Brain μ-Calpain Autolysis During Global Cerebral Ischemia

Abstract: 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 μ-calpain in the pathophysiology of ischemic neuronal injury, the evidence is indirect. A direct indicator of μ-calpain proteolytic activity is autolysis of its 80-kDa catalytic subunit, and therefore we examined the μ-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 μ-calpain catalytic subunit. In nonischemic brain homogenates the antibody identified an 80-kDa band, which migrated identically with purified μ-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 μ-calpain during cerebral ischemia. Further work is needed to characterize the extent, duration, and localization of μ-calpain activity during brain ischemia and reperfusion as well as its role in the causal pathway of postischemic neuronal injury.     

Multiple molecular interactions implicate the connectin/titin N2A region as a modulating scaffold for p94/calpain 3 activity in skeletal muscle

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.     

μ-Calpain Activation and Calpain-Mediated Cytoskeletal Proteolysis Following Traumatic Brain Injury

Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-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 μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-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 μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-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 α-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 μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-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 μ-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.     

Effect of Cathepsin D Treatment on ATPase Activity of Rabbit Myofibril

The effect of cathepsin D and pepsin treatment on rabbit myofibril was studied by measuring the amount of proteolytic products and Mg-enhanced ATPase activity.

When myofibril was treated with cathepsin D at 3°C and pH 5.0 or 5.5, a little but detectable amount of nonprotein nitrogenous compounds was released. However, there was no change in ATPase activity of myofibril, though treated with cathepsin D of higher units than assumed to be in muscle.

When myofibril was treated with pepsin under the same condition as used above, there was an increase in KCl-concentration dependence of ATPase activity followed by a decrease in the maximal value of ATPase activity.

From the present results, it was concluded that cathepsin D might not take a main role on the post-mortem degradation of myofibril.     

Cytosolic Proteolysis of τ by Cathepsin D in Hippocampus Following Suppression of Cathepsins B and L

Abstract: Incubation of cultured hippocampal slices with an inhibitor [ N -CBZ- l -phenylalanyl- l -alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of τ protein and the production of a 29-kDa τ fragment (τ₂₉). A τ antibody that is sensitive to the phosphorylated state of its epitopes did not recognize τ proteins or the τ₂₉ fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the τ fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. τ₂₉ exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal τ-tubulin interactions. Three lines of evidence indicated that ZPAD-induced τ proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and τ at neutral pH resulted in a loss of intact τ proteins and production of a 29-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced τ proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The τ fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features.     

Spectrophotometric Determination of L-Malic Acid with a Malic Enzyme

Purified β-connectin solutions were treated with hydrostatic pressures of 300 and 600 MPa for 10 min, and irreversible changes in the proportions of the secondary structure of connectin were estimated from CD spectra. The CD spectra showed that pressure had little effect on the secondary structure.     

Calcium-induced splitting of connectin filaments into beta-connectin and a 1,200-kDa subfragment.

When rabbit skeletal muscle myofibrils were treated with a solution containing 0.1 mM Ca2+ and 30 micrograms of leupeptin/ml, alpha-connectin, which forms very thin filaments in myofibrils, was split into beta-connectin and a 1,200-kDa subfragment. A part of beta-connectin located near the junction between beta-connectin and the subfragment seems to have an affinity for calcium ions and to be susceptible to the binding of large amounts of calcium ions. The calcium-binding site on beta-connectin is localized near the N2 line in the I band, and the subfragment is localized adjacent to the Z disk. It is possible that connectin filaments change their elasticity during the contraction-relaxation cycle of skeletal muscle at the physiological concentration of calcium ions. Because postmortem skeletal muscles lose their elasticity and become plastic in association with the calcium-specific splitting of connectin filaments, the splitting is considered to be a factor in meat tenderization during postrigor ageing.     

Immunohistochemical localization of cathepsin D and a possible role of melanocytes during tail resorption in tadpoles of a tropical toad

Programmed cell death during anuran tail resorption is primarily brought about by apoptosis. Cathepsin D, a lysosomal aspartyl protease, is involved in the death of tail tissues. Thus, anuran tail resorption presents an ideal model to study cathepsin‐mediated cell death during vertebrate development. Present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D in the tail tissues of the common Asian toad, Duttaphrynus melanostictus. Cathepsin D was involved in programmed cell death in epidermis, muscle, spinal cord, and blood cells in the resorbing tail. Interestingly, it was also involved in the pre‐resorbing tail before visible tail resorption which indicates initiation of cell death even before actually the tail resorbs. Melanocytes were found to be one of the causative agents in degrading tail tissues and were associated with the degradation of muscle, epidermis and spinal cord of the resorbing tail. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Mode of degradation of myofibrillar proteins by rabbit muscle cathepsin D

The mode of degradation of myofibrillar proteins by the action of highly purified rabbit muscle cathepsin D (EC 3.4.23.5) was studied using SDS-polyacrylamide gel electrophoresis. Cathepsin D optimally degraded myosin heavy chain, α-actinin, tropomyosin, troponin T and troponin I at around pH 3. It did not degrade actin or troponin C. Degradation of myosin heavy chain produced four major fragments of 155 000, 130 000, 110 000 and 90 000 daltons. Troponin T was hydrolyzed to 33 000-, and 20 000- and 11 000-dalton fragments. Troponin I was degraded into fragments of 13 000 and 11 000 daltons. Degradation of α-actinin and tropomyosin was not as rapid as that of mysoin and troponins T and I. Tropomyosin gave a fragment of 30 000 daltons, but α-actinin showed no distinct band of this fragment on gels.     

