Results TRB3 is Cleaved by Caspases in vitro and in the Apoptotic Process We previously reported that TRB3 is cleaved by CASP3 at Asp338 [21]. To analyze the biological consequence of TRB3 cleavage by caspase, we constructed three recombinant plasmids for expressing the wild type (WT), CASP3 cleavage-site mutant (D338A) and CASP3-cleaved form (DC20) of TRB3, respectively, cartoon diagrams of which are shown in Figure 1A. As was shown in our previous report [21], the WT-TRB3 was cleaved by CASP3 in vitro (Figure 1B). In contrast, the D338A-TRB3 and DC20- TRB3 were not cleaved by CASP3. This result suggests that TRB3 was cleaved at a single site by CASP3. We also tested CASP2, CASP6, CASP7, CASP8, CASP9 and CASP10 for their ability to cleave TRB3 in this in vitro assay (Figure 1C). Surprisingly, all caspases used in this assay cleaved the WT-TRB3, and all of them, except for CASP2, were unable to cleave the D338A mutant. As judged from the mobility of the cleaved fragment on the SDS/ PAGE (Figure 1C), the CASP2 cleavage site must be more close to the C-terminal end of TRB3 than the Asp338 residue, and we predicted that this site could be either at Asp343 and/or at Asp351. These results suggest that TRB3 is cleaved by multiple caspases, and the primary cleavage site is located at Asp338. Sequence alignment analysis of the TRB3 caspase-cleavage sites from multiple species raised a possibility that TRB3 is cleaved in various species and the cleavage has biological significance (see Figure S1 for details). To examine whether TRB3 is cleaved in apoptosis-induced cells, we transfected HeLa and Jurkat cells separately with the recombinant TRB3 plasmids expressing WT-TRB3, D338A- TRB3, and DC20-TRB3. Cleavage of WT-TRB3 was observed in cells that were made apoptotic by treatment with tumor necrosis factor-a and cycloheximide (TNFa/CHX) (HeLa cells) or anti-Fas antibody (Jurkat cells), and this process was blocked by z-VAD- FMK, a caspase inhibitor (Figure 1D). As a result of in vitro experiment, the cleavage fragment derived from D338A-TRB3 and DC20-TRB3 was not detected in the apoptotic cells. Although the reduction of TRB3 levels was observed upon TNFa/CHX treatment, it may be due to proteasomal degradation and the rescue of protein levels seems to be the unexpected effect of z- VAD-FMK [21,22]. However, it could not be denied that lack of DC20-TRB3 stability is caused by the deletion. Taken together, these results suggested that TRB3 was cleaved at Asp338 by caspases during apoptosis. Cleavage of TRB3 Promotes Apoptosis along with CASP3 Activation TRB3 has been shown to be involved in the regulation of apoptosis under stress conditions. For example, overexpression of TRB3 under ER stress conditions produced pro-apoptotic effect [6]. Exact contribution of TRB3 to apoptosis, however, remains unclear at present. To determine how TRB3 cleavage affects apoptosis, we transiently overexpressed the cleavable TRB3 (WT) and non-cleavable TRB3 (D338A) in HeLa and Jurkat cells. Accordingly, both cells were separately transfected with the recombinant plasmids expressing WT-TRB3 or D338A-TRB3, or with the pcDNA3.1 empty vector (control vector), transfected cells were then treated with TNFa/CHX (HeLa cells) or anti-Fas antibody (Jurkat cells) to induce apoptosis and subsequently dead cells were counted. As shown in Figure 1D, expression levels of WT-TRB3 and D338A-TRB3 in both cells were very similar. Interestingly, the percentage of dead cells was higher in the WT- TRB3 expressing cells, but not in the D338A-TRB3 expressing Nuclear Translocation of Procaspase-3 by TRB3 cells, as compared to that in the control cells (Figure 2A and B). In both cells, the reagent-induced cell death was rescued back to the untreated control level when the stimuli were performed in the presence of the caspase inhibitor z-VAD-FMK (Figure S2), suggesting that cell death was due to apoptosis. These results suggest that the pro-apoptotic effect of TRB3 depends on its cleavage by caspases. It is possible that the cleavage of TRB3 is an important event in the pro-apoptotic process. Because CASP3 activation is a critical step in the execution of apoptosis, we next investigated the effect of TRB3 cleavage on CASP3 activation. We used a luminescence- based assay to measure the CASP3/7 activity in cells. As shown, the CASP3/7 activity was found to be higher in the WT-TRB3 expressing cells, but not in the D338A-TRB3 expressing cells, than that in the control cells (Figure 2C and D). Taken together, these results suggest that cleavage of TRB3 is required for inducing further activation of CASP3 and/or CASP7, and in turn might promote apoptosis. TRB3 Inhibits CASP3 Activation and Subsequent Apoptosis under ER Stress Condition Tunicamycin treatment, which causes ER stress in cells, has been shown to induce TRB3 expression in several cell lines [6,16]. TRB3 expression was indeed induced in HeLa cells treated with 5 mM tunicamycin for 8 hours (Figure 3A), and the disappearance of endogenous TRB3 was clearly observed in apoptotic cells, although cleaved fragment was not observed due to use of C- terminus recognition antibody. Other antibodies could not also detect the cleaved fragment (Figure S3). Therefore, we next used tunicamycin treatment to examine how TRB3 cleavage affects CASP3/7 activation and subsequent apoptosis under ER stress conditions. D338A-TRB3 expressing cells were resistant to CASP3/7 activation and death (Figure 3B and C). Surprisingly, we also observed anti-apoptotic effect, rather than pro-apoptotic effect, in WT-TRB3 expressing cells, although this anti-apoptotic effect was lower than that in D338A-TRB3 expressing cells (Figure 3B and C). As previously reported [6], a major portion of the tunicamycin-induced cell death also appears to be apoptotic cell death, because cell death was strongly inhibited in the presence of z-VAD-FMK (Figure S4). Unlike in the case of TNFa/CHX- or anti-Fas antibody-induced apoptosis (as shown in Figure 2), WT-TRB3 actually rescued cells from death under ER stress condition. To explain this result, we examined the cleavage of WT-TRB3 during ER stress. We found hardly any cleavage of WT-TRB3 in tunicamycin-treated cells (Figure 3D), although it was clearly seen in TNFa/CHX- and anti-Fas antibody-treated cells (Figure 1D and 3D). Most of the subsequent reduction of WT-TRB3 after tunicamycin treatment may be not resulting consequence by cleavage because TRB3 is rapidly degraded through the ubiquitin-proteasome pathway as previously described [22] and non-cleavable TRB3 (D338A) was also reduced in a similar manner. Moreover, ER stress regulates protein synthesis via the unfolded protein response (UPR). Therefore, the global reduction of protein levels could be caused by ER stress condition itself. Next, to clarify the observed difference in susceptibility to cleavage, we measured the CASP3/7 activity in TNFa/CHX- or tunicamycin-treated cells. The increase in CASP3/7 activity was very slow during the tunicamycin-induced ER stress, and was significantly lower than in cells undergoing apoptosis induced by TNFa/CHX treatment (Figure 3E). This result suggests that the ER stress-induced CASP3/7 activity, at least in this condition, is not so high as to actively cleave TRB3. To assess whether TRB3 itself is required for the modulation of CASP3/7 activation, we used siRNA to knockdown the TRB3 PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42721