Calpain – Wikipedia

Protease enzyme present in mammals and other organisms

A calpain (;[1]EC, EC is a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases (proteolytic enzymes) expressed ubiquitously in mammals and many other organisms. Calpains constitute the C2 family of protease clan CA in the MEROPS database. The calpain proteolytic system includes the calpain proteases, the small regulatory subunit CAPNS1, also known as CAPN4, and the endogenous calpain-specific inhibitor, calpastatin.


The history of calpain’s discovery originates in 1964, when calcium-dependent proteolytic activities caused by a “calcium-activated neutral protease” (CANP) were detected in brain, lens of the eye and other tissues. In the late 1960s the enzymes were isolated and characterised independently in both rat brain and skeletal muscle. These activities were caused by an intracellular cysteine protease not associated with the lysosome and having an optimum activity at neutral pH, which clearly distinguished it from the cathepsin family of proteases. The calcium-dependent activity, intracellular localization, and the limited, specific proteolysis on its substrates, highlighted calpain’s role as a regulatory, rather than a digestive, protease. When the sequence of this enzyme became known,[2] it was given the name “calpain”, to recognize its common properties with two well-known proteins at the time, the calcium-regulated signalling protein, calmodulin, and the cysteine protease of papaya, papain. Shortly thereafter, the activity was found to be attributable to two main isoforms, dubbed μ (“mu”)-calpain and m-calpain (or calpain I and II), that differed primarily in their calcium requirements in vitro. Their names reflect the fact that they are activated by micro- and nearly millimolar concentrations of Ca2+ within the cell, respectively.[3]

To date, these two isoforms remain the best characterised members of the calpain family. Structurally, these two heterodimeric isoforms share an identical small (28 kDa) subunit (CAPNS1 (formerly CAPN4)), but have distinct large (80 kDa) subunits, known as calpain 1 and calpain 2 (each encoded by the CAPN1 and CAPN2 genes, respectively).

Cleavage specificity[edit]

No specific amino acid sequence is uniquely recognized by calpains. Amongst protein substrates, tertiary structure elements rather than primary amino acid sequences are likely responsible for directing cleavage to a specific substrate. Amongst peptide and small-molecule substrates, the most consistently reported specificity is for small, hydrophobic amino acids (e.g. leucine, valine and isoleucine) at the P2 position, and large hydrophobic amino acids (e.g. phenylalanine and tyrosine) at the P1 position.[4] Arguably, the best currently available fluorogenic calpain substrate is (EDANS)-Glu-Pro-Leu-Phe=Ala-Glu-Arg-Lys-(DABCYL), with cleavage occurring at the Phe=Ala bond.

Extended family[edit]

The Human Genome Project has revealed that more than a dozen other calpain isoforms exist, some with multiple splice variants.[5][6][7] As the first calpain whose three-dimensional structure was determined, m-calpain is the type-protease for the C2 (calpain) family in the MEROPS database.

Gene Protein Aliases Tissue expression Disease linkage
CAPN1 Calpain 1 Calpain-1 large subunit, Calpain mu-type ubiquitous
CAPN2 Calpain 2 Calpain-2 large subunit, Calpain m-type ubiquitous
CAPN3 Calpain 3 skeletal muscle retina and lens specific Limb Girdle muscular dystrophy 2A
CAPN5 Calpain 5 ubiquitous (high in colon, small intestine and testis) might be linked to necrosis,
as it is an ortholog of the C. elegans necrosis gene tra-3
CAPN6 Calpain 6 CAPNX, Calpamodulin
CAPN7 Calpain 7 palBH ubiquitous
CAPN8 Calpain 8 exclusive to stomach mucosa and the GI tract might be linked to colon polyp formation
CAPN9 Calpain 9 exclusive to stomach mucosa and the GI tract might be linked to colon polyp formation
CAPN10 Calpain 10 susceptibility gene for type II diabetes
CAPN11 Calpain 11 testis
CAPN12 Calpain 12 ubiquitous but high in hair follicle
CAPN13 Calpain 13 testis and lung
CAPN14 Calpain 14 ubiquitous
CAPN17 Calpain 17 Fish and amphibian-only
SOLH Calpain 15 Sol H (homolog of the drosophila gene sol)
CAPNS1 Calpain small subunit 1 Calpain 4
CAPNS2 Calpain small subunit 2


