Detection Of Interacting Proteins Of LIM Protein FHL1C

At present, the rate of morbidity and mortality of tumor patients is beingfound in more and more increasing trend. Statistics data shows that cancer hasjumped to the first place causing death among Chinese urban residents. Therefore,the prevention and treatment of malignant tumors became the focus of our study.Because of its biological characteristics, such as rapidly growth and metastaticspreading to other parts of the body, it is not only the cause of death in patients,also, the figures mentioned above would be the biggest obstacle in cancertreatment.Then, the research on this field will mainly focused on thedevelopmental mechanisms in order to hunting for a breakthrough for thetreatment of cancer.EMT is an important participant in tumor metastasis and migration as newconnections between the cell and tumor. In the EMT process, there occur a dysfunction of tight junctions between cell-cell adhesion molecules includinglosing of cell polarity and increasing cell migration and invasion ability. Thereare multiple signaling pathways involved, in which Notch signaling is one of themajor signaling pathways inducing EMT. But the exact mechanism has not beenfully elucidated. Notch/RBP-Jk signaling pathway is a highly conservedpathway, which closely related to a variety of physiological and pathologicalprocesses through interactions between adjacent cells. The results of studies showthat activation of Notch signaling pathway can not only promote the occurrenceof the EMT process, but also down regulation the Notch signaling pathwayleading to the opposite process through inhibition of the cell migration andinvasion. But its exact mechanism is still unclear. RBP-J is the major downstreameffector of Notch signaling pathway, and activation of Notch signaling pathway isdependent on RBP-J-mediated transcriptional activation of downstream genes. Itfound that LIM domain protein KyoT2can interact with RBP-J complexformation, competitivly inhibit the transcription of the downstream genes ofNotch pathway.FHL1C, homologue of KyoT2in human and a subtype of FHL1C, belongs tothe LIM protein family. It contains two and a half LIM domain of the N-terminaland carboxy-terminal of RBP-Jk binding sites.Protein containing the LIM domainhas an important role not only in different cellular processes, such as cytoskeletalorganization, signal transduction, gene expression and cell differentiation, butalso as a protein interaction motif. This interaction can display different functionsin the maintenance of cell structure and signaling pathway. Our research groupfound that human tight junction protein ZO-2can interact with KyoT2by yeasttwo-hybrid assay in former. We want to verified their interaction in vivo bymammalian two-hybrid assay. Then, we used co-immunoprecipitation experiments to pull down the protein of ZO-1with FHL1C as target protein,ZO-1and ZO-2, two proteins within the same family, plays an important role inconnection between the epithelial and endothelial cells. However during the EMT,the function of ZO-1and ZO-2are unclear. Based on the above study, wehypothesized that ZO-1may be involved in the regulation of Notch signaling bythe LIM domain protein FHL1C mediated EMT process. Based on this idea,up tonow, we conducted the following experiment.Major findings:1.The full-length gene of the mouse tight junction protein ZO-2open readingframe were acquired, and the mammalian two-hybrid system eukaryoticexpression vector of pCMX-VP16-F1R1, pCMX-VP16-F2R2,pCMX-VP16F3R3and pCMX-VP16-ZO were constructed.2. In order to confirm the interaction between KyoT2and tight junction proteinZO-2in vivo and to determine their sites of action, we verified by mammaliantwo-hybrid assay in Cos7cell lines. Analysis shows that they failed to activatethe activity of downstream reporter gene, KyoT2and ZO-2may not have theinteraction in vivo.Analysis of experimental results, the reasons may be:1. Yeast two-hybridtests have some false positive results.2. The interaction between the proteinverification by GST-pull-down does not necessarily have a role in vivo.LIMdomain protein is an interaction interface, we assume that KyoT2through itsRBP-J binding sites for competitive inhibition of the NIC transcriptionalactivation may also raise other transcriptional regulatory elements to the RBP-Jthrough its LIM domain. Therefore, we want to find a new protein interactingwith FHL1C in order to elucidate the mechanism of blocking the Notch signalingpathway more clearly and for design optimization of a new type Notch signaling blocker. We next conducted the following experiment:The main research results1. The vector pCMV-myc-FHL1C was transfected into293T48hours,andthe protein of FHL1C was successfully expressed testing by Western-blot.2. By co-immunoprecipitation and silver staining we found that in theFHL1C and RBP-J co-transfected group new proteins ZO-1were successfullypull down testing by mass spectrometry.Based on the above analysis, ZO-1and ZO-2belong to the same family oftight junction proteins, they combined with each other and co-located within thetight junctions at the plasma membrane of epithelial cells. Studies have shownthat ZO-1which no longer localize at the plasma membrane induced a dramaticepithelial tomesenchymal transition (EMT) of Madin-Darby caninekidney1(MDCK1) cells. It will be significant and creative for us to discover theirrelationships and mechanisms, and leading to a firmly foundaton for furtherresearch.