Calpains are a family of non-lysosomal cysteine proteases that are activated by calcium. Calcium-dependent protease activity has already been detected in 1964 in brain tissue from rats, and this activity was later related to calpain. Yet, the identification of the protein started off with structural analysis of muscle tissue and its alteration by post mortem degradation (history broadly reviewed by Goll et al. 1990, 2003): In the late 1960s, Wayne Busch and Darrel Goll studied the physiological effects of Ca2+ in tissue specimen of rabbit muscle. When muscle strips were left overnight in a 1 mM Ca2+ buffer solution, it became apparent that the Z-line (a structural element separating sarcomers in skeletal muscle) had completely disappeared. Busch and Goll subsequently incubated the stripes in EGTA, thereby preventing degradation of the Z-line. With the help of Marvin Stromer, they performed electron microscopy studies showing that the degradation process was completely restricted to the Z-line, leaving other structural elements unaltered. In 1972, Busch and colleagues isolated a protein fraction from muscle fibers which caused removal of the Z-disk in a preparation of rabbit skeletal muscle at Ca2+ concentrations higher than 0.1 mM. The Z-line remained intact when Ca2+ was substituted by EGTA, strengthening the view that this activity required calcium. In 1976, Bill Dayton joined this group to isolate the protein (at that time called “Ca2+-activated factor”). There was need for larger tissue quantities which required a switch of species from rabbit to pigs. From this preparation, Dayton and colleagues could isolate a sufficient amount of protein with Ca2+-dependent proteolytic activity, which showed Z-disk removing characteristics. They also revealed that this protease contained two subunits, an 80-kDa subunit and a 30-kDa subunit. The protein was later named m-calpain, and is now mainly referred to as calpain 2. The name calpain was given to indicate that this is a calcium-dependent protease with papain-like activity.
Ron Mellgren identified a second Ca2+-dependent protease eluting at a different salt concentration than m-calpain. The Ca2+ concentration necessary for activation was in the μM range, and this protein was later named μ-calpain (now called calpain 1). This protein is also a heterodimer consisting of a catalytic 80-kDa subunit and a small 30-kDa subunit. The cDNAs for the large subunit of the two calpains have been cloned in the 1990s, and described as gene products of CAPN1 (coding for calpain 1) and CAPN2 (coding for calpain 2). The gene encoding the 30-kDa subunit has also been cloned and referred to as CAPN4. In 1989, further calpains beyond calpain 1 and calpain 2 were discovered, and over the last two decades 15 calpain-like genes have been identified in various tissues, and implicated with a variety of diseases. Only nine of them have the typical calmodulin-like EF-hand sequence and are commonly called “typical calpains,” as opposed to the “atypical calpains,” which do not contain an EF-hand domain (Saez et al. 2006).
Since the mid-1970s it was suspected that there must be a natural inhibitor of calpain in purified fractions of the enzyme and, in 1982, the first detailed report on an intrinsic inhibitor of calpain from human erythrocytes, called calpastatin, was published. Calpastatin is specific for calpain 1 and calpain 2, and does not appear to inhibit any other protease (Rachel et al. 2008). A number of synthetic calpain inhibitors, mostly of peptidic nature, are now commercially available. However, none of them is specific to calpain (Pietsch et al. 2010).
In the late 1980s, it was discovered that calpain dysfunction underlies muscular dystrophies. Since then, calpains have been suggested to be involved in a number of different pathologies, and the information on the pathophysiology of the calpain system has been increasing tremendously in the last two decades (Zatz and Starling 2005). Yet, the physiological role of calpains remains to a large extent unclear.
