Calpain regulates actin remodeling during cell spreading.

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites 17beta-estradiol.

The interactions in vitro between [3H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins is serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17beta-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing 3H-labeled steroid-protein complexes or with serum protein-steroid complexes relative to [3H]estradiol. Nuclei preferentially accumulated 3H-labeled steroids from liver cytosol protein-steroid complexes relative to [3H[estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17beta-[3H]estradiol and the 3H-labeled water-soluble metabolite were retained in vitro by nuclei.U

Comparison of two calcium-dependent proteinases from bovine heart.

We have purified two calcium-dependent proteinases from bovine heart. Each enzyme was a heterodimer. One proteinase (designated CDP-I) contained subunits of 84 and 26 kDa. The other proteinase (designated CDP-II) contained subunits of 80 and 26 kDa. The large subunit of each proteinase accounted for the calcium-dependent proteolytic activity of the respective enzyme and could be isolated from the small subunit by casein-Sepharose affinity chromatography. The large subunits of CDP-I and CDP-II appeared to be distinctly different proteins, based on differences in peptide maps and on the lack of detectable immunologic cross-reactivity. On the other hand, the small subunits of the proteinases appeared to be identical peptides, based on peptide mapping and on two-dimensional gel electrophoresis. The function of the small subunit is unknown. The two calcium-dependent proteinases share many catalytic properties, including the nature of proteinase activity against several myofibrillar proteins. However, the proteinases were distinguished by different calcium-concentration requirements. CDP-II required 300 microM Ca2+ for half-maximal activity and 750 microM Ca2+ for full activity. CDP-I required 30 microM Ca2+ for half-maximal activity and 100 microM for full activity.

Cleavage of caldesmon and calponin by calpain: substrate recognition is not dependent on calmodulin binding domains.

The calmodulin binding proteins, caldesmon and calponin, are cleaved by both major isoforms of calpain in vitro. The patterns of fragments generated by each enzyme are essentially identical for a given substrate. Qualitatively, the cleavage pattern of each substrate is unchanged by the presence or absence of calmodulin suggesting that the interaction between calmodulin and these calmodulin-binding proteins does not alter substrate recognition by calpain. However, calmodulin (at microM concentrations) does have a small, but significant, inhibitory effect directly on calpain as evidenced by slower rates of cleavage of alpha-casein, a protein that does not bind calmodulin. Inhibition is more pronounced with mu-calpain (15-25%) than with m-calpain (6-10%). In order to demonstrate, unequivocally, that substrate recognition does not require an interaction between calpain and a substrate's calmodulin-binding domain, recombinant, full-length caldesmon and a mutant lacking the calmodulin binding domain were tested as substrates for calpain in the presence and absence of calmodulin. Calpain produced similar cleavage patterns of the baculovirus expressed caldesmon and the truncated mutant. Competition experiments demonstrated that calpain does not discriminate between the truncated mutant and full length caldesmon. This suggests that substrate recognition by calpain was not altered significantly by the absence of the calmodulin-binding domain. Cleavage of a second calmodulin-binding protein, calponin was also examined. The rate of calponin cleavage was increased in the presence of calmodulin, an observation that is also inconsistent with any requirement for calpain to bind to its calmodulin-binding site. These results demonstrate that calmodulin-binding domains do not provide substrate recognition sites for calpains. It seems likely that the calmodulin-like regions of calpain function to bind calcium and to regulate enzyme conformation as required for activity and that they do not interact directly with most substrates.

An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts.

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.

The high molecular weight multicatalytic proteinase, macropain, exists in a latent form in human erythrocytes.

The high molecular weight multicatalytic proteinase, macropain, has been purified from human erythrocytes in two forms that differ in caseinolytic activity up to 100-fold. Each form has a native molecular weight of 600,000 and is composed of a number of subunits ranging in molecular weights from 35,000 to 21,000. Although the two proteinase forms share a number of electrophoretically indistinguishable subunits, there are also subunits unique to the respective forms. The less active proteinase represents a latent enzyme because it was fully activated by two procedures including dialysis against water and pretreatment with low concentrations of sodium dodecyl sulfate. These procedures caused differential changes in the caseinolytic and two peptidase activities of the proteinase. An Mr 35,000 subunit, characteristic of latent macropain, is immunologically related to at least one of the other components of active macropain and disappeared after proteinase activation by dialysis. Nevertheless, loss of this subunit was not the cause of the increased activity. These results suggest that the proteolytic activity of cells may be regulated by the activation of the latent form of macropain.

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.

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.

Polyclonal antisera specific for the proenzyme form of each calpain.

Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes are exposed to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. We have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-terminal epitope of milli-calpain was used to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitope of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of mu-calpain in rat erythrocytes treated with ionomycin and calcium.

Calcium-dependent affinity purification of transglutaminase from rat liver cytosol.

Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases.

m-Calpain subunits remain associated in the presence of calcium.

The hypothesis that calpain subunits dissociate in the presence of Ca2+ has been tested by methods which avoid interference by Ca2+-induced aggregation and large subunit autolysis. Inactive Cys105Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca2+, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN2, also bound to immobilized casein and was eluted as a heterodimer. The Ca2+ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca2+. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca2+ both before and after autolysis.

Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments.

Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca(2+)-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca(2+)-regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71.5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47-48 and bPTH69-84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca(2+)-dependent degradation of PTH to secreted C-terminal fragments.

