitor (calpastatin) were measured using real-time polymerase chain reaction and casein zymography.Results: Carcass and meat traits were significantly different between feeding systems. Supplemented steers showed higher hot carcass weight (p<0.01), fat content (p = 0.02), and Warner-Bratzler shear force (p = 0.03). Furthermore, the control group showed higher protease:inhibitor ratios, at mRNA (p = 0.01) and protein levels (p<0.10). Warner-Bratzler shear force and mRNA calpains:calpastatin ratio were associated in both feeding systems (p<0.01).Conclusion: Based on the results obtained in the study, beef tenderness differences among finishing strategies could be modulated through differential expression of the calpain system proteins.
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Talbert, Erin E., Ashley J. Smuder, Kisuk Min, Oh Sung Kwon, and Scott K. Powers. "Calpain and caspase-3 play required roles in immobilization-induced limb muscle atrophy." Journal of Applied Physiology 114, no. 10 (May 15, 2013): 1482–89. http://dx.doi.org/10.1152/japplphysiol.00925.2012.
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Prolonged skeletal muscle inactivity results in a rapid decrease in fiber size, primarily due to accelerated proteolysis. Although several proteases are known to contribute to disuse muscle atrophy, the ubiquitin proteasome system is often considered the most important proteolytic system during many conditions that promote muscle wasting. Emerging evidence suggests that calpain and caspase-3 may also play key roles in inactivity-induced atrophy of respiratory muscles, but it remains unknown if these proteases are essential for disuse atrophy in limb skeletal muscles. Therefore, we tested the hypothesis that activation of both calpain and caspase-3 is required for locomotor muscle atrophy induced by hindlimb immobilization. Seven days of immobilization (i.e., limb casting) promoted significant atrophy in type I muscle fibers of the rat soleus muscle. Independent pharmacological inhibition of calpain or caspase-3 prevented this casting-induced atrophy. Interestingly, inhibition of calpain activity also prevented caspase-3 activation, and, conversely, inhibition of caspase-3 prevented calpain activation. These findings indicate that a regulatory cross talk exists between these proteases and provide the first evidence that the activation of calpain and caspase-3 is required for inactivity-induced limb muscle atrophy.