title: Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori.
abstract: Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Yan-Fen
authors.initials: YF
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Hua-Shan
authors.initials: HS
authors.lastname: Yi
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Song
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: La
authors.initials: L
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China. Electronic address: pmh0476@hotmail.com.
authors.forename: Min-Hui
authors.initials: MH
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China. Electronic address: lucheng@swu.edu.cn.
authors.forename: Cheng
authors.initials: C
authors.lastname: Lu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D012831:Bombyx / Q000378:metabolism*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D010539:Permeability; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D019476:Insect Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D045304:Cytochromes c; C028098:10-hydroxycamptothecin; D053148:Caspase 3; D002166:Camptothecin
keywords: Bcl-2 family; Bmbuffy; Bmp53; HCPT; Mitochondrial apoptotic pathway