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.

Changes in calpastatin localization and expression during calpain activation: a new mechanism for the regulation of intracellular Ca(2+)-dependent proteolysis.

The amount of calpastatin directly available in cytosol is under the control of [Ca2+] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca(2+)-dependent proteolysis.

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.

Extracellular processing of amphoterin generates a peptide active on erythroleukaemia cell differentiation.

The release of amphoterin by murine erythroleukaemia cells exposed to the chemical inducer hexamethylenebisacetamide represents an essential step for the process of their terminal differentiation. Once exported in the culture medium, amphoterin undergoes limited proteolysis, catalysed by a serine proteinase also secreted by stimulated cells. The isolated proteinase is responsible for degradation of amphoterin, with the production of a 10-amino-acid-residue fragment, specifically retaining the cell-differentiation-stimulating activity of the native protein molecule. This peptide does not express other properties of amphoterin, such as protein kinase C-stimulating activity or systemic toxicity. These findings define a selective mechanism accounting for extracellular amphoterin functional maturation.

Changes in intracellular calpastatin localization are mediated by reversible phosphorylation.

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

Human neuroblastoma cell differentiation requires protein kinase C-theta.

Neuroblastoma LAN-5 cells exposed to retinoic acid cease to multiply and extend neurite outgrowths acquiring a neuronal phenotype. We now report that protein kinase C-theta; (PKC-theta;) isozyme is involved in this differentiation process due to the following findings: (i) PKC-theta; is expressed by LAN-5 cells as a nuclear and perinuclear protein; (ii) cell stimulation with retinoic acid promotes in a large increase in the expression level of the kinase and its intracellular redistribution; and (iii) a PKC-theta; antisense oligonucleotide reduces at the same time the expression level of the kinase and the cell response to retinoic acid. Altogether these data are consistent with a specific role played by PKC-theta; in the differentiation program of neuronal cells.

Neuronal differentiation of PC12 cells involves changes in protein kinase C-theta distribution and molecular properties.

In this study we demonstrate that the rat pheochromocytoma PC12 cell line expresses the novel protein kinase C isozyme designated PKC-θ. The isozyme is almost completely localized in the nuclear compartment of proliferating cells. Following stimulation with the nerve growth factor, PKC-θ is redistributed into the cytoplasm and the outgrowing neurite processes, mostly as a cytoskeletal associated kinase. This event is accompanied by an eightfold increase in the expression level and by the appearance of specific modifications of PKC-θ molecule. Conversely, the kinase is down-regulated once cells reach the terminally differentiated state displaying a neuron-like phenotype. These data suggest a functional role for the kinase in the regulation of cytoskeletal modeling along the multistage differentiation process of PC12 cells.

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.

Properties and intracellular localization of calpain activator protein.

In this paper, we have further analyzed the properties of calpain activator (CA) in order to better define its physiological function. The activator shows a pH optimum approximately 7.8-8.0, independently of the nature of the buffer used. Although the maximal activity is observed with human acid-denatured globin, the effect of CA is detectable with other protein substrates, such as casein and insulin. A comparable activating effect is observed also with the synthetic substrate Succ-Leu-Tyr-AMC. The activatory effect has been evaluated in a reconstructed system, using plasma membrane Ca(2+)-ATPase as substrate. CA is localized in erythrocyte precursor cells on the inner surface of the plasma membrane in very high amount and its level profoundly decreases up to 10% of the original value when cells reach the terminal differentiated state.

Acyl-CoA-binding protein is a potent m-calpain activator.

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

Changes in intracellular localization of calpastatin during calpain activation.

Localization of the two main components of the Ca(2+)-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca(2+), which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

Protein kinase C-theta is specifically activated in murine erythroleukaemia cells during mitosis.

Protein kinase C-theta is a member of the n-protein kinase C subfamily that in mitotic cells translocates to centrosomes and kinetochores. Although this kinase is expressed in comparable amounts in murine erythroleukaemia cells during the interphase or metaphase, when localized in the mitotic structures, it selectively phosphorylates a 66 kDa protein, also associated to chromosomes. Moreover, protein kinase C-theta immunoprecipitated from cells at the metaphase results four times more active in the absence of lipid cofactors as compared with the kinase obtained from cells in the interphase. This activation is accomplished by interaction of protein kinase C-theta with a protein factor which also promotes an increased autophosphorylation of the kinase. These findings indicate that in the mitotic phase of the cell cycle, protein kinase C-theta recognizes a protein factor which operates as a positive modulator of the kinase activity in the absence lipids.

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.

Protein kinase C-theta is specifically localized on centrosomes and kinetochores in mitotic cells.

In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

Mechanism of action of a new component of the Ca(2+)-dependent proteolytic system in rat brain: the calpain activator.

Rat brain contains a calpain activator specific for the mu-form of the proteinase. We now report that this protein factor binds to the catalytic 80 kDa calpain subunit, promoting the dissociation of the heterodimer structure of the proteinase. The successive steps of the activation process, namely the two autoproteolytic steps producing the 78 kDa and the 75 kDa calpain forms, result in a 100 times faster rate. The activator competes with calpastatin and associates with the inner surface of plasma membranes. Based on its properties, the calpain activator can be visualised as the molecule indicating the sites for calpain activation at which the proteinase can also elude the negative control exerted by calpastatin.

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.

Molecular and functional properties of a calpain activator protein specific for mu-isoforms.

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the mu-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on mu-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.

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.

Stimulated astrocytes release high-mobility group 1 protein, an inducer of LAN-5 neuroblastoma cell differentiation.

Stimulated astrocytes specifically release large amounts of high-mobility group 1 protein into the extracellular medium. The identity of the released protein has been established on the basis of its biological activity on murine erythroleukaemia cells and by its immunoreactivity against a specific monoclonal antibody. High-mobility group 1 protein also plays an essential role in differentiation of LAN-5 neuroblastoma cells which, following stimulation with retinoic acid, express high-mobility group 1 protein on to the external surface of the plasma membrane. In retinoic acid-induced LAN-5 cells, high-mobility group 1 protein is not secreted but is accumulated in a membrane-bound form, particularly at the level of neurite outgrowths. These cells can also be induced to differentiate by high-mobility group 1 protein coated on the surface of the cell culture vessels. The specific function of the protein in this process is indicated by inhibition of cell differentiation by an anti-high-mobility group 1 protein antibody. The data are consistent with a role of high-mobility group 1 protein in promoting cell-cell interactions and in the development of nerve tissues.