calpain and Autolysis

Topics: Autolysis; Calpain; Catalysis; Humans

The intracellular calcium-dependent proteases (calpains) and their endogenous protein inhibitor (calpastatin) are present in many different mammalian cells. There is emerging evidence for their importance in the turnover of membrane-associated proteins. Accordingly, it is important to understand how these proteinases and their inhibitor interact within cells, in particular at membranes. Bovine myocardial calpastatin appears to be associated in part with intracellular membranes, where it may effectively block the activity of calpain II on membrane-associated proteins. Immuno-electron microscopic studies suggest that canine myocardial calpain and calpastatin are associated with a number of membranous organelles. During canine myocardial autolysis, the amount of calpain at various organelles decreased, but the amount of calpastatin decreased to an even greater extent. Thus there may be a high calpain to calpastatin balance during heart ischemia at these sites. Calpain II aggregation may contribute to localization of the proteinase at sites of high calcium concentration within cells. A model is presented for interaction of calpain II and calpastatin at cellular membranes in the presence of calcium.

Topics: Amino Acid Sequence; Animals; Autolysis; Blotting, Western; Calcium-Binding Proteins; Calpain; Cattle; Cell Membrane; Dogs; Erythrocytes; Molecular Sequence Data; Molecular Weight; Myocardium; Peptide Mapping; Phospholipids; Protein Binding; Sarcolemma; Sarcoplasmic Reticulum

The purpose of this study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise (PlyEx) both before and after PlyEx training, to understand if titin is affected differently after PlyEx training. Healthy untrained individuals (N = 11) completed the 1stPlyEx (10 × 10 squat-jumps, 1-min rest). Thereafter, six subjects completed 8 wk of PlyEx, while five controls abstained from any jumping activity. Seven days after the last training session, all subjects completed the 2ndPlyEx. Blood samples were collected before and 6 h and 1, 2, 3, and 4 days after each acute bout of PlyEx, and muscle biopsies 4 days before and 3 days after each acute bout of PlyEx. The 1stPlyEx induced an increase in circulating myoglobin concentration. Muscle sample analysis revealed Z-disk streaming, a stretch or a fragmentation of titin (immunogold), and increased calpain-3 autolysis. After training, 2ndPlyEx did not induce Z-disk streaming or calpain-3 activation. The previously observed post-1stPlyEx positional change of the titin COOH terminus was still present pre-2ndPlyEx, in all trained and all control subjects. Only two controls presented with Z-disk streaming after 2ndPlyEx, while calpain-3 activation was absent in all controls. Eccentric explosive exercise induced a stretch or fragmentation of titin, which presented as a positional change of the COOH terminus. Calpain-3 activation does not occur when titin is already stretched before explosive jumping. Enzymatic digestion results in titin fragmentation, but since an increase in calpain-3 autolysis was visible only after the 1stPlyEx acute bout, fragmentation cannot explain the prolonged positional change.

Topics: Adaptation, Physiological; Adult; Autolysis; Calpain; Connectin; Exercise; Female; Humans; Male; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Plyometric Exercise; Sarcomeres; Tissue Distribution

This study examined the potential influence of mitochondrial calcium sequestering ability on calpain-1 autolysis and proteolysis in vitro. We first tested whether mitochondria can sequester calcium in an in vitro setting. Isolated bovine mitochondria (0, 0.5, or 2 mg/mL) were incubated in a buffer containing varying calcium levels (0, 50, or 100 μM). An inverse relationship between mitochondrial content and measured free calcium was observed (P < 0.05), confirming that mitochondria can sequester calcium within the concentration range tested. In the first in vitro experiment, intact mitochondria (0, 0.5, or 2 mg/mL) were incorporated into an in vitro model simulating postmortem muscle conditions, and calpain-1 autolysis and proteolysis were evaluated over a 168-h period. Adding intact mitochondria to the in vitro model decreased calpain-1 autolysis and proteolysis during the first 4 h of incubation (P < 0.05), likely through reducing calcium availability. However, accentuated calpain-1 autolysis and proteolysis were observed at 24 h. To further explore these effects, mitochondrial integrity was evaluated at varying pH and calcium levels. Mitochondrial integrity decreased as pH declined (P < 0.05), especially in the presence of calcium. Based on these results, we conducted a second in vitro experiment involving disrupted mitochondria. Unlike intact mitochondria, which exerted a suppressive effect on calpain-1 autolysis and proteolysis early on, disrupted mitochondria increased both parameters at most time points (P < 0.05). Overall, it appears that intact mitochondria initially cause a delay in calpain-1 autolysis and proteolysis, but as their integrity diminishes, both processes are enhanced.

Topics: Animals; Autolysis; Calcium; Calpain; Cattle; Mitochondria; Muscle, Skeletal; Proteolysis

Apoptosis has been suggested as the first step in the process of conversion of muscle into meat. While a potential role of apoptosis in postmortem proteolysis has been proposed, the underlying mechanisms by which metabolome changes in muscles would influence apoptotic and proteolytic process, leading to meat quality variation, has not been determined. Here, apoptotic and proteolytic attributes and metabolomics profiling of longissimus dorsi (LD) and psoas major (PM) muscles in pigs from two different production cycles (July-Jan vs. Apr-Sep) were evaluated. PM showed higher mitochondrial membrane permeability (MMP), concurrent with less extent of calpain-1 autolysis and troponin T degradation and higher abundance of HSP27 and αβ-crystallin compared to LD (P < 0.05). Apr-Sep muscles showed concurrence of extended apoptosis (indicated by higher MMP), calpain-1 autolysis and troponin T degradation, regardless of muscle effects (P < 0.05). Metabolomics profiling showed Apr-Sep muscles to increase in oxidative stress-related macronutrients, including 6-carbon sugars, some branched-chain AA, and free fatty acids. Antioxidant AA (His and Asp) and ascorbic acid were higher in July-Jan (P < 0.05). The results of the present study suggest that early postmortem apoptosis might be positively associated with pro-oxidant macronutrients and negatively associated with antioxidant metabolites, consequently affecting meat quality attributes in a muscle-specific manner.

