The Mechanism Of Anti-apoptotic Protein C-FLIP Degradation By Ubiquitination

ObjectiveRecently, Cancer has been one of the major problems that threaten human’s health as the morbidity and the mortality of cancer increased quickly year by year all around the world.Nowadays radiation therapy, surgery, and chemotherapy are the main therapies for tumor treatment. Chemotherapy has been regarded as the most promising therapy, but the strong side effects and the low specificities of current anti-tumor drugs have been limitations of the development of chemotherapy. Therefore, as the main concern in the process of conquering cancer and a hotspot in scientific fields, searching for new drugs for cancer treatment and putting them into development become more and more important. As one key anti-apoptotic protein of death receptor signaling pathway, c-FLIP has been a promising target for cancer treatment.The over expression of apoptosis inhibition proteins and the tolerance of apoptosis in-tumor cells are two symbols of tumorigenesis and development. Recent studies have shown that c-FLIP is an important kind of apoptosis inhibite protein. In many different types of tumor cells c-FLIP over expression have been found. c-FLIP excessive expression suppress the combination of death ligands such as FasL with TRAIL and DISC and inhibition the activation of caspase-8to preventing tumor cell apoptosis. The down-regulation of c-FLIP can sensitizes the apoptosis of tumor cells. Studies show that small molecule drugs promote degradation of c-FLIP mainly through ubiquitin-proteasome pathway to induce the apoptosis of tumor cells. The specific identification of substrate by E3ubiquitin ligase can accurately regulate the degradation of substrate protein. Therefore, the identification of E3ubiquitin ligase of apoptosis inhibiting protein c-FLIP plays an important role in determining the new tumor therapeutic targets, understanding the regulatory mechanism of tumor cell apoptosis signaling pathway and the mechanism for search new anticancer drugs.Methods1. Cell culture.2. Co-Immunoprecipitation to detect the protein interactions3. The changes of proteins were examined by Western Blot.4. Search the protein subcellular location by the fluorescent microscope5. Genes over expression by Plasmid transfection6. Knockdown of target gene was achieved by transfection with specific siRNA.Results1. Cisplatin was used to treat the cancer cell line, and then we detected elevated protein level of BTBD6and the down regulation of c-FLIP protein by western blot.2. We detected BTBD6interacting with c-FLIP by Co-Immunoprecipitation. We detected their co-localization in the cytoplasm by fluorescence microscope.3. BTBD6promotes the degradation of c-FLIP. The over expression of BTBD6can promote the reduction of c-FLIP. To detect the role of BTBD6in induced degradation of c-FLIP we knock down it by siRNA. We search when the protein level of BTBD6drop down, the protein degradation of c-FLIP is restrained.4. BTBD6and c-FLIP are piecewise cloned. We found the interaction of those proteins by different domains. The BTB domain of BTBD6interacts with the Caspase-like domain of c-FLIP. BTBD6promote c-FLIP ubiquitination and degradation. BTBD6also promotes the ubiquitination and degradation of the c-FLIP cleavage.5. We detected the protein interaction of BTBD6and Cullin3with c-FLIP.Conclusions 1. BTBD6promotes cell apoptosis by ubiquitinaiton and degradation of c-FLIP.2. The BTB domain of BTBD6interact with the Caspase-like domain of c-FLIP.3. BTBD6can promote the degradation of c-FLIP and the ubiquitinaiton and degradation of the c-FLIP cleavages.4. BTBD6and c-FLIP co-localized in the cytoplasm. This co-localization inhibits the ubiquitinated proteins aggregation.5. Cullin3combined with BTBD6and promotes the degradation of c-FLIP and cell apoptosis.In summary, our study demonstrated that the degradation of c-FLIP is induced by its specific E3ligase BTBD6through ubiquitin-proteasome proteolytic pathway. We put forward a new hypothsis of how does the Cullin3promote apoptosis. The molecular mechanism is that:Cullin3promotes intracellular Caspase8ubiquitination to promote its cleavage activity while Cullin3interacts with BTBD6to promote the degradation of the anti-apoptotic protein c-FLIP and to suppress the aggregation of ubiquitinated c-FLIP to promote apoptosis mediated by caspase cascade.