Biological Properties and Regulation
The “classical” calpains (1 and 2) are heterodimers comprising an 80-kDa subunit (catalytic subunit) and a common 30-kDa subunit (regulatory subunit). The catalytic domains of both calpains share about 60% sequence homology, and are comprised of four domains (I–IV). Domain II is further divided into two subdomains, IIa and IIb. In the absence of calcium, the catalytic triad consisting of Cys105 in domain I and His262 and Asn286 in domain II is separated, indicating that a conformational change is required for activation. Domain IV shares some sequence identity with calmodulin and contains five EF-hand Ca2+-binding sites. The regulatory subunit constitutes two domains (V and VI), with EF-hand binding sites in domain VI (Strobl et al. 2000).
The cellular regulation of calpain and its inhibition by calpastatin is rather complex (Khorchid and Ikura 2002). Structural analysis revealed that the Ca2+-free enzyme is catalytically inactive. A rise in the Ca2+ concentration above 1 mM induces a conformational change in the calpain molecule, inducing its proteolytic properties. Calcium appears to bind to more sites of the protein than just the EF-hand motifs, and it is not yet resolved which binding sites are required for activation of proteolytic activity. Interestingly, the concentration of 1 mM for the activation of calpain is not reached under physiological conditions, as the total intracellular Ca2+ concentration normally does not exceed 500 nM. It is likely that calpain can only be activated in cellular subdomains with increased Ca2+ concentration (e.g., in proximity to Ca2+ channels, synaptic terminals, etc.). Phospholipids appear to modulate the Ca2+ requirements of calpain, and it is possible that localization of calpain to the plasma membrane would enhance catalytic activity. Recent studies in fibroblasts have revealed that calpain 2 can be activated independently of calcium. Mitogen-activated protein kinase ( MAP Kinases), for example, activates calpain 2 by phosphorylation. As a result, calpain 2 is rapidly activated in dendrites and dendritic spines, thereby participating in cytoskeletal reorganization. Calpain 2 is also phosphorylated by the serine/threonine protein kinase ERK, leading to an activation of calpain and subsequent changes in tau cleavage, cell motility, and adhesion.
The contribution of the small “regulatory” subunit to calpain regulation is not fully understood. It has initially been speculated that the catalytic subunit is activated by dissociation of the small subunit from the core molecule. However, it appears that proteolytic activity is also present when the subunits are associated. The 30-kDa subunit may also be involved in folding of the 80-kDa subunit (as a molecular chaperone), thereby regulating function.
The last decades have revealed the involvement of calpain in several pathological conditions. In contrast, less is known about the exact physiological function of calpain. It likely fulfills multiple roles, which is indicated by its great number of cellular substrates. It is therefore surprising that inhibition of calpain in animals by synthetic inhibitors is largely harmless.
Probably the best described function of calpain is its contribution to the (re)organization of the cytoskeletal system during a number of cellular events. The involvement of calpains in cell movement is well documented (Glading et al. 2002). Some cytoskeletal proteins including those assembling into intermediate filaments (vimentin, desmin, neurofilament protein) are cleaved by calpain and may allow adaptation of cellular architecture. Spectrin, a building block of the sub-membraneous cytoskeleton as well as cross-linkers of cytoskeletal filaments (e.g., talin) are also substrates for calpain, further underlining its involvement in structural modulation. Notably, a number of muscular elements including myosin and titin (which anchors actin and myosin to the Z-disk) are calpain substrates and possibly mediate the involvement of calpain in muscle diseases. Calpain also degrades dystrophin, a protein anchoring actin to the plasma membrane of muscle cells. Proteins of the microtubular system (MAP1 and MAP2) are also cleaved, as well as cell adhesion molecules (e.g., N-cadherin). In addition, calpain appears to be involved in cytoskeletal protein cleavage in platelet aggregation. Finally, calpain modulates cytoskeletal organization during myoblast fusion.
It is generally thought that calpain is central to a number of signaling pathways (Vosler et al. 2008). For example, RhoA activity is directly affected by calpain, thereby inhibiting cell spreading. Calpain also appears to be involved in apoptosis. Calpain inhibitors reportedly block proliferation at the G1 phase suggesting that calpain contributes to cell cycle. In some tissues calpains may have highly specialized functions. In neurons, for example, calpains appear to play a central role in regulating synaptic function and plasticity (Wu and Lynch 2006), and may mediate long-term potentiation (LTP), a cellular form of learning and memory.
