Role of calpain-1 in the early phase of experimental ALS.

Elevation in [Ca(2+)]i and activation of calpain-1 occur in central nervous system of SOD1(G93A) transgenic mice model of amyotrophic lateral sclerosis (ALS), but few data are available about the early stage of ALS. We here investigated the level of activation of the Ca(2+)-dependent protease calpain-1 in spinal cord of SOD1(G93A) mice to ascertain a possible role of the protease in the aetiology of ALS. Comparing the events occurring in the 120 day old mice, we found that [Ca(2+)]i and activation of calpain-1 were also increased in the spinal cord of 30 day old mice, as indicated by the digestion of some substrates of the protease such as nNOS, αII-spectrin, and the NR2B subunit of NMDA-R. However, the digestion pattern of these proteins suggests that calpain-1 may play different roles depending on the phase of ALS. In fact, in spinal cord of 30 day old mice, activation of calpain-1 produces high amounts of nNOS active species, while in 120 day old mice enhanced-prolonged activation of calpain-1 inactivates nNOS and down-regulates NR2B. Our data reveal a critical role of calpain-1 in the early phase and during progression of ALS, suggesting new therapeutic approaches to counteract its onset and fatal course.

Characterization of the single peptide generated from the amino-terminus end of alpha- and beta-hemoglobin chains by the Ca2+-dependent neutral proteinase.

Human erythrocyte Ca2+-dependent neutral proteinase catalyzes a limited proteolysis of isolated globin chains. The rate of hydrolysis is very rapid using heme-deprived alpha- or beta-globin chains and is reduced to one-fifth with their corresponding native forms. In both cases, the proteinase specifically cleaves a single peptide bond, this resulting in the removal from the amino-terminus end of an octapeptide in beta-globin and of an undecapeptide in alpha-globin. Both peptides have been isolated, their amino acid composition has been characterized and the susceptible site of cleavage has been identified. Hemoglobin variants show a different rate of digestion as compared to that of normal chains. The alpha-Hasharon [alpha 47(CE5) Asp----His] undergoes rapid digestion, while the beta-G San Josè chain [beta 7(A4) Glu----Gly], which carries the mutation near the site of cleavage, reveals a high degree of resistance to proteolytic degradation by the neutral proteinase.

Following association to the membrane, human erythrocyte procalpain is converted and released as fully active calpain.

When exposed to inside-out human erythrocyte vesicles, in the presence of micromolar Ca2+, the 80 kDa catalytic subunit of procalpain is processed through three successive and sequential steps. These include binding to the cytosolic surface of the membrane, followed by a very rapid conversion into the 75 kDa active subunit, and ultimately by spontaneous and complete release of this active proteinase form. Binding to the membranes is competitively inhibited by the endogenous natural inhibitor through the formation of the proteinase-inhibitor complex, in which form the 80 kDa subunit can no longer be associated to the membranes. Calcium ions and the natural endogenous inhibitor appear to be crucially involved in the modulation of this novel membrane-bound mediated activation of human red cell procalpain.

The calpastatin defect in hypertension is possibly due to a specific degradation by calpain.

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Differences and similarities among three acidic endopeptidases associated with human erythrocyte membranes. Molecular and functional studies.

The three acidic proteinases (designated I, II and III, respectively) associated with human erythrocyte membranes were solubilized and purified to an electrophoretically homogeneous state by conventional procedures. Comparative analysis of chemical properties, including amino acid composition and fragmentation by cyanogen bromide cleavage, revealed significant differences among proteinases I, II and III. On the other hand, complete identity among the three proteolytic enzymes was observed on the basis of the peptide bonds specifically hydrolyzed in both glucagon- and phenylalanine-deprived oxidized B chain of insulin. In fact, each of the three proteinases produced splitting of the glucagon molecule between phenylalanine-22 and valine-23, while the susceptible bonds in the oxidized B chain of insulin proved to be those between leucine-15 and tyrosine-16 and between phenylalanine-25 and tyrosine-26, respectively.

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging.

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sapartore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421-430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.

The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte.

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.

Erythrocyte deficiency in calpain inhibitor activity in essential hypertension.

The calpain-calpain inhibitor system was evaluated in erythrocytes of patients with essential hypertension and normotensive controls, either with or without a family history of hypertension. Calpain levels were similar in the controls and hypertensive patients, whereas the inhibitor activity level was significantly reduced in the latter (301.8 +/- 26.4 vs 220 +/- 14 U/mg hemoglobin, p less than 0.001). Borderline hypertensive patients and a few controls with a history of hypertension showed low inhibitor activity. Similar results have recently been reported in genetically hypertensive rats of the Milan strain. A significant inverse correlation (r = -0.43, p less than 0.001) was found between mean arterial pressure and calpain inhibitor. Although the pathophysiological significance of these observations is not yet clear, they suggest a new area of investigation into the molecular mechanisms underlying essential hypertension and its complications.