Although the physiological role of calpains is still poorly understood, they have been shown to be active participants in processes such as cell mobility and cell cycle progression, as well as cell-type specific functions such as long-term potentiation in neurons and cell fusion in myoblasts. Under these physiological conditions, a transient and localized influx of calcium into the cell activates a small local population of calpains (for example, those close to Ca2+ channels), which then advance the signal transduction pathway by catalyzing the controlled proteolysis of its target proteins.[8] Additionally, phosphorylation by protein kinase A and dephosphorylation by alkaline phosphatase have been found to positively regulate the activity of μ-calpains by increasing random coils and decreasing β-sheets in its structure. Phosphorylation improves proteolytic activity and stimulates auto-activation of μ-calpains. However, increased calcium concentration overruns the effects of phosphorylation and dephosphorylation on calpain activity, and thus calpain activity ultimately depends on the presence of calcium.[9] Other reported roles of calpains are in cell function, helping to regulate clotting and the diameter of blood vessels, and playing a role in memory. Calpains have been implicated in apoptotic cell death, and appear to be an essential component of necrosis. Detergent fractionation revealed the cytosolic localization of calpain.[8]

Enhanced calpain activity, regulated by CAPNS1, significantly contributes to platelet hyperreactivity under hypoxic environment.[10]

In the brain, while μ-calpain is mainly located in the cell body and dendrites of neurons and to a lesser extent in axons and glial cells, m-calpain is found in glia and a small number in axons.[11] Calpain is also involved in skeletal muscle protein breakdown due to exercise and altered nutritional states.[12]

Clinical significance[edit]


The structural and functional diversity of calpains in the cell is reflected in their involvement in the pathogenesis of a wide range of disorders. At least two well known genetic disorders and one form of cancer have been linked to tissue-specific calpains. When defective, the mammalian calpain 3 (also known as p94) is the gene product responsible for limb-girdle muscular dystrophy type 2A,[13][14] calpain 10 has been identified as a susceptibility gene for type II diabetes mellitus, and calpain 9 has been identified as a tumour suppressor for gastric cancer. Moreover, the hyperactivation of calpains is implicated in a number of pathologies associated with altered calcium homeostasis such as Alzheimer’s disease,[15] and cataract formation, as well as secondary degeneration resulting from acute cellular stress following myocardial ischemia, cerebral (neuronal) ischemia, traumatic brain injury and spinal cord injury. Excessive amounts of calpain can be activated due to Ca2+ influx after cerebrovascular accident (during the ischemic cascade) or some types of traumatic brain injury such as diffuse axonal injury. Increase in concentration of calcium in the cell results in calpain activation, which leads to unregulated proteolysis of both target and non-target proteins and consequent irreversible tissue damage. Excessively active calpain breaks down molecules in the cytoskeleton such as spectrin, microtubule subunits, microtubule-associated proteins, and neurofilaments.[16][17] It may also damage ion channels, other enzymes, cell adhesion molecules, and cell surface receptors.[11] This can lead to degradation of the cytoskeleton and plasma membrane. Calpain may also break down sodium channels that have been damaged due to axonal stretch injury,[18] leading to an influx of sodium into the cell. This, in turn, leads to the neuron’s depolarization and the influx of more Ca2+. A significant consequence of calpain activation is the development of cardiac contractile dysfunction that follows ischemic insult to the heart. Upon reperfusion of the ischemic myocardium, there is development of calcium overload or excess in the heart cell (cardiomyocytes). This increase in calcium leads to activation of calpain.[19][irrelevant citation]
Recently calpain has been implicated in promoting high altitude induced venous thrombosis by mediating platelet hyperactivation.[10]

Therapeutic inhibitors[edit]

The exogenous regulation of calpain activity is therefore of interest for the development of therapeutics in a wide array of pathological states. As a few of the many examples supporting the therapeutic potential of calpain inhibition in ischemia, calpain inhibitor AK275 protected against focal ischemic brain damage in rats when administered after ischemia, and MDL28170 significantly reduced the size of damaged infarct tissue in a rat focal ischemia model. Also, calpain inhibitors are known to have neuroprotective effects: PD150606,[20] SJA6017,[21] ABT-705253,[22][23] and SNJ-1945.[24]

