- Calpain
Calpains (EC number|3.4.22.52, EC number|3.4.22.53) are a family of
calcium -dependent, non-lysosomalcysteine protease s (proteolytic enzymes ) expressed ubiquitously in mammals and many other organisms. Calpains constitute the C2 family of protease clan CA in theMEROPS database . The calpain proteolytic system includes the calpain proteases, the small regulatory subunit (CAPNS1 (former CAPN4, a.k.a. 30K)), and the endogenous calpain-specific inhibitor,calpastatin .Historical background
The history of calpain originates during the mid-1960s, 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 andskeletal muscle . These activities were caused by an intracellular cysteine protease not associated with thelysosome and having an optimum activity at neutralpH , which clearly distinguished it from thecathepsin family of proteases. The calcium-dependent activity, intracellular localization, along with the limited, specificproteolysis on its substrates, highlighted calpain’s role as a regulatory, rather than a digestive protease. When the sequence of this enzyme became known,Ohno S, Emori Y, Imajoh S, Kawasaki H, Kisaragi M, Suzuki K. (1984). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=6095110 Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?] "Nature". 312: 566-570.] it was given the name “calpain”, to recognize it as a hybrid of two well-known proteins at the time, the calcium-regulated signaling protein,calmodulin , and the cysteine protease ofpapaya ,papain . Shortly thereafter, the activity was found to be attributable to two main isoforms, dubbed μ("mu")-calpain and m-calpain (a.k.a. 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 millimolar concentrations of Ca2+ within the cell, respectively.Glass JD, Culver DG, Levey AI, and Nash NR. (2002). [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11959150&dopt=Citation Very early activation of m-calpain in peripheral nerve during Wallerian degeneration.] "Journal of the Neurological Sciences". 196(1-2): 9-20.] To date, these two isoforms remain the best characterised members of the calpain family. Structurally, these two heterodimeric isoforms share an identical small (30k) subunit (CAPNS1 (former CAPN4)), but have distinct large (80k) subunits.Recently, sequencing of the human genome has revealed
gene s for more than a dozen other calpainisoform s, some with multiplesplice variant s. As the first calpain whose almost complete three-dimensional structure was determined, m-calpain is the type-protease for the C2 (calpain) family in the MEROPS database.Physiological roles
Although the physiological roles of calpains are still poorly understood, they have been shown to be active participants in processes such as
cell mobility andcell cycle progression, as well as cell-type specific functions such aslong-term potentiation inneuron s andcell fusion inmyoblast s. 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. Other reported roles of calpains are in cell function, helping to regulateclotting and the diameter ofblood vessel s, and playing a role inmemory . Calpains have been implicated in apoptotic cell death, and appear to be an essential component ofnecrosis .In the brain, while μ-calpain is mainly located in the
cell body anddendrite s ofneuron s and to a lesser extent inaxon s andglial cell s, m-calpain is found in glia and a small amount in axons. Calpain is also involved in skeletal muscle protein breakdown due to exercise and altered nutritional states.Belcastro AN, Albisser TA, Littlejohn B (1996). [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8905185&dopt=Citation Role of calcium-activated neutral protease (calpain) with diet and exercise.] "Canadian Journal of Applied Physiology". 21(5):328-346. PMID 8905185. RetrievedJanuary 19 ,2007 .]Calpains in pathologies
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,Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Beckmann JS. (1995). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=7720071 Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A.] "Cell". 81:27-40.] Ono Y, Shimada H, Sorimachi H, Richard I, Saido TC, Beckmann JS, Ishiura S, Suzuki K. (1998). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9642272 Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A.] "J. Biol. Chem.". 273:17073-17078.] calpain 10 has been identified as a susceptibility gene for type II diabetes mellitus, and calpain 9 has been identified as a tumor 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, 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 theischemic cascade ) or some types oftraumatic brain injury such asdiffuse 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 thecytoskeleton such asspectrin ,microtubule subunits,microtubule-associated protein s, andneurofilament s. Castillo MR and Babson JR. (1998). [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9697120&dopt=Abstract Ca2+-dependent mechanisms of cell injury in cultured cortical neurons.] "Neuroscience". 86(4): 1133-1144. PMID 9697120 Retrieved onJanuary 19 ,2007 .] It may also damageion channel s, other enzymes,cell adhesion molecule s, andcell surface receptors .Lenzlinger PM, Saatman KE, Raghupathi R, Mcintosh TK. 2001. Overview of basic mechanisms underlying neuropathological consequences of head trauma. Chapter 1. In, "Head trauma: Basic, preclinical, and clinical directions". Miller LP and Hayes RL, eds. Wiley Liss, (a John Wiley and Sons publication) New York. ] This can lead to degradation of the cytoskeleton andplasma membrane . Calpain may also break downsodium channel s that have been damaged due to axonal stretch injury,Iwata A, Stys PK, Wolf JA, Chen XH, Taylor AG, Meaney DF, and Smith DH. (2004). [http://www.jneurosci.org/cgi/content/full/24/19/4605 Traumatic Axonal Injury Induces Proteolytic Cleavage of the Voltage-Gated Sodium Channels Modulated by Tetrodotoxin and Protease Inhibitors.] "The Journal of Neuroscience". 24(19): 4605-4613. PMID 15140932 Retrieved onJanuary 19 ,2007 .] leading to an influx ofsodium into the cell. This, in turn, leads to the neuron'sdepolarization and the influx of more Ca2+. A significant consequence of calpain activation is the development ofcardiac 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. 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 inhibitorAK275 protected against focal ischemic brain damage in rats when administered after ischemia, andMDL28170 significantly reduced the size of damaged infarct tissue in a rat focal ischemia model.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.White V. (1999). [http://www.napa.ufl.edu/99news/braintra.htm 'Biochemical Storm' Following Brain Trauma an Important Factor in Treatment, University of Florida Researcher Finds.] Retrieved on
January 19 ,2007 .] However, calpain may also be involved in a "resculpting" process that helps repair damage after injury.Protein structure
Calpain 1 and 2 are heterodimeric enzymes that share a common 28k regulatory subunit (encoded by the "CAPNS1" (former "CAPN4") gene).
Cleavage specificity
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. Amongstpeptide and small-molecule substrates, the most consistently reported specificity is for small,hydrophobic amino acids (e.g.leucine ,valine andisoleucine ) at the P2 position, and large hydrophobic amino acids (e.g.phenylalanine andtyrosine ) at the P1 position.Cuerrier D, et al (2005). [http://www.jbc.org/cgi/content/full/280/49/40632 Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions.] "Journal of Biological Chemistry". 280(49):40632-40641. PMID 16216885 Retrieved onJanuary 19 ,2007 . ] Arguably, the best currently availablefluorogenic calpain substrate is (EDANS )-Glu-Pro-Leu-Phe=Ala-Glu-Arg-Lys-(DABCYL ), with cleavage occurring at the Phe=Ala bond.References
External links
* [http://www.tlc-aaea.org/calpains/index.html The Calpain Family of Proteases.] (2001). University of Arizona.
* Wang, K.K.W. and Yuen, P.-w. (Eds.) (1999) [http://www.amazon.com/dp/1560327138/ Calpain: The Pharmacology and Toxicology of Calcium-Dependent Protease.] Taylor and Francis, PA; 22 Chapters, 441 pages.
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