Topics: Animals; Apoptosis; Autolysis; Calpain; Cytochromes c; Female; Heat-Shock Proteins; Male; Metabolic Networks and Pathways; Metabolome; Metabolomics; Mitochondria, Muscle; Mitochondrial Membranes; Muscle, Skeletal; Postmortem Changes; Proteolysis; Red Meat; Swine; Troponin T

This study aims to investigate the changes in mitochondrial apoptotic factors and proteolysis of two porcine muscles (psoas major - PM and longissimus dorsi - LD) during aging. Results found that during 2-168 h postmortem mitochondrial membrane permeability, mitochondrial lipid peroxidation, Ca

Topics: Animals; Apoptosis; Autolysis; Calpain; Desmin; Mitochondria; Muscle, Skeletal; Myofibrils; Proteolysis; Red Meat; Swine; Time Factors; Troponin T

Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+- dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2-specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulation of calpain-1, a mechanism that may be applicable to other calpain species as well.

Topics: Autolysis; Calpain; Enzyme Activation; Enzyme Stability; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Proteolysis; Substrate Specificity; Time Factors; Troponin T

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Myofibrils; Nitric Oxide; Nitro Compounds; Proteolysis; Swine

The objective of this study was to investigate the differential role of cathepsin B, L, D and calpain in degradation and disassembly of myofilament. Myofibrillar protein of grass carp (Ctenopharyngodon idella) was incubated with proteases monotonously, simultaneously or sequentially. Subsequently, protein degradation were detected using SDS-PAGE and myofilament disassembly induced by changes of non-covalent interactions were measured through SDS-PAGE using l-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) as a zero length cross-linker. Additionally, content of heat shock proteins which functioned in stabilizing assembly architecture of myofibrillar protein was determined. Results showed that calpain and cathepsin B, calpain and cathepisn L could act in a stepwise and complimentary manner to synergistically dissociate and degrade myofibrillar protein. In synergistic action, cathepsin B disrupted the thick filament assembly through lowering the UNC45 and HSP90 concentration in myofibrillar protein, facilitating the degradation of dissociated MHC by calpain. Meanwhile, Cathepsin L was shown to preferentially remove the actin from thin filament via lowering the content of HSP27 and αb-crystallin, to create dissociated actin as substrate supply for calpain.

Topics: alpha-Crystallin B Chain; Animals; Autolysis; Calpain; Carps; Cathepsin B; Cathepsin D; Cathepsin L; Fish Proteins; HSP27 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Muscle Proteins; Myofibrils; Proteolysis; Seafood; Substrate Specificity; Time Factors

Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of μ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in μ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake.. SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine μ-calpain autolysis as well as μ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR.. The amounts of the autolysed isoform (78 kDa) of full-length μ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats.. In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of μ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Topics: Alcoholism; Animals; Autolysis; Calpain; Ethanol; Male; Muscle, Skeletal; Rats; Rats, Wistar

The study investigated the effects of alkaline phosphatase (AP) dephosphorylation and protein kinase A (PKA) phosphorylation on μ-calpain activity and its sensitivity to temperature. The purified μ-calpain was treated with AP or PKA for 30min at 30°C to modulate its phosphorylation level. Samples were then incubated at controlled freezing point (-1), 4, 25 and 37°C, respectively. The results showed that PKA and AP had no influence on pH values of incubation solution. At -1 and 4°C, the degradation rate of μ-calpain was maximum in AP group and minimum in control group. Low temperature of controlled freezing point prevented dephosphorylation and phosphorylation progression and delayed μ-calpain degradation. Increased incubation temperature of 4, 25 and 37°C increased μ-calpain degradation. Two about 50kDa degradation products from μ-calpain were identified, of which the intensity was also lower in control group than in the other two groups. These observations demonstrated that AP dephosphorylation and PKA phosphorylation of μ-calpain promoted μ-calpain autolysis and activation.

Topics: Alkaline Phosphatase; Autolysis; Calpain; Cyclic AMP-Dependent Protein Kinases; Food Analysis; Food Handling; Food Quality; Freezing; Hydrogen-Ion Concentration; Meat; Phosphorylation; Postmortem Changes; Proteolysis; Temperature

Topics: Animals; Autolysis; Calcium; Calpain; Caseins; Chickens; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Food Quality; Food Storage; Meat; Phosphoprotein Phosphatases; Postmortem Changes; Tandem Mass Spectrometry

The effect of S-nitrosylation on the autolysis and catalytic ability of μ-calpain in vitro in the presence of 50μM Ca(2 +) was investigated. μ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa μ-calpain subunit significantly decreased as GSNO increased from 0 to 300μM, but increases of GSNO to 300, 500 and 1000μM did not result in further inhibition. The catalytic ability of nitrosylated μ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300μM. The cysteine residues of μ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased μ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of μ-calpain resulting in decreased proteolytic ability on myofibrils.

Topics: Amino Acid Sequence; Animals; Autolysis; Calpain; Catalysis; Desmin; Muscle Proteins; Myofibrils; Protein S; Proteolysis; Swine; Troponin T

Bull Musculus longissimus dorsi (n=63) were categorised into high (pH≥6.2), intermediate (pH 5.8-6.19) and low (≤5.79) ultimate pH (pHu) and aged up to 28 days post mortem at -1°C. High pHu samples were acceptably tender at 1 day post mortem and significantly more tender than low pHu meat at all ageing timepoints (p<0.05). Rapid autolysis of μ-calpain in high pHu meat was linked with the more rapid degradation of titin, nebulin and filamin in this pHu group. Desmin degraded faster in low pHu meat and was concurrent with an increase of cathepsin B levels. The results from this study support the hypothesis that beef tenderisation is pHu compartmentalised with tenderness in high and low pHu meat characterised by variable rate of degradation of high and low molecular weight myofibrillar proteins during ageing, which are in turn regulated by μ-calpain and cathepsin B activities.

