Calcium-dependent proteases: an enzyme system active at cellular membranes?

Proteases having a neutral pH optimum and an absolute requirement for calcium ion are found in virtually all mammalian cells. Association of calcium-dependent proteases and a specific inhibitor protein with biological membranes seems to be an important regulatory feature of this proteolytic system, and it is likely that membranes are preferred sites for calcium-dependent protease action. Several recent hypotheses for the physiological function of calcium-dependent proteolysis are consistent with a membrane-associated protease action. Calcium-dependent proteases may participate in cell membrane fusion: the proteolysis of membrane proteins, which is required for the efficient fusion of erythrocytes, may be catalyzed by these enzymes. There is also evidence for the involvement of calcium-dependent proteolysis in postsynaptic membrane remodeling in the hippocampus after long-term potentiation. Although the relationship of the proteolysis to synaptic function is not known, it could have important physiological or pathophysiological consequences. Finally, it has recently been suggested that calcium-dependent proteolysis may be a physiologically significant mechanism for activating membrane-associated protein kinase C after exposure of some cell types to phorbol esters or other mitogens. Further pursuit of these hypotheses may reveal a novel role for intracellular calcium-regulated proteolysis in membrane-associated cell functions.     

Calpains and their endo- and exogenous regulators in various models of neurodegeneration

Based on the data obtained in a series of experiments with laboratory animals, conclusions about changes in the activity of calcium-dependent proteases in the brains of rats with induced neurodegeneration have been drawn. The properties of the proteolytic and regulatory components of the calpain system under the action of neurotoxic agents: amyloid β-peptide and glutamate have been characterized, and the main endogenous regulatory mechanisms of changes in the intensity of calcium-dependent proteolysis have been established. The neuroprotective properties of exogenous calpain regulators acting by different mechanisms: sex steroids and calcium channel regulators have been tested on degeneration models examined.     

Calpains and muscular dystrophies

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.     

Domain structure and phosphorylation of protein kinase C

The phospholipid- and calcium-dependent protein kinase C has been shown to autophosphorylate on both the catalytic and the regulatory domains. The autophosphorylation displays zero-order kinetics, indicating that it is an intramolecular event. Autophosphorylation increases the activity of protein kinase C by decreasing the Km for the substrate H1 histone. The catalytic fragment obtained by limited proteolysis can no longer autophosphorylate and has a reduced affinity for H1 histone, exhibiting a Km 5-fold higher than that of the intact enzyme. Monoclonal antibodies produced against the enzyme can distinguish between the catalytic fragment and the intact enzyme by inhibiting their activities in a specific manner. Evidence suggesting that dimerization of protein kinase C occurs upon activation is presented.     

DNA ligase III is degraded by calpain during cell death induced by DNA-damaging agents

A yeast two-hybrid screen identified the regulatory subunit of the calcium-dependent protease calpain as a putative DNA ligase III-binding protein. Calpain binds to the N-terminal region of DNA ligase III, which contains an acidic proline, aspartate, serine, and threonine (PEST) domain frequently present in proteins cleaved by calpain. Recombinant DNA ligase III was a substrate for calpain degradation in vitro. This calpain-mediated proteolysis was calcium-dependent and was blocked by the specific calpain inhibitor calpeptin. Western blot analysis revealed that DNA ligase III was degraded in human fibrosarcoma HT1080 cells following exposure to gamma-radiation. The degradation of DNA ligase III was prevented by pretreatment with calpeptin, which protected irradiated cells from death. Calpeptin treatment also blocked 9-amino camptothecin-induced DNA ligase III proteolysis and simultaneously protected the cells from death. HT1080 clones expressing a modified DNA ligase III that lacked a recognizable PEST domain were significantly more resistant to killing by gamma-radiation or 9- amino camptothecin than were cells that overexpressed the wild-type form of DNA ligase III. These data show that calpain-mediated proteolysis of DNA ligase III plays an essential role in DNA damage-induced cell death in human cells.     

Characterization of human recombinant transglutaminase 1 purified from baculovirus-infected insect cells

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.     

Characterization of Human Recombinant Transglutaminase 1 Purified from Baculovirus-infected Insect Cells

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both μ- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.     

Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms.

A novel method to observe the autolytic activation of a mammalian cytoplasmic calcium protease, mu-calpain, was developed using a set of antipeptidic antibodies capable of distinguishing between the pre- and post-autolysis forms of the enzyme. Antibodies raised against synthetic peptides designed to match the N-terminal sequences of the pre- and post-autolysis forms of the mu-calpain large subunit reacted specifically with the corresponding form of calpain and not with the other. The antibodies were specific and sensitive enough to detect the antigens in crude cell lysates. The relevance of the immunochemical detection of calpain activation was confirmed by the observation that proteolysis of a substrate protein by purified mu-calpain paralleled autolysis at various pCa as probed by these antibodies and that autolysis preceded substrate proteolysis. We also observed calcium-dependent autolysis of calpain accompanying subsequent proteolysis of substrate in intact cells using the antibodies. The method will provide a novel approach to assess the physiological targets of the enzyme by determining the local intracellular sites of calpain activation.     

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 micromol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in approximately 3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Calpain-mediated proteolysis of talin regulates adhesion dynamics

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.     

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo

Neuronal activity regulates the catabolism of specific structural proteins in adult mammalian brain. Pharmacological stimulation of rat hippocampal neurons by systemic or intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate induces selective loss of brain spectrin and the microtubule-associated protein MAP2, as determined by quantitative immunoblotting, but not of actin, the high molecular weight neurofilament polypeptide, or glial fibrillary acidic protein. The spectrin decrease occurs primarily by enhanced proteolysis, as levels of the major breakdown products of the alpha-subunit increase more than 7-fold. This proteolysis may occur from activation of the calcium-dependent neutral protease calpain I. The immunopeptide maps produced by alpha-spectrin degradation, selective loss of spectrin and MAP2, and decrease in calpain I levels are all consistent with calpain I activation accompanied by autoproteolysis. We propose that calcium influx and calpain I activation provide a mechanism by which neuronal activity regulates the degradation of specific neuronal structural proteins and may thereby modify neuronal morphology.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

Calpain activation in apoptosis

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley-Liss, Inc.     

Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion

Adhesion plaques, specialized regions of the plasma membrane where a cell contacts its substratum, are dynamic structures. However, little is known about how the protein-protein interactions that occur at adhesion plaques are controlled. One mechanism by which a cell might modulate its associations with the substratum is by selective, regulated proteolysis of an adhesion plaque component. Here we show that the catalytic subunit of the calcium-dependent protease type II (CDP-II) is localized in adhesion plaques of several cell types (BS-C-1, EBTr, and MDBK). We have compared the susceptibility of the adhesion plaque constituents vinculin, talin, and alpha-actinin to calcium-dependent proteolysis in vitro and have found talin to be the preferred substrate for CDP-II. The colocalization of a calcium-requiring proteolytic enzyme and talin in adhesion plaques raises the possibility that calcium-dependent proteolytic activity provides a mechanism for regulating some aspect of adhesion plaque physiology and function via cleavage of talin.     

Aurintricarboxylic Acid Protects Hippocampal Neurons from NMDA-and Ischemia-Induced Toxicity In Vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium-dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia-or excitotoxin-induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA-and ischemia-induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.     

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.