Proteomics analysis of starved cells revealed Annexin A1 as an important regulator of autophagic degradation

The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)–PIN1 fusion protein. Adding GST–PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine–proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.     

Dietary level of protein regulates glyceraldehyde-3-phosphate dehydrogenase content and synthesis rate in mouse liver cytosol

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a Mr 36 000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The Mr 36 000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.     

Age Peculiarities of the Calpain/Calpastatin Cerebral System in Rats: Relation to the Hypothesis of Brain Aging

We studied age-related changes in the activity of calpain, those in the activity of its endogenous inhibitor calpastatin, and the ratio of these indices in the brain of rats of four age groups (2-3 weeks, 2-3, 5-6, and 24 months). The activity of calpain was estimated using FITC casein as the substrate. In a soluble fraction of the brain homogenate, the enzyme activity in general increased with age. In mature rats (5 to 6 months old), this index exceeded 3.65 times the corresponding index in juvenile (2 to 3 weeks old) animals, while in old animals this index somewhat decreased. In the fraction obtained after separation of calpain from other components using DEAE-cellulose chromatography, the age-related trend toward an increase in the activity of calpain was preserved, but it was much more moderate. The activity of calpastatin demonstrated an opposite direction of age changes: it was the maximum in 2-3-week-old animals and gradually decreased (by 27%) in old rats. We also found that the efficacy of inhibitory action of calpastatin in the cerebral tissue with respect to the activity of calpain was, as a rule, redundant. In this case, the ratio of inhibitor/enzyme activities decreased with age; this index was 1.65, 1.33, 1.1, and 1.0 or less in 2-3-week-old, 2-month-old, mature, and old animals. Therefore, we found that the intensity of calpain-mediated proteolysis in the rat brain increases from the juvenile period to the mature age and somewhat decreases in old individuals. Such alterations are developed at the expense of both an increase in the activity of the enzyme and weakening of the action of its inhibitor (calpastatin).     

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.     

Calpain,calpastatin activities and ratios during myocardial ischemia-reperfusion

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a calpain burst impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). As expected no detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without affect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain–calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downegulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of I/R changes, although the role of calpastatin downregulation remains to be elucidated.     

Effect of dietary level of protein on the metabolism of mouse liver nuclear proteins.

The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.     

Age-dependent degradation of calpastatin in kidney of hypertensive rats

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.     

Calpain and calpastatin levels in dystrophic hamster skeletal muscles

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.     

Participation of the conventional calpains in apoptosis

The conventional calpains, m- and micro-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms. However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing micro-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells, which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity protected beta-spectrin, but not alpha-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other treatments. Calpain protected against TNF-alpha induced apoptosis. In contrast to previous studies, we found no evidence that calpains proteolyze I kappa B-alpha in TNF-alpha-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective.     

Calpain and calpastatin levels in different organs of the rabbit

1. The levels of Ca-independent and Ca-dependent proteolytic activity as well as the activities of calpains and calpastatin in different organs of the rabbit was examined at various developmental stages. 2. Calpain and calpastatin levels were highest in the lung and in the kidney. 3. In all organs examined except the thymus the total level of calpain was higher than that of calpastatin. 4. In the thymus the levels of calpains and calpastatin decreased markedly with age.     

Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.     

Epinephrine upregulates calpain activity in cultured C2C12 muscle cells

C2C12 cells were grown to confluence at 37 degrees C under a continuous 5% CO(2) stream and myotube formation was stimulated. The cultures were then incubated with or without 2 microg/mL epinephrine for 18 h prior to harvesting and calpain extraction. Epinephrine treatment resulted in a three-fold increase in extractable mu-calpain activity (P < 0.05), a three-fold increase in extractable m-calpain activity (P < 0.05), a 36% increase in calpastatin activity (P < 0.001), and a 16% decrease (P < 0.05) in the total protein content in the C2C12 cell homogenate. These results suggest that calpains may play a role in protein metabolism and that the hormone epinephrine may be directly involved in the regulation of their cellular expression.     

Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart

A cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpains in rat heart after ischemia and reperfusion. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band of 140kDa. Both the expression and the activity of HMWCaMBP were decreased by ischemia reperfusion. Immunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactivity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of micro-calpain and m-calpain in ischemia-reperfused heart was increased as compared to normal heart. The calpain inhibitory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic perfusion of hearts with a cell-permeable calpain inhibitor suppressed the increase in calpain expression but increased the HMWCaMBP expression. In-vitro HMWCaMBP was proteolyzed by micro-calpain and m-calpain. We also measured apoptosis in normal and ischemia-reperfused tissues. An increase in the number of apoptotic bodies was observed with increased duration of ischemia and reperfusion. Bcl-2 expression did not change in any of the groups, whereas Bax expression increased with ischemia-reperfusion and correlated well with the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptible to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury.     

Decreased expression of calpain and calpastatin mRNA during development is highly correlated with muscle protein accumulation in neonatal pigs

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain–calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2–4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain–calpastatin system.     

Down-regulation of calpain 9 is linked to hypertensive heart and kidney disease.

Calpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of the different calpain isoforms in hypertensive target organ damage. Using real-time PCR, we found calpain 6 and 9 down-regulated by more than 50% and the endogenous calpain inhibitor calpastatin up-regulated by 225%, respectively, in the hearts of Dahl salt-sensitive rats on a high salt (4% NaCl) compared to normal salt diet. On the protein level, calpain 9 but not calpastatin was regulated in the hypertensive target organs heart and kidney. Moreover, the myocardial expression of calpain 9 protein was inversely linked to left ventricular mass (r= -0.93, p<0.01), and renal expression of calpain 9 protein correlated inversely with albuminuria (r= -0.82, p<0.05). In the aorta, there was no regulation of calpain 9 on the protein level. We conclude that differential regulation of calpain 9 may play a role in hypertensive target organ damage.     

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

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

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

Activity of Calpain in the Guinea Pig Brain under Conditions of Experimental Allergic Encephalomyelitis

We studied the activity of calpain in the brain tissue of guinea pigs at different stages of the development of experimental allergic encephalomyelitis (EAE). Eleven days after inoculation of a mixture containing myelin main protein into the experimental animals, we observed a drop in the calpain activity (on average, by 27% with respect to the control), whereas on the 20th and 27th days the activity of the enzyme under study exceeded the norm (by 12%). The calpain/calpastatin ratio also altered at the different stages of development of EAE: the amount of calpastatin increased significantly on the 11th and 27th days, while on the 20th day the level of calpastatin was close to that typical of the control animals. Therefore, we found that the state of calpain/calpastatin system in the guinea pig brain demonstrates some correlation with the dynamics of development of EAE.     

Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.