Calpains and Disease
Calpains have been suggested to be involved in a number of CNS diseases, especially those accompanied by chronic neurodegenerative processes (Liu et al. 2008). Excessive stimulation of NMDA receptors leads to calpain-mediated neurodegenerative cascades, which can be prevented by inhibition of calpain in several in vivo models. Calpain is activated in a number of CNS disorders, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. It was recently shown that amyloid-β-induced nucleus basalis degeneration (a brain region affected during Alzheimer’s disease) can be prevented by calpain inhibition in rats. At the cellular level, calpain mediates amyloid-β-induced cleavage of the presynaptic protein dynamin, thereby causing deficits in synaptic function. Calpain exacerbates tau pathology in Alzheimer’s disease by cleaving p35, thereby activating cdk5, one of the kinases hyperphosphorylating tau protein. Calpain-mediated cdk5 activation also contributes to dopaminergic cell death in Parkinson’s disease. A dysfunctional calpain system is also present in Huntington’s disease, where active calpain directly cleavages the protein huntingtin, leading to a toxic accumulation of a protein fragment.
Beside chronic neurodegenerative diseases, calpain contributes to cell death in traumatic brain injury (Saatman et al. 2010) and cerebral ischemic processes (Bevers and Neumar 2008). Both pathologies involve excitotoxic cascades, which are prevented by calpain inhibition. For example, knockdown of calpain 1 increases long-term survival and protects hippocampal function in transient forebrain ischemia. Calpain is activated in the penumbra of the ischemic injury site and some calpain inhibitors have been shown to be neuroprotective. Similarly, calpain inhibition was effective in rat models of traumatic brain injury. Interesting, brain damage in both ischemia as well as traumatic brain injury can be prevented, when calpain inhibitors are given after the insult, indicating that calpain is involved in part of the downstream cascade of the excitotoxic process.
Calpain activation also facilitates degeneration of cardiac tissue after myocardial ischemia/reperfusion injury (Inserte et al. 2009). Both calpain 1 and calpain 2 are increased in ventricular muscle after coronary artery ligation in rats, and inhibition of calpain protects from ischemic cardiac muscle degeneration in animal models of myocardial infarction or in isolated rabbit heart. In an ischemia/reperfusion model in pigs, calpain inhibition decreased infarct size and improved ventricular contractility, and improved overall hemodynamic function.
Finally, there is strong evidence for the involvement of calpains in myopathies of skeletal muscle (Zatz and Starling 2005). Mutations in the gene encoding calpain 3 are responsible for the autosomal recessive disorder limb-girdle muscular dystrophy type 2A. Recent data suggest that mitochondrial abnormalities in calpain 3 deficient muscles may be the underlying mechanism.
Ubiquitously expressed and with numerous known substrates, calpain plays a significant role in cellular physiology. When activated, calpain cleaves various membrane components, cytosolic enzymes, and regulatory and structural proteins. Interaction with its substrates may lead to rapid changes in cellular functioning. While calpain activation is critically involved in numerous diseases including degenerative changes in the brain, muscles, retina, kidney, and other tissues, the physiological role is less understood. For an activity-regulated system like the calpain system, parameters like duration, intensity, and localization set the course for either a physiological or a pathological process. For example, activation of NMDA receptors activates calpain, leading to a cleavage of synaptic NMDA receptor subunits thereby preventing overexcitation. In contrast, excessive stimulation (possibly involving extrasynaptic NMDA receptors) induces calpain overactivation leading to downstream excitotoxic cascades and neurodegeneration. Analysis of the conditions which turn calpain activation from physiological function into pathophysiological offense is important not only for a full appreciation of the signaling role of this molecule, but also for the development of novel therapeutics exploiting the biological significance of the calpain system.
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