Study On The Regulation Of Apoptosis By Rap2b Via Its Interaction With A Mitochondrial Protein

A mitochondrion is an important energy-producing organelle in eukaryotic cells,which is involved in many cell physiological activities,such as cell proliferation,metabolism,differentiation,signal transduction,apoptosis,and so on.Apoptosis is closely related to the development of an embryo,the maintenance of dynamic balance of the cell number in vivo,and the occurrence and development of important human diseases,such as tumors.Generally,the mitochondrial pathway of apoptosis can be divided into three successive stages,and the mitochondria play an important role in the central regulatory stage by releasing cytochrorne C into the cytosol.It has been reported that Bax and Bak,two members of the Bcl-2 apoptotic protein family,are involoved in the process of apoptosis by localization in the mitochondrial membrane.Therefore,the study of mitochondrion-related proteins is of great significance for understanding the molecular mechanism of apoptosis and tumorigenesis.Rap2b is a target gene of p53 and belongs to the Ras oncogene superfamily.Our previous study found that downregulation of Rap2b expression makes tumor cells highly sensitive to DNA damage and prone to apoptosis.We speculate that Rap2b may participate in apoptosis by interaction with proteins on the mitochondrial membrane.In this study,we first constructed two recombinant lentiviral plasmids derived from pLKO.1-TetOn to package lentiviruses for controllable downregulation of Rap2b expression in HCT116 colorectal cancer cells.The mRNA and protein levels of Rap2b and pro-apoptotic genes such as Bax were examined by quantitative real-time PCR and immunoblotting,respectively.The results showed that there was no significant change in the total protein levels of Bax and PUMA after down-regulation of Rap2b,but the level of Bax protein in the mitochondria increased significantly.Next,we studied the subcellular localization of Rap2b in cells.We detected the isolated cytoplasm and mitochondria by immunoblotting and found that Rap2b mainly existed in the mitochondria.Immunofluorescence staining assay further demonstrated that Rap2b was localized in the mitochondria.Then,we performed GST pulldown and silver staining-mass spectrometry assays to look for mitochondrial proteins interacting with Rap2b.The results showed that Rap2b might interact with HSPA9 which is specifically localized in the mitochondia.Finally,we validated the interaction between Rap2b and HSPA9 in vivo and in vitro.The HSPA9 and Rap2b genes were cloned into the prokaryotic expression vectors pET-32a and pGEX-5X-1 and two proteins His-TrxA-HSPA9 and GST-Rap2b were expressed in Escherichia coli BL21(DE3),followed by purification using affinity chromatography.These two purified fusion proteins were used for GST pulldown.The results showed that Rap2b could bind to HSPA9 directly in vitro.In the meanwhile,the Rap2b and HSPA9 genes were also cloned into the plasmids pBiFC-mCherryN159 and pBiFC-mCherryC160,respectively.Subsequently,these two recombinant plasmids were cotransfected into HCT116 cells and the fluorescence images were visualized under a confocal laser scanning microscope.The data indicated that Rap2b interacts directly with HSPA9 in vivo.In conclusion,Rap2b is localized in the mitochondria through interaction with the mitochondrion-specific HSPA9 protein and involved in the process of apoptosis by downregulation of the level of Bax protein in the mitochondria.These findings were of great importance for a better understanding of the biological role of Rap2b and the molecular mechanism involved.