Purification of calpain by affinity chromatography on reactive red-agarose or on casein-sepharose.
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Animals↗
Biomedical subjects
D E Croall
Publications and source records attributed to D E Croall.
Crystallization and X-ray crystallographic analysis of m-calpain, a Ca2+-dependent protease.
The absolute requirement of Ca(2+) for proteolytic activity is a feature unique to the calpains, a family of heterodimeric cysteine proteases. Conditions are described which give rise to diffraction-quality crystals of m-calpain in two crystal forms, P1 and P2(1). Data have been collected from native crystals of m-calpain in both P1 and P2(1) forms, to 2.6 and 2.15 A, respectively. Selenomethionine-containing crystals have been grown in both forms, and anomalous data from the P2(1) selenomethionine enzyme provided the location of 17 of the 19 Se atoms in the protein.
Animals↗
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.
Alanine↗
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.
3T3 Cells↗
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.
Animals↗
Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation.
Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.
Adenosine Triphosphate↗
Calpains are activated in necrotic fibers from mdx dystrophic mice.
Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.
Age Factors↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Proteolytic cleavage of the integrin beta 4 subunit.
The integrin beta 4 subunit often undergoes proteolytic cleavage within its long cytoplasmic tail to yield a characteristic protein pattern of 205, 165, and 125 kDa. The results in this study suggest that beta 4 cleavage often occurs during or after cell lysis, where it was readily inhibitable by calcium chelators (EDTA, EGTA) and inhibitors of cysteine proteases (E64c, leupeptin). The cleavage of beta 4 is catalyzed by a calpain-like enzyme because (i) it requires calcium, (ii) it is mimicked by purified milli-calpain, and (iii) it is inhibited by several calpain inhibitors including the calpain-specific inhibitor calpastatin. Within intact cells, cleavage of beta 4 was cell type-specific and observed only when the cells were made permeable to calcium. Substantial cell viability was retained during beta 4 cleavage induced by ionomycin plus calcium, indicating that cleavage within intact cells was not necessarily a consequence of cell death. However, manipulations of cells including suspension, synchronization, and stimulation with serum, phorbol esters, or other agents all failed to induce cleavage, suggesting that if cleavage is physiologically relevant, it is not easily duplicated in vitro. Analysis of multiple cell types showed a wide variation in beta 4 sensitivity to proteolytic cleavage, suggesting that this process might be differentially regulated depending on the cellular environment.
Antigens, Surface↗
The insulin receptor substrate (IRS-1) is a PEST protein that is susceptible to calpain degradation in vitro.
The insulin receptor substrate 1 (IRS-1) contains at least 11 sequence motifs that are rich in proline (P), glutamic acid (E), serine (S), and threonine (T), i.e., PEST regions. Proteins with PEST regions turn over rapidly. IRS-1 is degraded rapidly in vivo upon exposure of 3T3-L1 adipocytes to insulin. The intracellular, calcium-dependent, neutral proteases known as calpains are one possible mechanism by which IRS-1 may be degraded. To begin to investigate this possibility, purified exogenous calpain was shown to degrade IRS-1 in cell-free extracts from basal and insulin-treated cells and rat recombinant IRS-1 in vitro. Only two proteolytic fragments could be detected. One had a mol wt of approximately 79 kDa, arising from the C-terminus end, and the second had a mol wt of approximately 90 kDa arising from near the N-terminus, possibly a product of the same cleavage event, since the mol wt of IRS-1 from insulin-treated cells was approximately 170 kDa. These results suggest that IRS-1 may serve as a substrate for calpain in vivo, accounting for its rapid degradation.
3T3 Cells↗
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.
Amino Acid Sequence↗
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.
Adenosine Triphosphate↗
Calcium-activated neutral protease (calpain) system: structure, function, and regulation.
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Animals↗
The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes.
Eukaryotic initiation factor 4F (eIF-4F) is a multisubunit protein that functions in the first step of the binding of capped mRNAs to the small ribosomal subunit. Its largest polypeptide component, p220, is cleaved following poliovirus infection. This is thought to inactivate eIF-4F function, thereby preventing cap-dependent initiation of translation of cellular mRNAs. In this report, we show that p220 in extracts of uninfected HeLa cells is specifically lost in the presence of calcium. The responsible activities have been partially purified and identified as the calcium-dependent, neutral, cysteine proteases calpains I and II. In addition, a third calcium-dependent activity was resolved from the calpains and also results in the loss of p220. This activity has properties similar to a transglutaminase and copurifies with tissue transglutaminase through several chromatographic steps. None of these calcium-dependent activities appears to mediate p220 cleavage in poliovirus-infected cells.
Calcium↗
Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I.
The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin.
Animals↗
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.
Animals↗
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.
Caseins↗