Topics: Animals; Autolysis; Calpain; Cathepsin B; Cattle; Connectin; Desmin; Dietary Proteins; Electrophoresis, Polyacrylamide Gel; Filamins; Hydrogen-Ion Concentration; Meat; Muscle Proteins; Muscle, Skeletal; Myofibrils; Proteolysis

The present study investigated changes in autolysis of three calpain isoforms in skeletal muscles undergoing eccentric contractions (ECC), leading to prolonged force deficits. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC in situ, excised immediately after or 3 or 6 days after cessation of ECC, and used for measures of force output and for biochemical analyses. Full restoration of tetanic force in ECC-treated muscles was not attained until 6 days of recovery. Maximal calpain activity determined by a fluorogenic substrate was unaltered immediately after ECC, but increased to 313 and 450 % after 3 and 6 days, respectively. Increases in the amount of autolyzed calpain-3 were apparent immediately and developed progressively with recovery time, whereas elevations of autolyzed μ- and m-calpain occurred after 3 and 6 days, respectively. The protein content was augmented only in m-calpain. It is suggested that the three calpain isoforms may be involved in the dismantling, repair, remodeling and/or regeneration processes in ECC-treated muscles.

Topics: Animals; Autolysis; Calpain; Male; Muscle Contraction; Muscle Fibers, Fast-Twitch; Protein Isoforms; Rats; Rats, Wistar

This study aimed to determine how small heat shock proteins (sHSPs) protect myofibrillar proteins from μ-calpain degradation during ageing. Immunoprecipitation experiments with M. longissimus dorsi (LD) from Angus heifers (n = 14) examined the interaction between αβ-crystallin, desmin, titin, HSP20, HSP27 and μ-calpain. Results showed that αβ-crystallin associated with desmin, titin, HSP20, HSP27 and μ-calpain. Exogenous αβ-crystallin reduced desmin and titin degradations in myofibrillar extracts and attenuated μ-calpain activity. In a second experiment, bull LD (n = 94) were aged at -1.5°C for up to 28 days post mortem. μ-Calpain autolysed faster in high ultimate pH (pH(u)) meat (pH(u)≥6.2) and this was concomitant with the more rapid degradation of titin and filamin in this pH(u) group. Desmin stability in intermediate pH(u) meat (pH(u) 5.8 to 6.19) may be due to the protection of myofibril-bound sHSPs combined with the competitive inhibition of μ-calpain by sHSPs.

Topics: Animals; Autolysis; Breeding; Calpain; Cattle; Connectin; Crystallins; Desmin; Female; Filamins; Heat-Shock Proteins, Small; HSP20 Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Hydrogen-Ion Concentration; Male; Meat; Muscle Proteins; Muscle, Skeletal; Myofibrils; Postmortem Changes; Proteolysis

Bull M. longissimus dorsi (n=94) categorised into high (n=28), intermediate (n=14) and low (n=52) ultimate pH (pHu) were aged at -1.5°C for 28days. Shear force was higher and more variable (p<0.05) in intermediate pHu samples during ageing. Titin, filamin and desmin degradation was also less extensive in intermediate pHu samples compared to the other two pH categories. The extent of the decline of HSP20, HSP27 and αβ-crystallin concentrations during post mortem ageing was pHu related such that high pHu meat maintained the highest concentration of small heat shock proteins followed by intermediate and low pHu meat. μ-Calpain autolysis was slowest in intermediate pHu and cathepsin B activities remained consistently low during ageing in this group (p<0.05). Meat toughness in the intermediate pHu group may be attributed to the combination of a larger pool of sHSP with a sub-optimal cathepsin B activity and intermediary μ-calpain activities.

Topics: Animals; Autolysis; Calpain; Cathepsin B; Cattle; Connectin; Crystallins; Desmin; Diet; Filamins; Food Quality; HSP20 Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Hydrogen-Ion Concentration; Male; Meat; Muscle Proteins; Muscle, Skeletal; Postmortem Changes; Stress, Mechanical

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.

Topics: Animals; Autolysis; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Food Handling; Meat; Muscle, Skeletal; Myofibrils; Particle Size; Proteolysis; Sarcomeres; Sus scrofa; Temperature; Time Factors

Two simultaneous trials were conducted to determine the effects of electrical input [electrical stunning and stimulation (ES)], wrapping, pre rigor temperature (15 °C and 38 °C) and different post rigor chilling rates on beef quality using M. longissimus lumborum (n=100). The high pre rigor temperature induced a faster pH decline than ES. The loins at 38 °C had significantly greater protein denaturation, more purge and drip loss, higher shear force values and less desmin degradation compared with the loins at 15 °C. No difference in sarcomere length was determined between the pre rigor temperatures regardless of ES and wrapping. Different post rigor chilling rates did not play a substantial role in water-holding capacity, proteolysis, or shear force values during ageing. These results suggest that high pre rigor temperature induces temperature-related toughness of muscle due to protein denaturation with subsequent limitation of proteolysis by μ-calpain, regardless of ES and wrapping treatments.

Topics: Animals; Autolysis; Calpain; Cattle; Desmin; Electric Stimulation; Female; Food Handling; Hydrogen-Ion Concentration; Male; Meat; Mechanical Phenomena; Muscle, Skeletal; Protein Denaturation; Protein Stability; Proteolysis; Quality Control; Sarcomeres; Temperature; Water

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

Topics: Adrenergic beta-Agonists; Animals; Autolysis; Calpain; Cell Death; Clenbuterol; Hypertrophy; Male; Muscle Cells; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Rats; Rats, Wistar; Regeneration

The skeletal muscle-specific calpain-3 protease is likely involved in muscle repair, although the mechanism is not known. Physiological activation of calpain-3 occurs 24 h following eccentric exercise in humans. Functional consequences of calpain-3 activation are not known; however, calpain-3 has been suggested to be involved in nuclear signaling via NF-κB. To test this and help identify how/where calpain-3 acts, we investigated whether calpain-3 autolysis (hence, activation) following eccentric exercise results in translocation from its normal myofibrillar location to the nucleus or the cytosol. In resting human skeletal muscle, the majority (87%) of calpain-3 was present in myofibrillar fractions, with only a small proportion (<10%) in an autolyzed state. Enriched nuclear fractions contained ∼8% of the total calpain-3, which was present in a predominantly (>80%) autolyzed state. Using freshly dissected human muscle fibers to identify freely diffusible proteins, we showed that only ∼5% of the total calpain-3 pool was cytosolic. At 3 and 24 h following eccentric step exercise, there was an ∼70% increase in autolysis in whole muscle samples (n = 11, P < 0.05, by 1-way ANOVA with repeated measures and Newman-Keuls post hoc analysis). This exercise-induced autolysis was attributed to myofibrillar-bound calpain-3, since neither the amount of calpain-3 nor the proportion autolyzed was significantly changed in enriched nuclear or cytosolic fractions following the exercise intervention. We present a model for calpain-3 localization at rest and following activation in human skeletal muscle and suggest that the functional importance of calpain-3 remains predominantly tightly associated with its localization within the myofibrillar compartment.