Calpain may be released in the brain for up to a month after a head injury, and may be responsible for a shrinkage of the brain sometimes found after such injuries.[25] However, calpain may also be involved in a “resculpting” process that helps repair damage after injury.[25]

See also[edit]


  1. ^ “the definition of calpain”. Retrieved 23 April 2018.
  2. ^ Ohno S, Emori Y, Imajoh S, Kawasaki H, Kisaragi M, Suzuki K (1984). “Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?”. Nature. 312 (5994): 566–70. Bibcode:1984Natur.312..566O. doi:10.1038/312566a0. PMID 6095110. S2CID 4359635.
  3. ^ Glass JD, Culver DG, Levey AI, Nash NR (April 2002). “Very early activation of m-calpain in peripheral nerve during Wallerian degeneration”. J. Neurol. Sci. 196 (1–2): 9–20. doi:10.1016/S0022-510X(02)00013-8. PMID 11959150. S2CID 22674283.
  4. ^ Cuerrier D, Moldoveanu T, Davies PL (December 2005). “Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions”. J. Biol. Chem. 280 (49): 40632–41. doi:10.1074/jbc.M506870200. PMID 16216885.
  5. ^ Thompson V (2002-02-12). “Calpain Nomenclature”. College of Agriculture and Life Sciences at the University of Arizona. Retrieved 2010-08-06.
  6. ^ Huang Y, Wang KK (August 2001). “The calpain family and human disease”. Trends Mol Med. 7 (8): 355–62. doi:10.1016/S1471-4914(01)02049-4. PMID 11516996.
  7. ^ Suzuki K, Hata S, Kawabata Y, Sorimachi H (February 2004). “Structure, activation, and biology of calpain”. Diabetes. 53. Suppl 1: S12–8. doi:10.2337/diabetes.53.2007.s12. PMID 14749260.
  8. ^ a b Jaguva Vasudevan, AA; Perkovic, M; Bulliard, Y; Cichutek, K; Trono, D; Häussinger, D; Münk, C (August 2013). “Prototype foamy virus Bet impairs the dimerization and cytosolic solubility of human APOBEC3G”. Journal of Virology. 87 (16): 9030–40. doi:10.1128/JVI.03385-12. PMC 3754047. PMID 23760237.
  9. ^ Du, Manting; Li, Xin; Li, Zheng; Shen, Qingwu; Wang, Ying; Li, Guixia; Zhang, Dequan (2018-06-30). “Phosphorylation regulated by protein kinase A and alkaline phosphatase play positive roles in μ-calpain activity”. Food Chemistry. 252: 33–39. doi:10.1016/j.foodchem.2018.01.103. ISSN 0308-8146. PMID 29478550. S2CID 3538480.
  10. ^ a b Tyagi, T.; Ahmad, S.; Gupta, N.; Sahu, A.; Ahmad, Y.; Nair, V.; Chatterjee, T.; Bajaj, N.; Sengupta, S.; Ganju, L.; Singh, S. B.; Ashraf, M. Z. (Feb 2014). “Altered expression of platelet proteins and calpain activity mediate hypoxia-induced prothrombotic phenotype”. Blood. 123 (8): 1250–60. doi:10.1182/blood-2013-05-501924. PMID 24297866.
  11. ^ a b Lenzlinger PM, Saatman KE, Raghupathi R, Mcintosh TK (2000). “Chapter 1: Overview of basic mechanisms underlying neuropathological consequences of head trauma”. In Newcomb JK, Miller LS, Hayes RL (eds.). Head trauma: basic, preclinical, and clinical directions. New York: Wiley-Liss. ISBN 978-0-471-36015-5.
  12. ^ Belcastro AN, Albisser TA, Littlejohn B (October 1996). “Role of calcium-activated neutral protease (calpain) with diet and exercise”. Can J Appl Physiol. 21 (5): 328–46. doi:10.1139/h96-029. PMID 8905185.
  13. ^ Richard I, Broux O, Allamand V, et al. (April 1995). “Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A”. Cell. 81 (1): 27–40. doi:10.1016/0092-8674(95)90368-2. PMID 7720071. S2CID 17565219.
