The Fhc, Of ​​bim Interaction In Apoptosis Role And ¦Õc31 Of Integration Of The Enzyme Structure

There are two parts in this thesis. One of them is on research of interaction between FHC and Bim and the anti-apoptosis function of FHC, the other is about some basic research on the structure ofΦC31 integrase.One important strategy of tumor gene therapy is to induce apoptosis in tumor cells. Members of bcl-2 family are good targets. The proapoptotic gene bim has more than ten splicing variants. Dr. Chen JZ has identified two novel variants of bim. We figured out several proteins by two-hybrid screening with Bim L, among which we are especially interested in FHC (Ferritin Heavy Chain). FHC polymers can bind Fe iron and contribute to the homeostasis in cells, and its expression is transcriptional regulated by the Fe level in cell.FHC has been found involved in the protection mechanism against cytotoxity of many stimuli and involved in the anti-apoptotic function of malignant transformation of normal cells. For example, in asbestos induced malignant transformation in human Mesothelial cell and in TNF-αinduced apoptosis, FHC is upregulated; low temperature can also induce FHC expression in rainbow trout cell. It's generally believed that FHC is one of the protection mechanisms of body to antagonize ROS. Recent study of NF-κB mediated protection against TNF-αinduced apoptosis indicated that ROS is the upstream event of FHC. In T-cell, ROS seems also to be the key mediator of NID and Bim EL, Bim L, caspase activity and apoptosis. It's obvious that both FHC and Bim are involved in pro-apoptotic signals induced ROS and subsequent apoptosis, but the relationship of FHC and Bim in those processes is still unkown.We constructed eukoryotic plasmids pEGF-Bim L, pcDNA-Bim L pCMV-MYC-fhc,pCMV-HA-fhc,pDsRed2-N1-fhc. To verify the interaction between FHC and Bim, We cotransfected 293T cell with pEGF-Bim L and pDsRed2-Nl-fhc,and found Bim L and Fhc were collocated in the cytoplasml of cells.Besides, co-IP assay verified that Bim L and FHC interacted in AD293 co-transfected with pEGF-Bim L and pCMV-MYC-fhc.Another co-IP was conducted in pCMV-MYC-fhc tranfected MCF-7 cells,in which Bim L is expressed at physiological level. Once again, the interaction between Bim L and FHC was confirmed. We also conducted experiments of yeast mating test. The results showed that the interaction domain on Bims was located to BH3 domain. And all isoforms with BH3 domain interact with FHC. Co-transfection HEK293 with FHC and Bim expressed plasmid protect the cells from cytotoxity of Bim. FHC also protection HEK293 cells from hydrogen peroxide treatment. We used the oxidants H2O2/diamide to treat cells. Many studies have reported that H2O2 introduces ROS and cytotoxity in many types of cells.We investigated the effects of FHC overexpression and knockdown by siRNA in H2O2 treated MCF-7 cells and found that FHC showed protection against the cytotoxity of oxidants.We also checked the effect of FHC overpression on Bim L overexpression in 293T. FHC exerted protective function as well.Previous research indicated that Bim L binds with microtubule protein DLC8 in normal cells. After cells are stabled by taxol and then lysed by Triton X-100, up to 80-90% Bim L are located in the insoluble frcation of treated cell lysis, asα-tubulin is. But for cells undergoing apoptosis induced by UV/TNF-a, all Bim L dropped with LC8 and appeared in soluble fraction in 4-6 hours. In sucrose gradient fraction, Bim L and DLC8 migrated from high density part to low density part during the process of apoptosis. So, we also tried to monitor FHC. Bim L, LC-8 andα-tubulin's migration that might be shown by sucrose gradient fraction in different situation: normal MCF-7 cells, H2O2 treated cells and FHC's protection from H2O2 induced cytoxity. But because of technical reasons.we didn't aquire convincing information about that till now.Our work identified a novel Bim interacting protein FHC and suggested that FHC play a role in Bim mediated apoptosis and oxidative stress. FHC is the first cellular protein interacting with Bim BH3 domain and presents anti-apoptotic activity.Insertion of gene transfer vectors into the human genome results in long-term gene expression usually, but uncontrolled insertion is raising significant safty concerns for their clinical use. Streptomyces phageΦC31 integrase is able to mediate site-specific integration between exogenous DNA with wild att sequences and pseu-att sites on mammalian cellular genome. This integrase system has great potential to be a tool in gene manipulation and gene therapy.A non-viral protein transduction technique was developed by Dr. Zhang MX. In that method, purifiedΦC31 integrase protein could get into mammalian cells in a dose-dependent manner and could mediate recombination successfully in mammalian cells according to our quantative assay. But we are always trying to further improve the specificity and efficiency ofΦC31 integrase mediated site-specif recombination. We made some improvement in the concentration conditions of the protein and explored various conditions to try to grow cystal of the integrase. We failed to get a crystal of full lengthΦC31 integrase protein with a proper size enough for X-ray analysis. In fact. till now, there is litter knowledge about its structure except that its short N-terminal is the catalytic domain and the left long C-terminal is thought to function in DNA recognization and binding. But the long C-terminal is still too long to be a fitful candidate (less than 30KD) to grow its cystal alone or complexed with its DNA substrate, attB or attP sequence.With little homologue to that of other large-serine integrase members, bioinfomatic analysis didn't give us useful information to decide the fragment either.We partially digested C31 integrase with trypsin at low concentration and found there was a regular digestation pattern. So we cut the fragment specific bands from PVDF blot and tried to decide the molecule weight and then the specific digesting site of the bands. But we can't get enough amount of protein from a single peak from HPLC analysis.So we can't do the planned mass spectrum analysis or Edman degradation to find out the specific digest site and then to decide the proper fragment.