Topics: Adult; Autolysis; Calpain; Cell Nucleus; Cytosol; Exercise; Humans; Male; Muscle Proteins; Muscle, Skeletal; Myofibrils; NF-kappa B; Protein Transport; Young Adult

This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation. From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline. The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24h. Calpain activity and autolysis from 1 to 72h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation. Colour and drip loss were measured. A faster decrease in pH resulted in reduced level of mu-calpain activity and increased autolysis of the enzyme, and hence an earlier loss of activity due to activation of mu-calpain in muscles with a fast pH decline. Paralleling the mu-calpain activation in muscles with a fast pH decline a higher myofibril fragmentation at 24h post-mortem was observed, which was no longer evident in the later phase of the tenderization process. In conclusion, the rate of early pH decline influenced mu-calpain activity and the rate but not the extent of myofibrillar degradation, suggesting an early effect of proteolysis on myofibril fragmentation that is reduced during ageing due to an earlier exhaustion of mu-calpain activity.

Topics: Animals; Autolysis; Calpain; Female; Hydrogen-Ion Concentration; Male; Meat; Muscle, Skeletal; Myofibrils; Postmortem Changes; Swine

Beef steaks from longissimus lumborum, semimembranosus, and adductor muscles (n=10; respectively) were cut at 24h postmortem, randomly assigned to either high-oxygen modified atmosphere packaging (HiOx-MAP; 80% O(2), 20% CO(2)) or vacuum (VAC), and displayed for 9days at 1 degrees C. HiOx-MAP packaged beef steaks had a rapid increase in lipid oxidation and a decrease in color stability during display. The steaks in HiOx-MAP had significantly lower tenderness and juiciness scores, and higher off-flavor scores compared to steaks in VAC. HiOx-MAP condition did not affect the postmortem degradation of troponin-T or desmin. Furthermore, autolysis of micro-calpain was not influenced by packaging. SDS-PAGE, immunoblotting, and diagonal-PAGE revealed oxidative cross-linking of myosin heavy chain in meat packaged in HiOx-MAP. These results suggest that the HiOx-MAP system may negatively affect meat quality characteristics by inducing lipid and myoglobin oxidation and cross-linking/aggregation of myosin by protein oxidation.

Topics: Animals; Autolysis; Calpain; Cattle; Color; Desmin; Electrophoresis, Polyacrylamide Gel; Food Handling; Food Preservation; Food Technology; Humans; Immunoblotting; Lipid Peroxidation; Meat; Muscle, Skeletal; Myoglobin; Myosin Heavy Chains; Oxidation-Reduction; Oxygen; Polymers; Postmortem Changes; Protein Carbonylation; Random Allocation; Taste; Troponin T; Vacuum

Because intracellular [Na(+)] is kept low by Na(+)/K(+)-ATPase, Na(+) dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca(2+)-activated intracellular "modulator proteases" that is responsible for a limb-girdle muscular dystrophy ("calpainopathy"), underwent Na(+)-dependent, but not Cs(+)-dependent, autolysis in the absence of Ca(2+). Furthermore, Na(+) and Ca(2+) complementarily activated autolysis of p94 at physiological concentrations. By blocking Na(+)/K(+)-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na(+)/Ca(2+)-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca(2+)-binding sequence in the protease acted as a Na(+) sensor. Proteomic analyses using Cs(+)/Mg(2+) and p94CS-KI mice as negative controls revealed that Na(+) and Ca(2+) direct p94 to proteolyze different substrates. We propose three roles for Na(+) dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca(2+)], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na(+)-dependent enzyme.

Topics: Amino Acid Sequence; Animals; Autolysis; Binding Sites; Calcium; Calpain; Enzyme Activation; Humans; Intracellular Space; Mice; Molecular Sequence Data; Muscle, Skeletal; Mutation; Organ Specificity; Protein Structure, Tertiary; Rats; Sodium; Substrate Specificity

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Topics: Animals; Autolysis; Blotting, Western; Calpain; Cell Fractionation; Cytosol; Disease Models, Animal; Dogs; Electrophoresis, Polyacrylamide Gel; Intracellular Membranes; Leupeptins; Male; Muscle, Skeletal; Myocardial Reperfusion Injury; Myocardium; Oligopeptides; Rabbits; Rats; Rats, Sprague-Dawley

Topics: Autolysis; Calpain; Exercise; Gene Expression; Humans; Muscle Fatigue; Muscle Proteins; RNA, Messenger; Sarcomeres

Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

Topics: Animals; Autolysis; Calpain; Catalytic Domain; Chromatography, Liquid; Disulfides; Hydrogen Peroxide; In Vitro Techniques; Mercaptoethanol; Models, Molecular; Muscle, Skeletal; Oxidation-Reduction; Swine; Tandem Mass Spectrometry

Calpains have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. However, limited data are available about the specific involvement of each calpain in the early stages of muscle atrophy. The aims of this study were to determine whether calpains 1 and 2 are autolyzed after a short period of muscle disuse, and, if so, where in the myofibers the autolyzed products are localized. In the rat soleus muscle, 5 days of immobilization increased autolyzed calpain 1 in the particulate and not the soluble fraction. Conversely, autolyzed calpain 2 was not found in the particulate fraction, whereas it was increased in the soluble fraction after immobilization. In the less atrophied plantaris muscle, no difference was noted between the control and immobilized groups whatever the fraction or calpain. Other proteolytic pathways were also investigated. The ubiquitin-proteasome pathway was activated in both skeletal muscles, and caspase 3 was activated only in the soleus muscle. Taken together, our data suggest that calpains 1 and 2 are involved in atrophy development in slow type muscle exclusively and that they have different regulation and protein targets. Moreover, the activation of proteolytic pathways appears to differ in slow and fast muscles, and the proteolytic mechanisms involved in fast-type muscle atrophy remain unclear.

