Study On Pro-apoptotic Of Bax Mediated By Photosensitive Protein CRY2

Bax is a pro-apoptotic protein in the Bcl-2 protein family,and the formation and the redistribution of its dimers plays an important role in mitochondrial apoptosis pathways.Cryptochrome2(CRY2)is a blue light receptor in Arabidopsis plants(Arabidopsis thaliana),and its N-terminal photolyase homolog region(PHR)responds to blue light in vitro,which prompting CRY2 to produce dimers or polymers.This article constructed and obtained fusion protein CRY-Bax according to the characteristics of CRY2 aggregation to study the phenomena of Bax polymerization and pro-apoptotic mediated by photo-sensitive protein CRY2 stimulated by blue light.Construct CRY-Bax、Bax and CRY2PHR eukaryotic expression plasmids andtransientlytransfect into HEK293T/Hela cells,thensemi-quantitative RT-PCR and western blot were used to determine the expression,which resultsindicated that CRY-Bax、Baxand CRY2PHR well expressed in HEK293T/Hela cells,and the level of expression were significantly up-regulated in the groups transiently transfected with eukaryotic expression plasmid compared with that in un-transfected groups.The results of Hoechst 33258 staining showed that,after the stimulation of blue light,the apoptosis ratio was increased in the group transfected with CRY-Bax expression plasmid;and the results of Flow cytometry were the same as those of Hoechst 33258 staining,and found that the total apoptosis ratio was positively correlated with the time of stimulation of blue light(Normal cell group,CRY2PHR and Bax plasmid transfected groups work as control groups).Western blot revealed significant increased on expression levels of the cleaved PARP and cleaved caspase-3 in cells overexpressed CRY-Bax.In order to further determine how develops about apoptosis induced by CRY-Bax under the stimulation blue light,we employed Western blot after Native-PAGE(DSS cross-linking)and SDS-PAGEElectrophoresis,which indicated that blue light could induce recombinant protein CRY-Bax to form dimers;Western blot analysis showed that the distribution of CRY-Bax in mitochondrial was increased after blue light induction after separating cytosolic and mitochondrial proteins.In conclusion,this research successfully obtained the fusion protein CRY-Bax,within eukaryotic cells,after the stimulation of the blue light,CRY-Bax aggregates and transfers to mitochondrial and then initiates the caspase cascade reactions of apoptosis,ultimately induce apoptosis cells apoptosis,and the ratio of apoptosis positively correlated with the time of stimulation of blue light.The study of CRY2 mediated Bax polymerization and pro-apoptotic phenomena in this paper shows that light is hopefuil to be introduced into the field of tumor therapy,which uses light to regulate protein structures and further change protein functions,which has a certain development prospect in medical science research.