Calpains — An elaborate proteolytic system

Calpain is an intracellular Ca²⁺-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca²⁺via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Evolutionary and physical linkage between calpains and penta-EF-hand Ca2+-binding proteins

The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.     

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.     

Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimer's disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Impact of genetic insights into calpain biology

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.     

Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin?

Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.     

The crystal structures of human calpains 1 and 9 imply diverse mechanisms of action and auto-inhibition

Calpains are calcium activated cysteine proteases found throughout the animal, plant, and fungi kingdoms; 14 isoforms have been described in the human genome. Calpains have been implicated in multiple models of human disease; for instance, calpain 1 is activated in the brains of individuals with Alzheimer's disease, and the digestive tract specific calpain 9 is down-regulated in gastric cancer cell lines. We have solved the structures of human calpain 1 and calpain 9 protease cores using crystallographic methods; both structures have clear implications for the function of non-catalytic domains of full-length calpains in the calcium-mediated activation of the enzyme. The structure of minicalpain 1 is similar to previously solved structures of the protease core. Auto-inhibition in this system is most likely through rearrangements of a central helical/loop region near the active site cysteine, which occlude the substrate binding site. However, the structure of minicalpain 9 indicates that auto-inhibition in this enzyme is mediated through large intra-domain movements that misalign the catalytic triad. This disruption is reminiscent of the full-length inactive calpain conformation. The structures of the highly conserved, ubiquitously expressed human calpain 1 and the more tissue specific human calpain 9 indicate that although there are high levels of sequence conservation throughout the calpain family, isolated structures of family members are insufficient to explain the molecular mechanism of activation for this group of proteins.     

Calpains: targets of cataract prevention?

There is emerging evidence to suggest that the unregulated Ca(2+)-mediated proteolysis of essential lens proteins by calpains might be a major contributor to some forms of cataract in both animals and humans. Moreover, recently solved calpain structures have revealed molecular-level details of the activation mechanism used by these proteases, enabling the structure-based design of potent calpain inhibitors with the potential to act as anti-cataract agents. These agents offer the first real hope of an urgently needed alternative to the surgical treatment of at least some forms of cataract and relief from a life-depreciating condition on a global scale.     

Trigonella foenum graecum seed powder protects against histopathological abnormalities in tissues of diabetic rats

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca2+) and elevated levels of lens Ca2+ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3: Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca2+-mediated activation of these enzymes.     

Action of calpain on the basic estrogen receptor molecule of porcine uterus

Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.     

Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster.

A Ca(2+)-activated thiol protease was purified from Drosophila melanogaster. The procedure involves Phenyl-Sepharose, Reactive Red-Agarose and Q-Sepharose fast flow (or MonoQ) chromatographic steps. The enzyme eluting from Q-Sepharose fast flow seems to be homogeneous as judged by silver staining on SDS-PAGE: it consists of a single polypeptide chain of M(r),app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca(2+)-dependent, characterized by 0.6 mM free Ca2+ at half-maximal activity. Ca2+ ions cannot be replaced effectively by the divalent cations Mg2+, Mn2+, Zn2+, Ba2+, and Cd2+. The enzyme shows the inhibitor pattern of thiol proteases. Human recombinant calpastatin (domain I) completely inhibits the enzyme at a nearly 1:1 molar ratio. Several of these properties resemble those of vertebrate calpain II. However, various attempts to detect a small subunit of M(r) approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.     

The intriguing Ca2+ requirement of calpain activation

Mammalian ubiquitous micro- and m-calpains, as well as their Drosophila homologs, Calpain A and Calpain B, are Ca(2+)-activated cytoplasmic proteases that act by limited proteolysis of target proteins. Calpains are thought to be part of many cellular signaling pathways. These enzymes, however, require such high Ca(2+) concentration for half-maximal activation in vitro, [Ca(2+)](0.5), that hardly ever occurs in intact cells. This major dilemma has pervaded the literature on calpains for decades. In this paper several considerations are put forward that challenge the orthodox view and envisage mechanisms that may govern calpain action in vivo. The "unphysiologically" high Ca(2+) demand for activation may turn out to be an evolutionarily adjusted safety device.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).     

Mitochondrial localization of mu-calpain

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80kDa mu-calpain large subunit and 28kDa small subunit. In rat cortex, antibodies against domains II and III of the large mu-calpain subunit also detected a 40kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte mu-calpain with Ca(2+). Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of mu-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca(2+) released from mitochondrial stores.     

Stimulation of beta-amyloid precursor protein alpha-processing by phorbol ester involves calcium and calpain activation

Normal processing of Alzheimer's beta-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PDBu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP alpha-processing by PDBu is through a mechanism that involves the activation of Ca(2+) and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP alpha-processing.     

The atypical calpains: evolutionary analyses and roles in Caenorhabditis elegans cellular degeneration

The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.     

Activation of m-calpain is required for chromosome alignment on the metaphase plate during mitosis

Calpains form a superfamily of Ca(2+)-dependent intracellular cysteine proteases with various isoforms. Two isoforms, micro- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca(2+) concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not mu-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.