Topics: Animals; Autolysis; Calpain; Caspase 3; Disease Models, Animal; Enzyme Activation; Hindlimb Suspension; Male; Muscle Fibers, Fast-Twitch; Muscle Fibers, Skeletal; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Muscular Atrophy; Myofibrils; Phenotype; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Time Factors; Ubiquitin

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.

Topics: Animals; Autolysis; Calcium; Calpain; Humans; Isoenzymes; Male; Muscle Proteins; Muscle, Skeletal; Physical Endurance; Physical Exertion; Rats; Rats, Long-Evans; Time Factors

Calpain 3, commonly called p94 in the literature, is the abundant skeletal muscle-specific calpain that is genetically linked to limb girdle muscular dystrophy type 2A. Recently, we showed that p94's insertion sequence 1 (IS1) is a propeptide that must be autoproteolytically cleaved to provide access of substrates and inhibitors to the enzyme's active site. Removal of IS1 from the core of p94 by recombinant methods produced a fully active enzyme. Here we have resolved the discrepancies in the literature about the Ca(2+) requirement of p94 using the protease core. Even at substoichiometric levels of Ca(2+), and in competition with EDTA, autoproteolyzed enzyme slowly accumulated. Because the initial autoproteolytic cleavage is an intramolecular reaction, transient binding of two Ca(2+) ions to the core would be sufficient to promote the reaction that is facilitated by having the scissile peptide lying close to the active site cysteine. The second autolytic cleavage was much slower and required higher Ca(2+) levels, consistent with it being an intermolecular reaction. Other metal ions such as Na(+), K(+), and Mg(2+) cannot substitute for Ca(2+) in catalyzing the intramolecular autoproteolysis of the p94 core or in the subsequent hydrolysis of exogenous substrates. These metal ions increase moderately the activity of this enzyme but only at very high concentrations. Thus, the proteolytic activity of the core of p94 and its deletion mutant lacking NS and IS1 was shown to be strictly Ca(2+)-dependent. We propose a two-stage model of activation of the proteolytic core of p94.

Topics: Autolysis; Calcium; Calpain; Catalytic Domain; Coumarins; Dipeptides; DNA Transposable Elements; Dose-Response Relationship, Drug; Edetic Acid; Enzyme Activation; Humans; Hydrolysis; Magnesium; Models, Biological; Mutation; Potassium; Recombinant Proteins; Sodium

Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.

Topics: Animals; Annexin A5; Apoptosis; Autolysis; Calpain; Cell Differentiation; Cell Line; Cell Nucleus; Enzyme Activation; Fluorescein-5-isothiocyanate; Hippocampus; I-kappa B Proteins; Ionomycin; Isoenzymes; Muscle Proteins; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; PC12 Cells; Protein Processing, Post-Translational; Protein Transport; Rats

The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P(4) to P(7)'. In compliance with earlier data, preferred residues in the P(2) position are Leu, Thr, and Val, and in P(1) Lys, Tyr, and Arg. In position P(1) ', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P(2)-P(1)' segment, i.e. positions P(3) and P(2)'-P(4)'; most notable is its occurrence 5.59 times above chance in P(3)'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.

Topics: Amino Acid Sequence; Amino Acid Substitution; Autolysis; Base Sequence; Calpain; Cysteine Endopeptidases; DNA Primers; Isoenzymes; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Substrate Specificity

Uncontrolled activation of calpain has been linked to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, and multiple sclerosis and Alzheimer's disease. In vivo, the activity of calpain is regulated by its endogenous inhibitor calpastatin. The pathological role of calpain has been attributed to an imbalance between the activities of the protease and its inhibitor. Thus, it is possible that by reimposing functional control on the protease, the progression of calpain-mediated diseases could be slowed or eliminated. B27-WT is a 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from calpastatin that was previously shown to be a potent inhibitor of mu- and m-calpain. Recently, we identified two hot spots (Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19)) within which the amino acid residues that are key to B27-WT's bioactivity are clustered. In the work described here, the most critical residues of B27-WT, Leu(11) and Ile(18), were further probed to determine the nature of their interaction with calpain. Our results demonstrate that the side chains of both residues interact with hydrophobic pockets in calpain and that each of these interactions is indispensable for effective inhibition of calpain. Direct interactions involving the beta- and gamma-CH(2)- of the Leu(11) and Ile(18) side chains, respectively, rather than the degree of side chain branching or hydrophobicity, seemed to play a significant role in the peptide's ability to inhibit calpain. Furthermore, the minimum peptide sequence that still retained the calpain-inhibitory potency of B27-WT was found to be MSSTYIEELGKREVTIPPKYRELL.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Isoleucine; Leucine; Molecular Sequence Data; Peptide Fragments; Peptide Library; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship; Swine

Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.

Topics: Animals; Autolysis; Calpain; Cattle; Chromatography, Gel; Molecular Weight; Osmolar Concentration; Potassium Chloride; Protein Subunits; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The finding that phospholipid micelles lowered the Ca2+ concentration required for autolysis of the calpains led to a hypothesis suggesting that the calpains are translocated to the plasma membrane where they interact with phospholipids to initiate their autolysis. However, the effect of plasma membranes themselves on the Ca2+ concentration required for calpain autolysis has never been reported. Also, if interaction with a membrane lowers the Ca2+ required for autolysis, the membrane-bound-calpain must autolyze itself, because it would be the only calpain having the reduced Ca2+ requirement. This implies that the autolysis is an intramolecular process, although several studies have shown that autolysis of the calpains in an in vitro assay and in the absence of phospholipid is an intermolecular process. Inside-out vesicles prepared from erythrocytes had no effect on the Ca2+ concentration required for autolysis of either mu- or m-calpain, although phosphatidylinositol (PI) decreased the Ca2+ concentration required for autolysis of the same calpains. The presence of a substrate for the calpains, beta-casein, reduced the rate of autolysis of both mu- and m-calpain both in the presence and in the absence of PI, suggesting that mu- and m-calpain autolysis is an intermolecular process in the presence of PI just as it is in its absence. Because IOV have no effect on the Ca2+ concentration required for calpain autolysis, association with the plasma membrane, at least with erythrocyte plasma membranes, does not initiate calpain autolysis by reducing the Ca2+ concentration required for autolysis as suggested by the membrane-activation hypothesis. Interaction with a membrane may serve to bind calpains to their substrates rather than promoting autolysis.

