Ivity in pWPItransduced cells. However, caspase activity was inhibited in thepresence of a-crystallins, with around 50 and 20 reduction in 661W cells overexpressing aA- and aB-crystallins, respectively (Fig. 6B).The C-terminal extension domain of aA-crystallin was sufficient to prevent Bax-induced apoptosisa-Crystallins are characterized by a conserved a-crystallin domain flanked by a N-terminal domain and a short C-terminal extension [2?]. To identify the domain of aA-crystallin sufficient to protect against Bax-induced apoptosis, we generated Title Loaded From File deletion mutants corresponding to distinct domains of aA-crystallin (Fig. 7A). Fusion proteins were created in which full-length and mutant Title Loaded From File aA-crystallins were fused in frame at the N-terminus of luciferase.a-Crystallin Cytoprotective ActionFigure 4. STS-induced apoptosis in 23115181 661W cells. Dose-dependent induction of apoptosis in 661W cells exposed to increasing amounts of STS (25 to 200 nM) for 24 h, as depicted by (A) increased TUNEL-positive apoptotic cells and (B) decreased level of intracellular ATP content. Data are the mean 6SE of at least four independent experiments, each performed in triplicates. doi:10.1371/journal.pone.0055372.gAs 661W cells are low-efficient transfectable cells, we evaluated the anti-apoptotic properties of aA-crystallin mutants in 293T cells transiently transfected with the different constructs. Ectopic expression of full-length and mutant aA-crystallins was verified by western blot analysis (Fig. 7B). Bands of the expected size were detected for wt and mutant proteins. A 34-kDa band corresponding to luciferase was also observed in cells over-expressing the aAcrystallin proteins, as well as in cells transfected with the empty pRluc plasmid. This may be explained by translational leakiness as the pRluc plasmid contains an internal ATG start codon at the Nterminus of luciferase. As a control, no signal was detected in cells transfected with the pcDNA3.1 plasmid. Immunofluorescence analysis confirmed the cytoplasmic localization of the different mutants, similarly to wt aA-crystallin (Fig. 7C). The anti-apoptotic activity of the different aA-crystallin mutants against Bax-induced apoptosis was then assessed in 293T cells cotransfected with Bax and with wt or mutant aA-crystallins. Twenty-four hours post-transfection, TUNEL assay was performed to detect and count TUNEL-positive apoptotic cells. As shown in Fig. 8, wt aA-crystallin (Bax/aA_wt) inhibited Baxtriggered apoptosis (Bax/pRluc). Moreover, aA_90-143 mutant was as efficient as wt protein to prevent apoptosis, while the Cterminal extension aA_144-173 1662274 mutant significantly displayedbetter protection than the full-length aA-crystallin. However, we can not exclude that it may reflect differences in the levels of expression of the corresponding proteins. In addition, N-terminal aA_1-89 and aA_1-116 mutants, along with aA_64-143 mutant containing the a-crystallin domain, did not protect against Baxinduced apoptosis. We further investigated whether the C-terminal extension domain of aA-crystallin retained its capacity to bind Bax in vivo. The interaction of full-length aA-crystallin or aA_144-173 mutant with Bax was assessed by co-immunoprecipitation in 293T cells over-expressing the aA-crystallin constructs and treated for 3 h with 100 nM STS. As observed for full-length aA-crystallin (aA_wt), the C-terminal extension domain (aA_144-173) was sufficient to bind Bax, whereas no immunoprecipitated proteins were observed in.Ivity in pWPItransduced cells. However, caspase activity was inhibited in thepresence of a-crystallins, with around 50 and 20 reduction in 661W cells overexpressing aA- and aB-crystallins, respectively (Fig. 6B).The C-terminal extension domain of aA-crystallin was sufficient to prevent Bax-induced apoptosisa-Crystallins are characterized by a conserved a-crystallin domain flanked by a N-terminal domain and a short C-terminal extension [2?]. To identify the domain of aA-crystallin sufficient to protect against Bax-induced apoptosis, we generated deletion mutants corresponding to distinct domains of aA-crystallin (Fig. 7A). Fusion proteins were created in which full-length and mutant aA-crystallins were fused in frame at the N-terminus of luciferase.a-Crystallin Cytoprotective ActionFigure 4. STS-induced apoptosis in 23115181 661W cells. Dose-dependent induction of apoptosis in 661W cells exposed to increasing amounts of STS (25 to 200 nM) for 24 h, as depicted by (A) increased TUNEL-positive apoptotic cells and (B) decreased level of intracellular ATP content. Data are the mean 6SE of at least four independent experiments, each performed in triplicates. doi:10.1371/journal.pone.0055372.gAs 661W cells are low-efficient transfectable cells, we evaluated the anti-apoptotic properties of aA-crystallin mutants in 293T cells transiently transfected with the different constructs. Ectopic expression of full-length and mutant aA-crystallins was verified by western blot analysis (Fig. 7B). Bands of the expected size were detected for wt and mutant proteins. A 34-kDa band corresponding to luciferase was also observed in cells over-expressing the aAcrystallin proteins, as well as in cells transfected with the empty pRluc plasmid. This may be explained by translational leakiness as the pRluc plasmid contains an internal ATG start codon at the Nterminus of luciferase. As a control, no signal was detected in cells transfected with the pcDNA3.1 plasmid. Immunofluorescence analysis confirmed the cytoplasmic localization of the different mutants, similarly to wt aA-crystallin (Fig. 7C). The anti-apoptotic activity of the different aA-crystallin mutants against Bax-induced apoptosis was then assessed in 293T cells cotransfected with Bax and with wt or mutant aA-crystallins. Twenty-four hours post-transfection, TUNEL assay was performed to detect and count TUNEL-positive apoptotic cells. As shown in Fig. 8, wt aA-crystallin (Bax/aA_wt) inhibited Baxtriggered apoptosis (Bax/pRluc). Moreover, aA_90-143 mutant was as efficient as wt protein to prevent apoptosis, while the Cterminal extension aA_144-173 1662274 mutant significantly displayedbetter protection than the full-length aA-crystallin. However, we can not exclude that it may reflect differences in the levels of expression of the corresponding proteins. In addition, N-terminal aA_1-89 and aA_1-116 mutants, along with aA_64-143 mutant containing the a-crystallin domain, did not protect against Baxinduced apoptosis. We further investigated whether the C-terminal extension domain of aA-crystallin retained its capacity to bind Bax in vivo. The interaction of full-length aA-crystallin or aA_144-173 mutant with Bax was assessed by co-immunoprecipitation in 293T cells over-expressing the aA-crystallin constructs and treated for 3 h with 100 nM STS. As observed for full-length aA-crystallin (aA_wt), the C-terminal extension domain (aA_144-173) was sufficient to bind Bax, whereas no immunoprecipitated proteins were observed in.
