Sivia Barnoy

On the involvement of calpains in the degradation of the tumor suppressor protein p53

A crude fraction that contains ubiquitin–protein ligases contains also a proteolytic activity of ∼100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous ∼250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N‐myc, c‐Fos and c‐Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

Calpain and calpastatin in myoblast differentiation and fusion: effects of inhibitors.

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca(2+)-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-beta and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of mu-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.

The calpain-calpastatin system and protein degradation in fusing myoblasts

Calpain (Ca(2+)-activated cysteine protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously found that calpastatin (the endogenous inhibitor of calpain) diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing Ca(2+)-induced calpain activation at that stage. Here, we show that a limited degradation of some proteins occurs within the myoblasts undergoing fusion, but not in proliferating myoblasts. The protein degradation is observed at the stage when calpastatin is low. Protein degradation within the myoblasts and myoblast fusion are inhibited by EGTA, by the cysteine protease inhibitors calpeptin and E-64d and by calpastatin. The degradation appears to be selective for certain myoblast proteins. Integrin beta 1 subunit, talin and beta-tropomyosin are degraded in the fusing myoblasts, whereas alpha-actinin, beta-tubulin and alpha-tropomyosin are not. A similar pattern of degradation is observed in lysates of proliferating myoblasts when Ca2+ and excess calpain are added, a degradation that is inhibited by calpastatin. The results support the notion that degradation of certain proteins is required for myoblast fusion and that calpain participates in the fusion-associated protein degradation. Participation of calpain is made possible by a change in calpain/calpastatin ratio, i.e., by a diminution in calpastatin level from a high level in the proliferating myoblasts to a low level in the differentiating myoblasts. Degradation of certain proteins, known to be responsible for the stability of the membrane-skeleton organization and for the interaction of the cell with the extracellular matrix, would allow destabilization of the membrane and the creation of membrane fusion-potent regions.

Association of calpain (Ca2+-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts

Calpain isozymes (intracellular, Ca2+‐dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of μ‐calpain and m‐calpain (activated by μM and mM Ca2+, respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti‐calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti‐calpastatin). Calpastatin was co‐precipitated when calpain was immunoprecipitated; calpain was co‐precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca2+‐dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca2+levels. It is proposed that the status of cellular calpain‐calpastatin association is modulated by cell constituents, for which some possibilities are suggested. J. Cell. Biochem. 74:522–531, 1999.

Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability.

Calpain (Ca(2+)-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that mu-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor beta (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease.

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimers disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.

Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and calpain systems

Previously, we found that calpastatin diminished transiently prior to myoblast fusion (rat L8 myoblasts), allowing calpain‐induced protein degradation, required for fusion. Here we show that the transient diminution in calpastatin is due to its degradation by caspase‐1. Inhibition of caspase‐1 prevents calpastatin diminution and prevents myoblast fusion. Caspase‐1 activity is transiently increased during myoblast differentiation. Both calpain and caspase appear to be responsible for the fusion‐associated membrane protein degradation. Caspase‐1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase‐1 in L8 myoblast fusion represents a novel function for this caspase in a non‐apoptotic differentiation process, and points to cross‐talk between the calpain and caspase systems in some differentiation processes.

Calpain activates caspase-8 in neuron-like differentiated PC12 cells via the amyloid-β-peptide and CD95 pathways

The neurotoxic amyloid-beta-peptide (Abeta) is important in the pathogenesis of Alzheimers disease (AD). Calpain (Ca(2+)-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in AD/Abeta toxicity. We previously found that Abeta promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. We now report on the previously unrecognized caspase-8 activation by calpain. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the Abeta and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIP(S)). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIP(S) degradation in the Abeta-treated and CD95-triggered cells. Increased cellular Ca(2+) per se results in calpain activation but does not lead to caspase-8 activation or FLIP(S) degradation. The results suggest that procaspase-8 and FLIP(S) association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase-8, and CD95 pathway in AD/Abeta toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

Amyloid β peptide toxicity in differentiated PC12 cells: Calpain‐calpastatin, caspase, and membrane damage

Amyloid β peptide (Aβ) is implicated in the pathogenesis of Alzheimers disease (AD). The peptide is toxic to neurons, possibly by causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca2+. Calpain (Ca2+‐dependent protease) and caspase have also been implicated in AD. There is little information on communication between the two proteases or on the involvement of calpastatin (the specific calpain inhibitor) in Aβ toxicity. We studied the effects of Aβ25–35 (sAβ) on calpain, calpastatin, and caspase in neuronal‐like differentiated PC12 cells. sAβ‐treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide, without apparent nuclear changes of apoptosis, and little poly (ADP‐ribose) polymerase [PARP] degradation). The sAβ‐induced membrane damage is in contrast with staurosporine‐induced damage (nuclear apoptotic changes, PARP degradation, without membrane propidium iodide permeability). sAβ led to activation of caspase‐8 and calpain, promotion of calpastatin degradation (by caspase‐8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). The results support the idea that Aβ causes primarily neuronal membrane dysfunction, and point to cross‐talk between calpain and caspase (protease activation and degradation of calpastatin) in Aβ toxicity. Increased expression of calpastatin and/or decrease in calpain and caspase‐8 may serve as means for ameliorating early symptoms of AD.

Calpastatin overexpression attenuates amyloid-β-peptide toxicity in differentiated PC12 cells

Amyloid beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimers disease (AD). Abeta is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Abeta25-35 (sAbeta) on neuronal-like differentiated PC12 cells. We reported that sAbeta-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAbeta-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAbeta-promoted degradation of fodrin, protein kinase Cepsilon, beta-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAbeta-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca(2+)-promoted calpain activation and protein degradation; this is consistent with the notion that the Abeta-induced increase in calpain activity results from a rise in cellular Ca(2+), provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)-calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAbeta-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Abeta-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Abeta-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.