Topics: Animals; Autolysis; Calcium; Calpain; Caseins; Cattle; Erythrocyte Membrane; Muscle, Skeletal; Phosphatidylinositols

The calcium-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor and resides within caveolin-rich membrane domains in bovine parathyroid cells. The proenzyme of calpain 2 (m-calpain) is a heterodimeric calcium-dependent cysteine protease consisting of catalytic and regulatory subunits. The effects of calcium on the enzyme include activation, autolysis, and subunit dissociation. Here, we examine the potential role of caveolin-1 and caveolae in regulating the cellular distribution and function of m-calpain in parathyroid cells. We show that the inactive heterodimeric forms of m-calpain are concentrated in caveolin-rich membrane fractions prepared from parathyroid cells incubated with low extracellular calcium (Ca2+(o)). In contrast, in cells incubated with 3 mm Ca2+(o), which activates the CaR and increases intracellular calcium, there is a reduction in m-calpain in association with an increase in CaR protein and phosphorylated protein kinase C alpha and beta in caveolin-rich fractions. To assess the impact of activation of calpain on CaR protein in caveolar fractions, we analyzed the effects of m-calpain on the CaR. Activation of the CaR with high Ca2+(o) induced the release of lower molecular weight fragments of the receptor into the cell culture medium, and calpain inhibitors blocked this effect. Moreover, the fragments of the CaR as well as caveolin-1, m-calpain, and alkaline phosphatase were localized in membrane vesicles shed by parathyroid cells, supporting the association of these proteins in living cells. Treatment of CaR proteins in vitro with m-calpain also resulted in the appearance of lower molecular weight fragments of the CaR. Our data suggest that localization of m-calpain within caveolae may contribute to maintenance of the enzyme in an inactive state and that m-calpain may also contribute to the regulation of CaR levels.

Topics: Animals; Autolysis; Calcium; Calpain; Carcinogens; Cattle; Caveolae; Caveolin 1; Caveolins; Cell Line; Humans; Isoenzymes; Parathyroid Glands; Parathyroid Hormone; Phosphorylation; Protein Kinase C; Rabbits; Receptors, Calcium-Sensing; Receptors, Cell Surface; Substrate Specificity; Tetradecanoylphorbol Acetate

Various stimuli including anticancer drugs are capable of initiating the apoptotic death program in human tumor cells via activation of caspases. Mitochondria play an essential role for cell apoptotic commitment. Previous studies have shown a potential role of calpain activation in apoptosis, however, the involved molecular mechanisms remain to be defined. In the current study, we have examined the expression and activation of mitochondrial calpain in Jurkat T leukemia cells, MCF-7 breast carcinoma and LNCaP prostate cancer cells during apoptosis induced by an anticancer drug (VP-16, tamoxifen) or the specific p38 kinase inhibitor PD-169316. Our results suggest that increased expression and autolysis of the mitochondrial calpain small subunit are tightly associated with calpain activation in an early stage of apoptosis. In contrast, there were no correlations observed between the early calpain activation and changes in levels of mitochondrial calpain large subunit and the endogenous calpain inhibitor calpastatin. Furthermore, pretreatment with the specific pharmacological calpain inhibitor calpeptin blocked the drug-induced calpain small subunit autolysis and calpain activation in mitochondria and inhibited apoptosis-associated caspase-3 activation, demonstrating that mitochondrial calpain activation through small subunit cleavage is an essential step for inducing tumor cell apoptosis by various anticancer drugs.

Topics: Antineoplastic Agents; Apoptosis; Autolysis; Breast Neoplasms; Calpain; Cytochrome c Group; Enzyme Activation; Enzyme Inhibitors; Etoposide; Female; Humans; Imidazoles; Jurkat Cells; Male; Mitochondria; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Protein Subunits; Tamoxifen; Tumor Cells, Cultured

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Autolysis; Calpain; Catalytic Domain; Cells, Cultured; Connectin; Contractile Proteins; Cytoskeletal Proteins; Cytoskeleton; Enzyme Activation; Filamins; In Vitro Techniques; Mice; Microfilament Proteins; Models, Biological; Muscle Proteins; Muscle, Skeletal; NIH 3T3 Cells; Phosphoproteins; Protein Kinases; Protein Structure, Tertiary; Recombinant Proteins; Sarcolemma; Sarcomeres; Substrate Specificity; Talin

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.

Topics: Autolysis; Binding Sites; Calcium; Calpain; Cloning, Molecular; Cysteine Endopeptidases; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Humans; Muscle, Skeletal; Mutagenesis, Site-Directed; Peptides; Plasmids; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.

Topics: Amino Acid Sequence; Animals; Autolysis; Brain; Calcium-Binding Proteins; Calpain; Cattle; Chromatography; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Isoelectric Focusing; Isoelectric Point; Kinetics; Molecular Weight; Species Specificity; Struthioniformes; Swine; Temperature

Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.

Topics: Animals; Autolysis; Blotting, Western; Calpain; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Meat; Muscle, Skeletal; Postmortem Changes; Sensitivity and Specificity; Sheep

The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only mu- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT mu- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either mu- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either mu- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.

Topics: Acrylates; Animals; Anti-Bacterial Agents; Antimycin A; Autolysis; Biological Transport, Active; Calcium-Binding Proteins; Calpain; Caseins; Cell Death; Cell Membrane; Cysteine Proteinase Inhibitors; Cytosol; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression Regulation, Enzymologic; Immunoblotting; Isoenzymes; Kidney Tubules, Proximal; Mitochondria; Rabbits; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Sodium

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Autolysis; Calcium; Calpain; Catalytic Domain; Cell Line; Enzyme Activation; Enzyme Stability; Humans; Insecta; Models, Molecular; Molecular Weight; Mutagenesis, Site-Directed; Protein Subunits

Heterodimeric p-calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible four-step purification procedure. Obtained material is more than 95% pure and has a specific activity of 6-7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at -80 degrees C the enzyme remains fully activatable by Ca2+, although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence 60RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In casein-zymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis.

