Transglutaminase 2 inhibits apoptosis induced by calcium- overload through down-regulation of Bax.

An abrupt increase of intracellular Ca(2+) is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca(2+) triggers apoptotic cell death through mitochondrial swelling and activation of Ca(2+)-dependent enzymes. Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca(2+)-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca(2+)-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca(2+)-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.

Monoclonal antibodies to human transglutaminase 4.

Transglutaminase 4 (TG4) is a member of the enzyme family that catalyzes the calcium-dependent post-translational modification of proteins via cross-linking, polyamination, or deamidation. TG4 exhibits prostate-specific expression pattern and plays a crucial role in the formation of the copulatory plug in rodents. However, the physiological function(s) of human TG4 remains speculative. Human TG4 has been postulated to participate in the maturation process of sperm by modifying its cell surface, which results in suppression of sperm antigenicity in the female genital tract. To better understand the pathophysiological role of TG4 in prostate tissue, we generated monoclonal antibodies (MAb) against human TG4 in mice by repeated injections with the recombinant human TG4. Western blot analysis demonstrated that the selected MAbs react specifically with TG4, but not with other isoenzymes of the TG family. Immunocytochemical and immunohistochemical analyses showed that specific staining is observed with the cells overexpressing TG4 and with the paraffin-embedded prostate tissue specimens obtained from the benign prostate hyperplasia and prostate cancer patients, respectively. Our results indicate that these MAbs are suitable for detecting TG4 in the cultured cells or prostate tissues for investigating the biological functions of human TG4.

Differential alternative splicing of human transglutaminase 4 in benign prostate hyperplasia and prostate cancer.

Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.

Degradation of transglutaminase 2 by calcium-mediated ubiquitination responding to high oxidative stress.

Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the transamidation reaction. There is conflicting evidence on the role of TG2 in apoptosis. In this report, we show that TG2 increases in response to low level of oxidative stress, whereas TG2 diminishes under high stress conditions. Monitoring TG2 expression, activity and calcium concentration in cells treated with A23187 revealed that the initial rise of calcium activates TG2 but subsequent calcium-overload induces the degradation of TG2 via calcium-mediated polyubiquitination. These results indicate that the role of TG2 in apoptosis depends on the level of calcium influx triggered by oxidative stress.

TGFbeta mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation.

Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFbeta, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFbeta-mediated activation of TGase2. These results indicate that TGFbeta mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFbeta-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.

Alteration of Rb binding to HPV 18 E7 modified by transglutaminase 2 with different type of polyamines.

High-risk human papillomavirus (HPV) E7 is a major oncoprotein that plays a crucial role in the development of cervical cancer. A previous study showed that transglutaminase (TGase) 2 catalyzes the incorporation of polyamines into HPV 18 E7 protein, and thereby diminishes its ability to bind Rb. Therefore, TGase 2 activity may be implicated in a suppressive function of host against HPV-induced carcinogenesis. To better understand the nature of polyamination of HPV 18 E7, we investigated the Rb binding of E7 polyaminated in vitro with different type of polyamines. The incorporation of spermine diminished the Rb binding of E7 more profoundly compared with that of spermidine, suggesting that either the additional positive charge or a steric effect or both may have altered the chemical or structural properties of the protein. In addition, the treatment of either spermidine or spermine in cultured cell system reduced the ability of E7 to inactivate Rb with a TGase activity-dependent manner. Spermine was more effective in inhibiting E7 activity than spermidine. These results may provide the basis for future investigation aiming at delineating the significance of polyamine metabolism on HPV E7 functions.

Different inhibition characteristics of intracellular transglutaminase activity by cystamine and cysteamine.

The treatment of cystamine, a transglutaminase(TGase) inhibitor, has beneficial effects in several diseases including CAG-expansion disorders and cataract. We compared the inhibition characteristics of cystamine with those of cysteamine, a reduced form of cystamine expect-ed to be present inside cells. Cystamine is a more potent inhibitor for TGase than cysteamine with different kinetics pattern in a non-reducing condition. By contrast, under reducing conditions, the inhibitory effect of cystamine was comparable with that of cysteamine. How-ever, cystamine inhibited intracellular TGase activity more strongly than cysteamine despite of cytoplasmic reducing environment, suggest-ing that cystamine itself inhibits in situ TGase activity by forming mixed disulfides.