Association of the calpain/calpastatin network with subcellular organelles

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.     

Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.     

Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.     

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.     

In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.     

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.     

The Calpain–Calpastatin System and Cellular Proliferation and Differentiation in Rodent Osteoblastic Cells

The calpain–calpastatin system, which consists of calpains I and II (two ubiquitously distributedcium-activated pa-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain–calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain–calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.     

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.     

Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.     

Changes in pulmonary calpain activity following treatment of mice with butylated hydroxytoluene

The antioxidant, butylated hydroxytoluene (BHT), causes lung toxicity in mice followed by regenerative repair, and can also modulate the development of carcinogen-induced lung adenomas. We are investigating changes in pulmonary biochemistry following BHT treatment in order to understand the mechanisms of BHT-induced pulmonary regenerative repair. BHT administration lowered cytosolic Ca2+-activated neutral protease (calpain) activity, increased the activity of the endogenous calpain inhibitor, calpastatin, increased the extent of photoincorporation of 8-N3-[32P]cAMP into a Mr 37,000 proteolytic product derived from cAMP-dependent protein kinase regulatory (R) subunits, and increased membrane-associated protease activity. All of these changes were dependent on the BHT dosage; the altered proteolytic activities occurred at a dose lower than that which caused observable lung toxicity as assessed by the lung weight/body weight ratio. Decreased cytosolic calpain activity was detectable within 1 day after BHT administration, was lowest at 4-7 days, and had not returned to control levels by Day 21, a time when normal lung morphology had been regained. The decrease in calpain activity cannot fully be accounted for by increased calpastatin activity; upon separation of these proteins by DEAE chromatography, the amount of calpain activity from BHT-treated mice remained lower than the corresponding peak from control mice. Increased photolabeling of the Mr 37,000 protein began at 1 day and continued to increase up to 4 days after BHT. All of the cytosolic changes preceded the increased particulate proteolytic activity by 1-2 days. R-subunits which have dissociated from their catalytic subunits are more susceptible to degradation by calpain, but BHT treatment did not enhance subunit dissociation as determined by the elution profile of 8-N3-[32P]cAMP-labeled R-subunits following DEAE chromatography. A large percentage of the particulate protease activity was inhibited by calpastatin, leupeptin, and E-64, all of which are known to inhibit calpain activity; this suggested that calpain accounted for most of this activity. Changes in the activities of proteases which catalyze limited proteolysis reactions may play an important role in the repair of acute lung injury.     

Calpain from rat intestinal epithelial cells: Age-dependent dynamics during cell differentiation

Micromolar and millimolar Ca²⁺-requiring neutral protease (calpain I and calpain II) along with their endogenous inhibitor calpastatin were isolated and partially purified from the same preparation of rat intestinal epithelial cells. Calpain I and II were partially purified by 1300 and 900-fold with 57 and 53 per cent yield, respectively. The optimum assay conditions revealed pH 7.5, 20 min incubation at 25° C and 0.24% casein substrate for both calpains. The optimum calcium concentration obtained for calpain I and II were 25 M and 4 mM, respectively. Distribution of rat intestinal epithelial cells calpain I and II along with calpastatin during cell differentiation stages in weanling to senescence age were studied. Calpain I in weanling rats was in an increasing order from villus to crypt regions. Adult rats indicated well expressed consistent calpain I throughout the differentiation stages. Whereas, significant lowering towards crypt region cells were evident in old rats. Calpain II in weanling and adult rats was found to be consistent throughout the differentiation stages. Old animals revealed an increasing trend from villus to crypt region with insignificant activity present in upper villus cells. Concomitantly, different concentrations of calpastatin were observed throughout the differentiation stages in all the age groups. Moreover, the levels of calpains exceeded that of calpastatin in most of the epithelial cell populations during developmental stages. In addition to casein, intestinal epithelial cell membranes were found to be equally good substrates for calpains. Proteolytic susceptibility of weanling, adult and old rat membrane proteins varied significantly all along the ageing process in rats. Simultaneous age-dependent calpastatin response were also evident. Taken together the results obtained provided strong evidence that calpain plays significant role in rat intestinal cell differentiation and ageing process with calpastatin as its specific regulatory protein.Abbreviations DEAE-cellulose O-(Diethylaminoethyl)-cellulose - EDTA Ethylene Diamine Tetra Acetic Acid - Tris Tris (hydroxymethyl) amino methane - KH₂PO₄ potassium dihydrogen orthophosphate - Na₂HPO₄ disodium hydrogen phosphate - CaCl₂ Calcium Chloride - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride     

Intracellular regulatory system involving calpain and calpastatin

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.     

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation

Background

To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.

Methods

To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.

Results and conclusions

By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.

General significance

The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Characterization of the calpain/calpastatin system in human hemopoietic cell lines

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.     

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.     

Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.