Role Of BmBuffy In Intrinsic Mitochondrial Apoptotic Pathway And Autophagy In Silkworm Cells, Bombyx Mori

Apoptosis is a highly conservative physiological death process regulated by genes, it play key roles in keeping stability in internal environment of organism and defending the harmful factors outside. Apoptosis plays important roles in growth, development and maintaining normal physiological functions. Some progresses have been made on researches of apoptotic regulating mechanisms in insect cells, but the researching scope are very narrow and restricted, needing to be constantly explored and investigated. As the representative of Lepidoptera and one kind of important economic insect, the silkworm, Bombyx mori, has increasing research and application value. Silkworm is an insect which undergoes complete metamorphosis. Apoptosis plays vital roles in the growth and development processes of metamorphosis and molting, and the apoptotic regular control is the basis for the normal mechanism of development. However, researches on the apoptotic mechanisms of silkworm are still in the initial stage of exploration, therefore, how to carry on a further research on investigating and demonstrating the elementary apoptotic mechanisms in silkworm is an urgent problem to be solved at this stage.Apoptotic approaches include three passage ways:mitochondrial pathway, ER-stress pathway, and death-receptor pathway. The three pathways can mediate apoptosis via the interrelations, in which the role of mitochondrial pathway is determinant. Study on mitochondrial pathway is the key for the revelation of apoptotic mechanisms. Bcl-2family members play important roles in mitochondrial apoptotic pathway, they interact with each other or other proteins to mediate mitochondrial apoptosis together. In silkworm, Bmbuffy has been identified as a key homologue of Bcl-2families in silkworm, however, its exact function is unknown. Therefore, we investigate the role of Bmbuffy in mitochondrial apoptotic pathway.As the type II programmed cell death, autophagy plays key roles in the growth and development process of living beings, cleaning waste up to complete metabolic turnover, defending outer environmental stress and the infections of harmful microorganisms, and keeping the stability of cell internal environment. Autophagy has become a hot spot in recent years. Autophagy and apoptosis are either mutually exclusive or interconnected. On one hand, autophagy occurs before apoptosis, and then transforms into apoptosis. On the other hand, autophagy inhibits apoptosis to keep cell living. Kinds of molecules participate in the process of autophagy, including the involvement of Bcl-2families. Investigating the role of Bmbuffy in the process of autophagy in silkworm is one important part of this article. The results of the paper are concluded as below:1. Research on hydroxycamptothecine (HCPT)-induced apoptosis in BmN-SWUlcells via mitochondrial apoptotic pathwayTreatment with HCPT in BmN-SWU1cells leads to the apoptotic features of cell nucleus, DNA fragmentation, decrease of cell viability, and the increase of cell apoptotic rate, showing that HCPT can induce apoptosis in BmN-SWU1cells. Then we investigated the mitochondrial apoptotic pathway initiated by HCPT in BmN-SWU1cells. By detecting the change of mitochondria distribution, mitochondrial membrane potential(MMP), and the cytochrome c release, we found that the even and concentrated distribution of mitochondria became diffused and disordered, MMP and cytochrome c release reduced. Treatment of CsA and Bip-V5in cells leads to the inhibition of cytochrome c release and apoptosis, indicating that the release of cytochrome c is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the promotion of mitochondrial outer membrane permeability (MOMP). By detecting Caspase activities, we found that the activities of Caspase-9and-3increased in HCPT-treated cells, whereas FSB A, an Apaf-1inhibitor, was able to inhibit Caspase-3activity, showing that the activation of Pro-Caspase-3depends on the formation of apoptosome. In conclusion:HCPT induces apoptosis in BmN-SWU1cells via intrinsic mitochondrial pathway.2. Research on the role of Bmbuffy in the endogenous apoptotic pathway of silkworm cellsResearch on the localization of Bmbuffy in silkworm cells indicates that it is located on the outer-membrane of mitochondria and ER, whereas Bmbuffy is randomly distributed within the cytoplasm and nucleus in the absence of the hydrophobic transmembrane domain at COOH terminus, showing that it is capable of anchoring Bmbuffy to the MOM and ER. Moreover, overexpression of Bmbuffy in HCPT-treated cells inhibits the release of cytochrome c from the mitochondria, the activation of Caspase-3, and cell apoptotic rate, indicating an anti-apoptotic role for Bmbuffy in the mitochondrial apoptotic pathway. In the absence of the hydrophobic membrane anchor, Bmbuffy has no effect on HCPT-induced apoptosis, showing that mitochondrial localization is required. In HCPT-treated cells, Bmp53gradually translocates from nucleus to mitochondria and interacts with Bmbuffy to inhibit its anti-apoptotic function, and then promotes apoptotic process. In addition, overexpression of Bmp53increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these, indicating the antagonistic relation between Bmbuffy and Bmp53.3. Research on the role of Bmbuffy in cell autophagy of silkwormAfter incubation with insect balanced salt solution(IBSS) for a while in BmN-SWU1cells, autophagy occurred which was detected by MDA staining, showing that starvation induced autophagy in IBSS-treated silkworm cells. Overexpression of Bmbuffy inhibits autophagy induced by IBSS, indicating that Bmbuffy can inhibit autophagy in silkworm cells. Study on the correlation of Bmbuffy and BmAtg6demonstrates that Bmbuffy interacts with BmAtg6in normal cells, whereas it dissociates away from BmAtg6. The results of site directed mutagenesis proves that Bmbuffy interacts with BmAtg6via the BH3domain and the amino acid located on the145site plays a key role for the interaction. PIZ-Bmbuffy-ActA-TM-dsRed and PIZ-Bmbuffy-cb5-TM-dsRed vectors were constructed to anchor Bmbuffy to the outer membrane of mitochondria and ER respectively. The results shows that mitochondrial Bmbuffy has anti-autophagic function, inferring that Bmbuffy interacts with BmAtg6on the outer membrane of ER.In conclusion, Bmbuffy plays important roles in the intrinsic mitochondrial apoptotic pathway and autophay, showing that it is a key gene to regulate apoptosis and autophay in silkworm cells. Therefore, research on the role of Bmbuffy contributes to know the interrelations between autophagy and apoptosis in silkworm cells, which provides theoretical basis for the further related researches, and offers theoretical references for studies on the regulatory mechanisms of growth and development in Lepidoptera insects.