Selection And Molecular Simulation Of Small Molecular Lead Peptides Targeting Human Fox Mlc Protein

Cancer is a major disease that is seriously harmful to human life and health. Among various types of tumor associated transcription factors, Fox Family is the most important with large members. Developmental malformation, metabolic disease and tumorigenesis are related to its mutation and abnormality expression. FoxMl is one of the gene family which controls the entire cell life cycle, tumor growth and metastasis as a special member of Forkhead family. It was closely related to the tumor. At present, Foxmlc transcription factor was less reported especially in bioinformatics analysis. The work is of great value and consequences for the further understanding of the bioinformatics bases of FoxM1c and the selection and finding of small molecular leading peptides targeting FoxMlc.This work mainly includes three parts:1) the bioinformatics analysis of FoxM1c; 2) the cloning and exprsssion of human FoxM1c protein; 3) the selection and molecular simulation of lead peptide targeting human FoxM1c protein.Homology among FoxM1c genes were found by searching eight representative sequences from different species by protein sequence query in NCBI, as well the finding of FoxMlc or its domain in other species, meanwhile, physico-chemical property, primary, secondary and tertiary structure and protein function of human FoxMlc were presented.The results showed that "fork head box" of FoxM1 protein was a conservative region. Homology between Fox family members such as C and S, J and K, H and Q, P and R, M and O, were respectively higher. Human FoxMlc was composed of a total of 748 amino acids. Human FoxM1c protein sequence had two domain:One is Fork head box (Forkhead, 236-314) and transcriptional activation domain (AD,688-748), to which p19ARF protein in the amino acid 26-46 binding and hence restraining human FoxM1c and reducing the transcription activity of human FoxM1c nuclear targeting. As we failed in obtaining the reliable tertiary structure of the AD, the precise mechanism of action requires further more study.Also, the human full-length FoxMlc cDNA was identified and cloned in the lab. The work offered great basis for the cloning and expression of human full-length FoxM1c cDNA or its functional domains which would be the important steps in the selection and finding of leading peptides targeting FoxM1c.Here, at first the full length, the DNA binding domain and the transcriptional activation domain of human FoxM1 cDNA were amplified by PCR, then they were genetically cloned into the prokaryotic expression vector pQE30 and confirmed by restriction analysis and sequencing, by IPTG induction and polyacrylamide gel electrophoresis the FoxMl proteins were successfully expressed and confirmed and later purified by Ni-NTA agarose gel packing and quantified up to 0.8mg protein/ml solution by BCA assay.By four rounds of selection of phage random dodecapeptide library against this expressed FoxMlc protein, the phages were enriched and eighteen different sequences were obtained by sequencing of different phages clones, importantly several clones showed higher affinities for FoxM1c protein with the highest near to P/N of 200. In addition, important consensus sequence were also found. It was the first report of the finding of lead peptides targeting FoxMlc by phages display technology.It was found that the binding peptides for FoxMl protein may contain at least one of the following sequence characteristics:the first group:WHL/Q/D/N; the second group:DLY, DLLY, DHYN, DLNY; the third group:SSLWN/E; the fourth group:HLDY, HLYE, YHLE, LYHDD, YHLEE; the fifth group:FYNL, NYFL; the sixth group:YPH, YPL, YPS. By molecular simulation of docking software MVD, it was predicted that the above described binding peptides may target FoxM1-binding domain mainly on the sites of Arg-Arg (RR, aa: 254,256), and Glu-Thr-Ser-Ala-Asn-Gly-Lys (E-T-S-A-N-G-K, aa:298-304). The further analysis and study of the peptides are still in progress.