Study Of Structure And Function Of FHL3

Angiogenesis is the key step of many physiological and pathological phenomena of the organism, such as embryogenesis, wound healing and cancer,which is a complicated process to be completed through the cooperation of cytokines of angiogenesis promotion or inhibition. Angiogenin(Ang) is an important factor that can initiate and promote the angiogenesis in tumor tissue effectively, and then accelerate the development and metastasis of tumor. Previous researches showed that Ang interacts with the LIM protein FHL3. Four and a half LIM domain protein 3(FHL3) is a member of the superfamily of LIM proteins and the LIM domain represents a cysteine-rich double zinc finger motif denoted by the sequence (CX2CX17–19HX2C)X2(CX2CX16–20CX2(H/D/C)). FHL3 plays an important role in myoblast differentiation, myogenesis and the actin stress fiber remodeling. Recent years, other functions of FHL3 were found and attached importance, such as the interaction with cell division cyclin 25B phosphatase. Our research set the interaction between Ang and FHL3 as the starting point and used the yeast two and three-hybrid systems and other assays to uncover the structural and functional characteristics in angiogenesis pathway of FHL3.In the study, the full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression plasmid pBV220. FHL3 was highly expressed in a prokaryotic system (E.coli JM109). The expressed products exist mainly in the form of inclusion body and were purified by the HisTrap FF chromatography, then the recombinant human FHL3 with high purity was obtained and recombinant protein was renatured. Then, the rhFHL3 was identified by Western blot analysis with polyclonal antibody of FHL3. The proliferation effect of purified protein was analyzed and the apoptosis effect was analyzed when the purified protein was added to HepG2 cells, which is a cell line of liver cancer cells. The mechanism needs further studies to be explained clearly.We screened the human fetal liver cDNA library using the bait vector pAS2-1-FHL3d4, and gained a positive protein of 68% AA homology with Metallothionein-1X subtype(MT1X). Then the full-length MT1X cDNA was amplified and the interaction between FHL3 and MT1X was defined. MT1X is one of ten function subtypes of MT. Metallothionein (MT) has wide functions in many pathological processes, such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, etc. Researches found that abnormal high expression of MT1X is closely related to the development of breast cancer, for whose mechanism we know a little. In the research of FHL3`s function, its activity of transcriptional activation was found. To locate the transcriptional activation domain in the LIM protein, the genes for five FHL3`s mutants each with one deleted LIM domain were gained by PCR and the expressions were confirmed in Yeast cells and analyzed by Western blot.β-Galactosidase assay was used to locate the fourth LIM domain(LIM4) in C-terminal of FHL3 which is related to the transcriptional activation. Futher removing each of the two zinc-fingers in LIM4, it is the first time that only the zinc-finger in C-terminal was found to be related to the transcriptional activation function.In contrast to the AAs in LIM4 domain of FHL3 and FHL2, the two domains was found to have have the same length and aminoacid homology up to 56%. Although the aminoacid residues of FHL3 and FHL2 in certain sites are different, their properties are similar. If these aminoacids were considered, the similarity between these two LIM domains reached to 76%. And reasearches on FHL2 show the LIM4 is also related to the transcriptional activation of FHL2.To analyze the function of FHL3 in angiogenesis pathway, we construct the recombinant plasimid pBridge-Ang-FHL3, and use yeast three-hybrid assay to screen the interactive protein with Ang/FHL3 complex, but after three screenings, no such proteins were found. In another side, we use a series of mutants of FHL3 to construct the AD recombinant plasmids. Using theβ-galactosidase assay, the interaction possibility of every LIM domain of FHL3 with Ang was analyzed, which would provide clues to analyzed the binding domain or the crucial aminoacid residues for binding between FHL3 and Ang in the protein modeling.In summary, this study has obtained the following results: 1.The full-length gene of FHL3 was successfully constructed, which had been highly expressed in E.coli. The recombinant protein with high activity was obtained after purification and renaturation; 2. The recombinant protein can promote the apoptosis of HepG2 cells; 3. The library was screened and the protein MT1X interacting with FHL3 was found; 4.A series of mutants of FHL3 had been construted, and the transcriptional activation domain was located; 5.The possibility of every LIM domain of FHL3 in the interaction with Ang was analyzed.Generally, our study provided new clues to reveal the interaction between FHL3 and Ang and the structure and function of FHL3 protein in angiogenesis, which suggested the new tumor therapy with anti-angiogenesis.