Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human μCANP and mCANP

We investigated the relative inhibition of purified human CANP and mCANP by five cysteine proteinase inhibitors including N-acetyl-Leu-Leu-nor-leucinal (C-I) and N-acetyl-Leu-Leu-methioninal (C-II), calpeptin, E64, and leupeptin. Based on IC₅₀ measurements, calpeptin and C-I were stronger inhibitors by one to two orders of magnitude than C-II, leupeptin or E64. None of the five inhibitors, however, exhibited greater specificity for human CANP or mCANP. These results indicate that, although the inhibition of a given cellular event by these compounds may suggest CANP involvement, effects on CANP cannot be discriminated from those on mCANP.     

The Effects of Calpain Inhibition on IkBα Degradation After Activation of PBMCs: Identification of the Calpain Cleavage Sites

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkB alpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkB alpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkB alpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkB alpha levels compared to nonactivated controls. Recombinant IkB alpha was incubated with purified porcine m-calpain in the presence of 0.1% Triton X-100, and the degradation products were monitored by SDS-PAGE and sequenced. Most of the degradation products were peptides derived from calpain, but one was derived from IkB alpha cleaved between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire signal response domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. The results suggest that calpain plays an important role in IkB alpha degradation, a crucial event in T cell activation.     

An insight into the mechanism of cytotoxicity of ricin to hepatoma cell: roles of Bcl-2 family proteins, caspases, Ca(2+)-dependent proteases and protein kinase C

The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.     

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na⁺/Ca²⁺ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 ± 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 μM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting α-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.     

Calpeptin suppresses tumor necrosis factor-α-induced death and accumulation of p53 in L929 mouse sarcoma cells

The cytokine tumor necrosis factor (TNF) induces caspase-dependent cell death in a subset of tumor cells. In this report, we show a novel suppressive effect of calpeptin, a calpain inhibitor, on TNF-induced cell death and accumulation of p53 in L929 mouse fibrosarcoma. Exposure to 10 ng/ml TNF induced cell death in >50% of L929 cells within 12 h and stimulated accumulation of p53 (8-fold). Preincubation of cells with calpeptin blocked both TNF-induced cell death and accumulation of p53 as examined with Western blot. TNF-induced accumulation of p53 was in part contributed by increase of p53 mRNA level (2.2-fold) in a calpeptin-insensitive manner. Interestingly, other calpain inhibitors tested did not show these effects like calpeptin and TNF treatment did not increase apparent calpain activity in L929 cells, suggesting that calpeptin may have another function besides targeting calpain. Expression of dominant negative mutant p53Val¹³⁵ reduced the incidence of TNF-mediated cell death. Taken together, our findings suggest that TNF induces calpeptin-dependent, but calpain-independent accumulation of p53 protein as a necessary step leading to death in L929 cells.     

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage

Mitochondrial impairment and calcium (Ca⁺⁺) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca⁺⁺-ATPase pumps are impaired causing cytoplasmic Ca⁺⁺ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser⁶⁵, and reduced calpain-cleavage of Parkin. Treatment with the Ca⁺⁺ ionophore A23187, which facilitates Ca⁺⁺ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca⁺⁺ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca⁺⁺-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.