Differential degradation of calpastatin by mu- and m-calpain in Ca(2+)-enriched human neuroblastoma LAN-5 cells.

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Rat brain contains multiple mRNAs for calpastatin.

This work was undertaken to establish the forms of the calpain inhibitor, calpastatin, expressed in the brain tissue. Five cDNA clones were obtained and the corresponding amino acid sequences were deduced. Three of these proteins contain an N-terminal domain (domain L) and four inhibitory repeats typical of the calpastatin molecule. The other two are truncated forms, containing the domain L, free or associated with a single inhibitory repeat. Other differences, due to exon skipping, produce calpastatin forms with different susceptibility to posttranslational modifications. The more represented mRNA form corresponds to a calpastatin molecule containing the four inhibitory domains. These results may be useful to understand the involvement of calpain in the onset of acute and degenerative disorders of the central nervous system.

Phosphorylation of rat brain calpastatins by protein kinase C.

Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr calpastatins are phosphorylated by protein kinase C at the same site. These calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca2+ required to induce the formation of the calpain-calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of calpastatins. We suggest that phosphorylation of calpastatin represents a mechanism capable to balance the actual amount of active calpastatin to the level of calpain to be activated.

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization.

The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to microM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca(2+)-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

Modulation of rat brain calpastatin efficiency by post-translational modifications.

Calpains, the thiol proteinases of the calcium-dependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.

Properties of calpastatin forms in rat brain.

Four recombinant calpastatin forms, deduced from rat brain mRNAs and differing in the number of inhibitory repetitive domains from zero to four, were expressed and characterized for their inhibitory efficiency on mu- and m-calpain. Although the most effective one is a truncated calpastatin form composed of the N-terminal region (domain L) and a single inhibitory domain, all inhibitors are more active against mu-calpain, but are preferentially degraded and inactivated by m-calpain. The protein form composed exclusively of a domain L is deprived of any inhibitory activity but prevents inhibition of calpain by the other calpastatin forms, indicating that this calpastatin region could be relevant in the recognition of the proteinase. A calpastatin form having molecular properties similar to those of the recombinant truncated calpastatin, has also been found in rat brain. It does not derive from proteolysis of a higher molecular mass precursor. The expression of multiple calpastatin forms may be relevant for the specific modulation of the different calpain isozymes normally present in a single cell type.

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.

Participation of protein kinase C in the activation of human neutrophils by phorbol ester.

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.

The alpha1-antitrypsin/elastase complex as an experimental model for hemodialysis in acute catabolic renal failure, extracorporeal blood circulation and cardiocirculatory bypass.

A modified polyethersulphone graft membrane was loaded with antiproteases, with the aim of reducing the active protease blood concentration during hemodialysis in acute catabolic renal failure or cardiopulmonary bypass. As protease/antiprotease system, elastase and alpha1-antitrypsin were used. The concentration of active elastase in aqueous solutions decreased as function of contact time with the membrane, approaching saturation. A 40% loss of elastase activity was obtained at pH 7.4, which was not due to autolysis, which accounted for 5% of the loss. The highest reduction was achieved at pH 9.0 (25% higher than at pH 7.4). The saturation level of elastase decrease, calculated by means of the Einstein equation, was reached after more than 47 minutes. We speculate that a time reduction might be achieved either increasing the concentration of immobilized antiproteases, or increasing the rate of elastase movement across the membranes by hydraulic, osmotic, or temperature gradients. This technology can be applied to hemodialysis, and in extracorporeal blood circulation to promote elastase release.

Protease removal by means of antiproteases immobilized on supports as a potential tool for hemodialysis or extracorporeal blood circulation.

This work studies protease concentration decrease in aqueous solutions in contact with a modified polyethersulphone graft membrane onto which antiproteases were immobilized. As a model of protease/antiprotease interaction, elastase and alpha1-antitrypsin were used. Experiments were carried out either under fixed amounts of immobilized antiproteases and variable protease concentration or under fixed protease concentration and variable amounts of immobilized antiproteases. In both cases, active protease concentrations decreased with increase in contact time with the membrane. Experimental conditions under which active elastase concentration becomes zero were also found. Occurrence of the same phenomenology has also been ascertained with protease solutions obtained from human blood neutrophils. The membrane activated with alpha1-antitrypsin showed differential inhibitory power on elastase and cathepsin G. This technology could open new perspectives in manufacturing new membranes to be used in hemodialysis and extracorporeal circulation when elastase is released.