  14. ^ Ono Y, Shimada H, Sorimachi H, et al. (July 1998). “Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A”. J. Biol. Chem. 273 (27): 17073–8. doi:10.1074/jbc.273.27.17073. PMID 9642272.
  15. ^ Yamashima T (2013). “Reconsider Alzheimer’s disease by the ‘calpain-cathepsin hypothesis’–a perspective review”. Progress in Neurology. 105: 1–23. doi:10.1016/j.pneurobio.2013.02.004. PMID 23499711. S2CID 39292302.
  16. ^ Liu J, Liu MC, Wang KK (April 2008). “Calpain in the CNS: from synaptic function to neurotoxicity”. Sci. Signal. 1 (14): re 1. doi:10.1126/stke.114re1. PMID 18398107. S2CID 21992464.
  17. ^ Castillo MR, Babson JR (October 1998). “Ca2+-dependent mechanisms of cell injury in cultured cortical neurons”. Neuroscience. 86 (4): 1133–44. doi:10.1016/S0306-4522(98)00070-0. PMID 9697120. S2CID 54228571.
  18. ^ Iwata A, Stys PK, Wolf JA, et al. (May 2004). “Traumatic axonal injury induces proteolytic cleavage of the voltage-gated sodium channels modulated by tetrodotoxin and protease inhibitors”. J. Neurosci. 24 (19): 4605–13. doi:10.1523/JNEUROSCI.0515-03.2004. PMC 6729402. PMID 15140932.
  19. ^ Wang KK, Larner SF, Robinson G, Hayes RL (December 2006). “Neuroprotection targets after traumatic brain injury”. Curr. Opin. Neurol. 19 (6): 514–9. doi:10.1097/WCO.0b013e3280102b10. PMID 17102687. S2CID 28119069.
  20. ^ Wang KK, Nath R, Posner A, Raser KJ, Buroker-Kilgore M, Hajimohammadreza I, Probert AW, Marcoux FW, Ye Q, Takano E, Hatanaka M, Maki M, Caner H, Collins JL, Fergus A, Lee KS, Lunney EA, Hays SJ, Yuen P (June 1996). “An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective”. Proc. Natl. Acad. Sci. U.S.A. 93 (13): 6687–92. Bibcode:1996PNAS…93.6687W. doi:10.1073/pnas.93.13.6687. PMC 39087. PMID 8692879.
  21. ^ Kupina NC, Nath R, Bernath EE, Inoue J, Mitsuyoshi A, Yuen PW, Wang KK, Hall ED (November 2001). “The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury” (PDF). J. Neurotrauma. 18 (11): 1229–40. doi:10.1089/089771501317095269. hdl:2027.42/63231. PMID 11721741.
  22. ^ Lubisch W, Beckenbach E, Bopp S, Hofmann HP, Kartal A, Kästel C, Lindner T, Metz-Garrecht M, Reeb J, Regner F, Vierling M, Möller A (June 2003). “Benzoylalanine-derived ketoamides carrying vinylbenzyl amino residues: discovery of potent water-soluble calpain inhibitors with oral bioavailability”. J. Med. Chem. 46 (12): 2404–12. doi:10.1021/jm0210717. PMID 12773044.
  23. ^ Nimmrich V, Reymann KG, Strassburger M, Schöder UH, Gross G, Hahn A, Schoemaker H, Wicke K, Möller A (April 2010). “Inhibition of calpain prevents NMDA-induced cell death and beta-amyloid-induced synaptic dysfunction in hippocampal slice cultures”. Br. J. Pharmacol. 159 (7): 1523–31. doi:10.1111/j.1476-5381.2010.00652.x. PMC 2850408. PMID 20233208.
  24. ^ Koumura A, Nonaka Y, Hyakkoku K, Oka T, Shimazawa M, Hozumi I, Inuzuka T, Hara H (November 2008). “A novel calpain inhibitor, ((1S)-1((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester, protects neuronal cells from cerebral ischemia-induced damage in mice”. Neuroscience. 157 (2): 309–18. doi:10.1016/j.neuroscience.2008.09.007. PMID 18835333. S2CID 29425598.
  25. ^ a b White V (1999-10-21). “– ‘Biochemical Storm’ Following Brain Trauma An Important Factor In Treatment, University of Florida Researcher Finds”. University of Florida News. Archived from the original on 2011-06-23. Retrieved 2010-08-07.

Further reading[edit]

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