GRIM-19 Interaction With The E6AP

[Background] Cervical cancer is one of the most common malignancy, the incidence rate is second in women malignancies . Every year about 20 million women die from the disease.Cause of cervical cancer has not yet fully understood, is a results of multi-factor, multi-step development.There are two main factors: First, the high-risk HPV continued infection; second, tumor suppressor gene p53 degradation and inactivation. When HPV infection in host cells, host DNA can be encoded through the integration of early cancer protein E6. E6 protein has not been found in enzyme activity, mainly through protein-protein interactions play an oncogenic function. Studies have shown that , E6 in the pathogenesis of cervical cancer, E6AP almost mediates all the features of E6. First ,E6 and E6AP complex to form E6/E6AP ,then combined with p53, E6AP ubiquitin will be transferred to the activation of p53 protein, p53 is ubiquitin degradation pathway to enter the final in the role of 26S proteasome degradation. Therefore, E6AP ubiquitin degradation of p53 function plays an important role in the incidence of cervical cancer development.E6AP (E6-associated protien) gene, also known as UBE3A (ubiquitin protein ligase E3A), located on chromosome 15q11-q13, a HECT E3 (E3 homology to the E6AP carboxy Terminus E3) protein family members. According to the different N-, E6-AP is divided into three transcripts,â… -type protein encoded 98.9 kDa;â…¡type codes 101.7 kDa;â…¢code 101.1 kDa, all three subtypes have the function of protein degradation. E6AP has complex functions, the study found, under stress, E6AP expression levels were significantly increased, and combine with the molecular chaperone Hsp70/Hsc70 , it can inhibit stress-induced apoptosis. In the course of cervical cancer, E6AP is the most important role in the degradation of tumor suppressor protein p53. E6 and E6AP complex to form E6/E6AP, combined with p53, E6AP ubiquitin will be transferred to the activation of p53 protein, causing degradation of p53 ubiquitination. The combination of E6AP and E6 another important role is to increase the expression of telomerase hTERT promoter, the mechanism has nothing to do with the degradation of p53. The first E6 combined with E6AP , the activation of the c-Myc promoter, the transcription of c-Myc promote further activation of hTERT promoter, hTERT promoter to promote and enhance the expression of hTERT telomerase activity by inducing acetylation of histone H3. This fully shows that E6AP can promote the development of cervical cancer.GRIMs (genes associated with retinoid-IFN-inducedmortality) is a recently discovered group of participating interferon (interferon, IFN) and retinoic acid (retinoic acid, RA) combination induced apoptosis gene. GRIM-19 is GRIMs family members, it involved in cell respiration and mitochondrial regulation of apoptosis in a novel gene, located in 19 p13.2, the region of multiple genes for prostate cancer suppressor gene.Encoding144 amino acid residues containing the protein, the molecular weight of about 16kD.It expressed in many normal human tissues, mainly located in mitochondria, the nucleus has a small amount of expression. GRIM-19 expression in normal cells to maintain normal life cycle, so that cells from old to enter apoptosis, while low expression of certain cells can not enter the cell cycle normally, apoptosis, giving rise to tumors. Such as thyroid cancer, renal cancer, oral cancer, colorectal cancer, such as in the expression of GRIM-19 were found to decrease. STAT3 is a new target for cancer therapy .It is a sustained activation state in many tumors. Current studies suggest that GRIM-19 is the expression and activation of STAT3 negative regulator, GRIM-19 and its binding, inhibit the activation of downstream gene transcription induced by this binding of GRIM-19 in turn activate the serine 727 site completed. The study also found that the HPV E6 protein of cervical cancer with GRI M-19 directly binds, and the combination of high-risk HPV was stronger than low-risk type of HPV, however, GRIM-19 in cervical cancer cells in the specific mechanism of action still unclear.In previous studies our group found that GRIM-19 expression was significantly reduced in cervical cancer tissue . Also found that, GRIM-19 can promote apoptosis by in vivo experiments; Further study also found that HPV-positive cervical cancer cell lines HeLa, SiHa and CaSki cells were found GRIM-19 can raised the expression of p53, while HPV-negative cells in ovarian cancer cell line H08910 was not found the phenomenon.Inference, GRIM-19 can induce cervical cancer cell lines p53 protein accumulation, which is due to play the role of inhibition of the p53 protein degradation and prolong itshalf-life to achieve. P53was mainly due to the degradation of E6AP ubiquitination degradation in cervical cancer cells . Studies have confirmed, GRIM-19 can be a direct binding of many cytokines and protein molecules. Therefore, we speculate, GRIM-19 may occur with the E6AP binding, thus inhibiting the degradation of E6AP ubiquitination function of p53. In this experiment, on the basis of previous experiments,we will further study whether the GRIM-19 and E6AP has protein binding.[Objective]Discussion whether the GRIM-19 and E6AP has the protein binding.Further elaborated GRIM-19 mechanisms in the tumor suppressor and pathogenesis of cervical cancer and Provide theoretical and experimental basis for the treatment of clinicalapplications.[Methods]â‘ RT-PCR analysis E6AP three transcripts in normal cervical tissue and cervical tissue expression;â‘¡Immunoprecipitation (CO-IP)detection of GRIM-19 and the E6AP protein in vivo binding;â‘¢E 6AP -â…¢the truncated body of prokaryotic expression plasmid: pET-22b (+)-his-E6AP-â…¢;â‘£The expression of Fusion protein pET-22b (+)-his-E6AP-â…¢truncated body in E. Coli. [Results]â‘ R T-PCR showed that E6AP exist in three subtypes in cervical tissues , and was higher than normal cervical tissue, which is very low expression levels of typeâ…¡, mainly in the typeâ… andâ…¢;â‘¡Immunoprecipitation (CO-IP) displayed in the cervical cancer cell line Hela, Siha,ovarian cancer cells with H08910 E6AP in GRIM-19 with protein binding;â‘¢E6AP-â…¢the truncated body of the prokaryotic expression vector wassuccessfully constructed;â‘£Fusion protein E6AP-â…¢the truncated body induce the expression successfully.[Conclusion]Successfully constructed the E6AP-â…¢of the truncated body of prokaryotic expression plasmid of fusion protein pET-22b (+)-his-E6AP-â…¢of the truncated body of inducedsuccessful, for further study whether GRIM-19 and E6AP has the direct binding in vitro and the role of specific mechanisms.Provide an experimental basis.