Topics: Autolysis; Calpain; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Fluorometry; Humans; Sequence Analysis, Protein

The free Ca(2+) concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 microM and are much higher than the 50-500 nM free Ca(2+) concentrations that exist in living cells. Consequently, a number of studies have attempted to find mechanisms that would lower the Ca(2+) concentration required for proteolytic activity of m-calpain. Although autolysis lowers the Ca(2+) concentration required for proteolytic activity of m-calpain, 90-400 microM Ca(2+) is required for a half-maximal rate of autolysis of m-calpain, even in the presence of phospholipid. It has been suggested that mu-calpain, which has a lower Ca(2+) requirement than m-calpain, might proteolyze m-calpain and reduce its Ca(2+) requirement to a level that would allow it to be active at physiological Ca(2+) concentrations. We have incubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain:m-calpain, in the presence of 50 microM free Ca(2+); this Ca(2+) concentration is high enough for more than half-maximal activity of mu-calpain, but does not activate m-calpain. Under these conditions, mu-calpain caused no detectable proteolytic degradation of the m-calpain polypeptide and did not change the Ca(2+) concentration required for proteolytic activity of m-calpain. mu-Calpain also did not degrade the m-calpain polypeptide at 1000 microM Ca(2+), which is a Ca(2+) concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1000 microM Ca(2+) and because the subsite specificities of mu- and m-calpain are very similar if not identical, failure of mu-calpain to rapidly degrade m-calpain at 1000 microM Ca(2+) suggests a unique role of autolysis in calpain function.

Topics: Animals; Autolysis; Calcium; Calpain; Cattle; Enzyme Activation; In Vitro Techniques; Kinetics; Muscle, Skeletal

Topics: Amino Acid Sequence; Animals; Autolysis; Base Sequence; Calpain; Cloning, Molecular; COS Cells; DNA Primers; DNA, Complementary; Gene Expression; Humans; Molecular Sequence Data; Muscular Dystrophies; Mutation; Recombinant Proteins; RNA; Transfection

Topics: Autolysis; Calpain; Enzyme Activation; Erythrocytes; Fluoresceins; Fluorescent Dyes; Humans; In Vitro Techniques; Isoenzymes; Kinetics; Microtubule-Associated Proteins; Protein Structure, Quaternary; Substrate Specificity

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca2+ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated alpha- and beta-paramyosins only beta-paramyosin is degraded by CaDP in the presence of 5 mM Ca2+ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous beta-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis.

Topics: Actins; Animals; Autolysis; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Enzyme Activation; Muscle Proteins; Muscles; Myofibrils; Myosin Heavy Chains; Octopodiformes; Substrate Specificity; Tropomyosin

Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Topics: Animals; Autolysis; Baculoviridae; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Chromatography, Affinity; Cysteine Proteinase Inhibitors; Enzyme Precursors; Humans; Mutation; Protein Binding; Protein Processing, Post-Translational; Recombinant Proteins; Spodoptera

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.

Topics: Animals; Autolysis; Calcium; Calpain; Cerebral Cortex; Enzyme Activation; Erythrocytes; Ionomycin; Ionophores; Isoenzymes; Neurons; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spectrin

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 microM. The lower value of [Ca2+]0.5 is therefore a property of the Delta9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.

Topics: Animals; Autolysis; Calcium; Calpain; Caseins; Dimerization; Enzyme Stability; Mutation; Protein Conformation; Rats; Recombinant Proteins; Sequence Deletion

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.

Topics: Amino Acid Sequence; Animals; Autolysis; Base Sequence; Calcium; Calpain; Cloning, Molecular; Dimerization; Enzyme Activation; Enzyme Stability; Hydrolysis; Lung; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Rats; Sequence Deletion

Proteolytic degradation of numerous calpain substrates, including cytoskeletal and regulatory proteins, has been observed during brain ischemia and reperfusion. In addition, calpain inhibitors have been shown to decrease degradation of these proteins and decrease postischemic neuronal death. Although these observations support the inference of a role for mu-calpain in the pathophysiology of ischemic neuronal injury, the evidence is indirect. A direct indicator of mu-calpain proteolytic activity is autolysis of its 80-kDa catalytic subunit, and therefore we examined the mu-calpain catalytic subunit for evidence of autolysis during cerebral ischemia. Rabbit brain homogenates obtained after 0, 5, 10, and 20 min of cardiac arrest were electrophoresed and immunoblotted with a monoclonal antibody specific to the mu-calpain catalytic subunit. In nonischemic brain homogenates the antibody identified an 80-kDa band, which migrated identically with purified mu-calpain, and faint 78- and 76-kDa bands, which represent autolyzed forms of the 80-kDa subunit. The average density of the 80-kDa band decreased by 25 +/- 4 (p = 0.008) and 28 +/- 9% (p = 0.004) after 10 and 20 min of cardiac arrest, respectively, whereas the average density of the 78-kDa band increased by 111 +/- 50% (p = 0.02) after 20 min of cardiac arrest. No significant change in the density of the 76-kDa band was detected. These results provide direct evidence for autolysis of brain mu-calpain during cerebral ischemia. Further work is needed to characterize the extent, duration, and localization of mu-calpain activity during brain ischemia and reperfusion as well as its role in the causal pathway of postischemic neuronal injury.

Topics: Animals; Autolysis; Blotting, Western; Brain Ischemia; Calpain; Female; Isoenzymes; Nerve Tissue Proteins; Rabbits

To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.