The N-terminal sequence of paratropomyosin binding fragments from beta-connectin

In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken beta-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from beta-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the I51 domain.     

Airborne limonene confers limited thermotolerance to Quercus ilex

The purpose of the study was to test the possible and controversial thermotolerance role of monoterpene production and emission and the related responses of antioxidants. Quercus ilex seedlings were exposed to a ramp of temperatures of 5°C steps from 25 to 50°C growing with and without limonene fumigation (7.5 µl l⁻¹). Net photosynthetic rates, maximal photochemical efficiency of PSII (F_v/F_m), oxidation state of ascorbic acid, and lipid peroxidation estimated by malondialdehyde concentrations of limonene-fumigated (LF) plants did not significantly differ from control (C) plants. No consistent changes in emissions of the other monoterpenes, α-pinene, β-phellandrene, β-pinene or β-myrcene were found. However, slight differences were found in the concentration of antioxidants. The amounts of α-tocopherol did not change or even tended to decrease at high temperatures in LF plants whereas they tended to increase by approximately 60% at 45 and 50°C relative to 25°C in C plants. Ascorbic acid reached its maximum concentration only at 45°C in LF plants whereas it reached its maximum at 35°C in C plants. β-Carotene did not decrease at high temperatures in LF plants whereas it decreased by approximately 15% at 45–50°C in C plants. Brown pigment index (BPI), an optical indicator of tissue oxidative processes, was lower in LF plants than in C plants. The photochemical reflectance index (PRI), an optical indicator of photosynthetic light use efficiency, was higher for LF plants than for C plants at elevated temperatures. Visual leaf damage (browning) tended to be less in LF plants than in C plans although not significantly (26.5 ± 8.5 versus 16.2 ± 4.8%). These results show that limonene does not confer clear and strong thermotolerance but might have some minor role. These results are in agreement with previous indications of weaker thermotolerance effect of monoterpenes than of isoprene.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.     

Membrane-bound tubulin: Resistance to cathepsin D and susceptibility to thrombin

In recent studies we found that cytoplasmic tubulin from brain was rapidly split by brain cathepsin D. Two pools could be established; the major portion was split at 18%/h, a minor portion at 2%/h, under our experimental circumstances. In the present work these experiments were extended to membrane-bound tubulin from brain. The membrane-bound form, in contrast to the cytoplasmic tubulin, was not degraded by cerebral cathepsin D under similar experimental conditions. This was not due to the presence of an inhibitory protein since added cytoplasmic tubulin was degraded. Several other protein components of membrane fractions (synaptosomal, mitochondrial) were degraded by cathepsin D, as measured on two-dimensional electropherograms. Thrombin degraded cytoplasmic tubulin, but the degradation products differed from those of cathepsin D degradation. Thrombin also hydrolyzed membrane-bound tubulin, but at a lower rate than the cytoplasmic form. Our results indicate great differences in the breakdown rate of a protein, which depend on its localization, in accord with the differences found in in vivo turnover rates.     

Effect of exercise on synthesis and degradation of muscle protein.

Several reports have shown that amino acid utilization via oxidation and gluconeogenesis is increased during exercise. The purpose of this study was to investigate whether these changes are accompanied by alterations in protein synthesis and degradation in the muscle of exercising rats. One group of rats was made in swim for 1h and then protein synthesis and protein degradation were measured in a perfused hemicorpus preparation. Protein synthesis was decreased and protein degradation was increased in exercised rats compared with sedentary control rats. Exercise also decreased amino acid incorporation by isolated polyribosomes from muscle. Measurement of several muscle proteinase activities demonstrated that exercise had no effect on alkaline proteinase or Ca2+-activated proteinase. However, the free (unbound) cathepsin D activity was elevated in muscle of exercised rats, whereas the total activity of catepsin D was unchanged. This increase in the proportion of free cathepsin D activity suggests that lysosomal enzymes may be involved in the increased protein degradation that was observed.     

Action of brain cathepsin B,cathepsin D,and high-molecular-weight aspartic proteinase on angiotensins I and II

The action of three previously isolated electrophoretically homogeneous brain proteinases—cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (M_r=90K; EC 3.4.23.−)—on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val³-Tyr⁴ bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions. Special Issue dedicated to Dr. Eugene Kreps.     

Acclimation by suboptimal growth temperature diminishes photooxidative damage in maize leaves

Leaves of Zea mays L. seedlings which developed at optimal (25°C) or suboptimal (15°C) temperature were exposed to high irradiance (1000 μmol m^?2 s^?1) and a severe chilling temperature (5°C) for up to 24 h to investigate their ability to withstand photooxidative stress. During this stress, the degradation of the endogenous antioxidants ascorbate, glutathione and α-tocopherol was delayed and less pronounced in 15°C leaves. Similarly, the decline in chlorophyll a, chlorophyll b, β-carotene and lutein was slower throughout the stress period. Faster development and a higher level of non-photochemical quenching (NPQ) of chlorophyll fluorescence, related to a stronger de-poxidation of the larger xanthophyll cycle pool in 15°C leaves, could act as a defence mechanism to reduce the formation of reactive oxygen species during severe chilling. Furthermore, plants grown at suboptimal temperature exhibited a higher amount of the antioxidants glutathione and α-tocopherol. The higher α-tocopherol content in leaves (double based on leaf area; 4-fold higher based on chlorophyll content) which developed at suboptimal temperature may play an especially important role in the stabilization of the thylakoid membrane and thus prevent lipid peroxidation.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.