Proteolytic modification of calcium-dependent protease 1 in erythrocytes treated with ionomycin and calcium.

In vitro, limited proteolytic cleavage of the subunits of the purified calcium-dependent proteases [also known as calpains (EC 3.4.22.17) or calcium-activated neutral proteinases (CANPs)] appears to be required for enzyme activity. It has not yet been demonstrated if similar processing of the protease subunits occurs in vivo. To directly assess proteolytic modification of these proteases in cells, we have measured the loss of the proenzyme form of the regulatory subunit (a 26-kDa protein) and/or the appearance of the modified regulatory subunit (a 17-kDa protein) by densitometric analysis of immunoblots. In rat erythrocytes, proteolytic modification of the endogenous calcium-dependent protease (calcium-dependent protease 1, mu CANP) occurs in vivo in response to ionomycin and calcium. The extent of enzyme modification was dependent on time, ionomycin concentration, and calcium concentration, suggesting that in this cellular model Ca2+ regulates proteolytic modification of the enzyme.

Purification and characterization of a calcium-dependent protease from rat liver.

A calcium-dependent protease, previously identified in rat liver and designated peak II [DeMartino, G. N. (1981) Arch. Biochem. Biophys. 211, 253-257], was purified and characterized. The calcium-dependent proteolytic activity was accounted for by an 80 000-dalton protein. Depending on the method of purification, we found that this protease could be associated with a 28 000-dalton subunit, which was devoid of protease activity. The catalytic characteristics of the two different forms of the protease were indistinguishable. Each was half-maximally activated by approximately 250 microM calcium.

Domain structure of calpain: mapping the binding site for calpastatin.

The peptide EKLGERDDTIPPEYRELLEKKTGV was synthesized to mimic the central consensus sequence of calpastatin, the specific, endogenous inhibitor of the calpains (EC 3.4.22.17). The peptide competitively inhibits hydrolysis of casein by either micro- or milli-calpain but does not affect the activity of other proteases. This inhibitory peptide was preferentially cross-linked to milli-calpain in the presence of calcium using the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Cross-linking of the peptide was blocked by calpastatin. The site of cross-linking for the peptide within milli-calpain was localized using random chemical cleavage of the enzyme-peptide complex at cysteine residues. Calpain fragments were identified as amino-terminal fragments through reactivity with a peptide-specific antiserum or as non-amino-terminal fragments through incorporation of 14C from 14CN. Analysis of the control and cross-linked fragments, from experiments using both milli-calpain and micro-calpain, maps the chemical cross-linking site to cysteine-497 and localizes the binding site for the calpastatin-like peptide to this highly conserved region of domain III of calpains catalytic subunit.

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver.

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent Mr = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with Mr = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.

Calcium-dependent protease in neuroblastoma cells.

Extracts of neuroblastoma cells contained calcium-dependent proteolytic activity. This activity was accounted for by two distinct proteases. These proteases were separated by ion-exchange chromatography. Although each was totally dependent on calcium, the calcium requirements of the enzymes were different; one enzyme required approximately 40 microM Ca2+ for half-maximal activity and the other enzyme required approximately 150 microM Ca2+ for half-maximal activity.

ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain.

Soluble extracts of cultured cells (BHK 21/C13) degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated this proteolytic activity up to 10-fold. The ATP effect was dependent on Mg2+ and was not elicited by nonhydrolyzable analogs of ATP. After the extract was fractionated on DEAE-cellulose, ATP-stimulated protease activity was in the fraction that bound to the resin and eluted in buffer containing 0.4 M NaCl. This activity had characteristics that were indistinguishable from those of the unfractionated extract but the degree of ATP stimulation was two- to three-fold lower. Although no protease activity was detected in the unbound fraction, reconstitution of this material with the bound fraction enhanced the ATP stimulation up to twofold. The component responsible for the enhancement of the ATP stimulation had properties similar to ubiquitin and purified ubiquitin enhanced the ATP-stimulated protease activity in the fractionated extract. Substrates whose amino groups were almost completely blocked by various chemical modifications were still degraded in an ATP-stimulated fashion, but the degradation of these substrates was not affected by ubiquitin. The protease activity isolated by ion-exchange chromatography was fractionated further by gel filtration chromatography on Sephacryl S-300. ATP-stimulated protease activity eluted with an apparent molecular weight of 750,000. Protease activity was enhanced up to eightfold by Mg2+-ATP but was not increased further by ubiquitin. An activity that hydrolyzed the synthetic peptide Z-Val-Leu-Arg-MNA coeluted with ATP-stimulated protease activity, but peptide hydrolysis was not affected by ATP. These and other catalytic and biochemical characteristics suggested that the protease might be related to the high-molecular-weight protease, macropain, recently purified by us from human erythrocytes (M. J. McGuire and G. N. DeMartino Biochim. Biophys. Acta (1986) 873, 279-289). Antibodies raised against macropain specifically reacted with proteins characteristic of macropain in the column fractions containing ATP-stimulated protease activity. These antibodies also specifically immunoprecipitated 70-100% of the ATP-stimulated protease activity as well as Z-Val-Leu-Arg-MNA hydrolyzing activity. Thus BHK cell extracts appear to contain both ubiquitin-mediated and ubiquitin-independent pathways for the ATP-stimulated degradation of proteins. Furthermore, at least one of these pathways appears to involve a high-molecular-weight, ATP-stimulated protease related to macropain.