Topics: Animals; Autolysis; Calpain; Cattle; Coumarins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Erythrocytes; Leupeptins; Microscopy, Phase-Contrast; Multienzyme Complexes; Neurites; Oligopeptides; PC12 Cells; Proteasome Endopeptidase Complex; Rabbits; Rats

The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Topics: Autolysis; Calpain; Enzyme Activation; Enzyme Precursors; Erythrocytes; Humans; In Vitro Techniques; Kinetics; Microtubule-Associated Proteins; Molecular Structure; Protein Conformation

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Topics: Analysis of Variance; Animals; Autolysis; Blotting, Western; Brain Injuries; Calpain; Cerebral Cortex; Cytoskeleton; Enzyme Activation; Functional Laterality; Isoenzymes; Male; Rats; Rats, Sprague-Dawley

The Ca-induced autoproteolysis calpain proceeds through the sequential formation of two forms of active enzyme with molecular masses of 78 kD and 75 kD, respectively. The autolysed calpains are produced by the cleavage of the peptide bond between Ser15-Ala16 and then between Gly27-Leu28. Calpastatin reduces with high efficiency the transition from 78 kD to 75 kD calpain forms. At higher concentration also the first autolytic event is blocked. The data are consistent with the presence of two calpain forms with different susceptibility to calpastatin. Furthermore, calpain, once bound to phospholipid vesicles, undergoes autoproteolysis which preferentially accumulates the 78 kD species. These data provide new information on the activation process of calpain, indicating that a Ca-induced conformational change is the triggering event, followed by the appearance of the active 78 kD calpain which can be considered the preferential form of calpain at the membrane level.

Topics: Amino Acid Sequence; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Erythrocytes; Humans; Liposomes; Models, Biological; Molecular Sequence Data; Phospholipids

The aim of this study was to investigate the process leading to Ca(2+)-activated neutral protease (CANP) activation in vivo. The unautolyzed form of CANP has been targeted to the erythrocyte membrane by increasing, in a controlled way, the Ca2+ concentration in the cells; this was achieved by incubating erythrocytes with the Ca2+ ionophore A23187 and fixed Ca2+ concentrations. After isolation of the CANP-bearing erythrocyte membrane, we could observe that CANP remained bound to the membrane in the 80-kDa unautolyzed form in the presence of low Ca2+ concentrations (1.75 microM); under these conditions, the preferred CANP substrates (the Ca(2+)-ATPase and Band 3) were cleaved. That the cleavage was due to CANP was shown by the finding that the two substrates were not degraded in the presence of a membrane-permeable irreversible CANP inhibitor, Cbz-Leu-Leu-Tyr-CHN2, nor when the free Ca2+ concentration was decreased to sub microM levels with EDTA. The findings suggest an activation mechanism of CANP based on its translocation to the membrane rather than on its autolysis. In this mechanism, CANP would become reversibly activated on the membrane and would return to the quiescent state after dissociating from it when the cell Ca2+ concentration has returned to the physiological, submicromolar level.

Topics: Anion Exchange Protein 1, Erythrocyte; Autolysis; Blood Proteins; Calcium-Transporting ATPases; Calpain; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Erythrocyte Membrane; Humans; Membrane Proteins; Molecular Weight

We examined the activation of mu-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration using antibodies specifically recognizing different activation states of mu-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (M(r) = 78 k) of mu-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic mu-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, mu-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellular conditions associated with the cell cycle may affect the activation of mu-calpain.

Topics: Amino Acid Sequence; Antibody Specificity; Autolysis; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Molecular Sequence Data; Tumor Cells, Cultured

Using a rapid FPLC procedure for the separation of calpain I, calpain II and the inhibitor calpastatin from pork meat, results of proteolytic and inhibitory activity were obtained. Duplicate measurements showed variation coefficients less than 0.13. Compared to others we found lower calpain I activity 1 h post-mortem. Three groups of pigs were randomly selected. One group was exposed to work immediately before slaughter, another group was exposed to work but allowed 30 min rest before slaughter, while the third group was a control group. Samples were taken from m longissimus dorsi 1 h and 6 h post-mortem. Work had no influence on the calpain/calpastatin activity, but a significant decrease in the calpain and calpastatin activity 6 h post-mortem was found. It should be noted that the results are preliminary and are part of an ongoing study. The method described allows separation of three extracts per instrument per day followed by activity determinations of calpains and calpastatin by the casein assay.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Chemical Fractionation; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Muscles; Reproducibility of Results; Swine

To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cattle; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Muscles; Postmortem Changes; Temperature

1. The scallop calpain-like proteinase is about five times more labile than the rabbit calpain II upon heat treatment at 35 degrees C. 2. By autolysis of the scallop proteinase of two 100 kDa subunits, 90, 45 and 30 kDa fragments were formed. Thereby the activity decreased monophasically in the presence of millimolar order of Ca2+, but did not increase in the presence of micromolar order of Ca2+ unlike the rabbit calpain II.

Topics: Animals; Autolysis; Calpain; Crustacea; Cysteine Endopeptidases; Enzyme Stability; Hot Temperature; Molecular Weight; Protein Conformation; Rabbits; Species Specificity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

Topics: Autolysis; Calcium; Calpain; Enzyme Activation; Globins; Kinetics; Phospholipids; Proteins; Vimentin

Ca2+-Activated neutral protease (CANP), that consists of 80K and 30K subunits, is converted to a low-Ca2+-requiring form by autolysis in the presence of Ca2+. Phosphatidylinositol greatly reduces the Ca2+-requirement for the autolysis of native CANP. However, this effect was not observed for CANP with a trimmed 30K subunit lacking the NH2-terminal hydrophobic and glycine-rich region. This suggests that the NH2-terminal hydrophobic region of the 30K subunit is important for the interaction of CANP with the cell membrane and that the calcium sensitivity of CANP is increased at the cell membrane through the effect of phosphatidylinositol.

Topics: Amino Acids; Animals; Autolysis; Calpain; Enzyme Activation; Muscles; Phosphatidylinositols; Rabbits; Structure-Activity Relationship

The noted loss of alpha-actinin from the Z-line of myofibrils during post-mortem autolysis, probably following the action of calcium-activated protease, has previously been attributed to its release without degradation. This report shows that in isolated myofibrils alpha-actinin is proteolysed in a Ca2+-sensitive manner presumably via the action of calcium-activated protease.

Topics: Actinin; Animals; Autolysis; Calcium; Calpain; Endopeptidases; Female; In Vitro Techniques; Myofibrils; Protease Inhibitors; Rats; Rats, Inbred Strains