Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r 2 ‫؍‬ 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

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 M). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 M 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 M), 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 downregulates 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.Although the role of caspases in the apoptotic cascade has been extensively characterized, the role of calpains is less clear. Ubiquitously expressed calpains I and II are reversibly activated by calcium and regulated by the specific endogenous inhibitor, calpastatin (1). Calpain activation during apoptosis has been observed in a number of cell culture and in vivo models of apoptosis (2-18). The role of calpain appears to depend on the cell type being studied. Calpain inhibitors reduce apoptotic cell death in CHO 1 cells, PC12 cells, U937 cells thymocytes, metamyelocytes, lymphocytes, auditory sensory cells, human lung carcinoma cells, cardiac myocytes, and neurons (2, 5, 11-13, 16, 17, 19 -27). In contrast, calpain inhibitors have been reported to induce apoptosis in Molt 4, HL-30 cells, human acute and chronic lymphocytic leukemia cells, and human prostate carcinoma cells (18, 28 -30). Even within the same cell line, the role of calpain can vary depending on type and severity of apoptotic stimulus (4,14,15,17). Determining the relative roles of calpains and caspases in apoptotic cell death is further complicated by the growing body of evidence for cross-talk between the two proteolytic systems. Calpain-mediated cleavage of caspases-3, -7, -8, -9, and -12 has been reported with varying functional consequences (5,8,12,(31)(32)(33). Calpain-mediated N-terminal truncation of caspase-3 to a p30 polypeptide enhanced activation in one study and inhibited activat...

Although activation of calcium-activated neutral protease (calpain) by the NMDA receptor has been suggested to play critical roles in synaptic modulation and neurologic disease, the nature of its substrates has not been completely defined. In this study, we examined the ability of calpain to cleave the NMDA receptor in cultured hippocampal neurons. Activation of the NMDA receptor by agonist application led to rapid calpain-specific proteolysis of spectrin and decreased levels of NR2A/2B subunits. Cleavage of the NR2A/2B subunit created a 115 kDa product that retained the ability to bind 125I-MK-801 and is predicted to be active. Increases in levels of this product appeared within 5 min of NMDA receptor activation and were stable for periods of >30 min. Subtype-specific antibodies demonstrated that the NR2B subunit was cleaved in these primary cultures, but the NR2A subunit was not. An inhibitor of calpain blocked both the decrease of intact NR2B and the increase of the low molecular weight form, whereas neither caspase nor cathepsin inhibitors had an effect on these events. Cell surface biotinylation experiments demonstrated that the 115 kDa fragment remained on the cell surface. This NR2B fragment was also found in the rat hippocampus after transient forebrain ischemia, showing that this process also occurs in vivo. This suggests that calpain-mediated cleavage of the NR2B subunit occurs in neurons and gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation. Such forms could contribute to excitotoxicity and synaptic remodeling.