Domain Of Selenoprotein P Identification And Its Mutant Function Study

Hepatocellular carcinoma (HCC) is one of the most common malignant tumor affecting people around the world. Chemotherapy play a key role in the combination therapy of HCC. After five decades of development, the directions of drug discovery that are currently pursuing have fource on design of novel analogs of natural leading compound which act against new molecular targets, and with greater anticancer activity and less toxicity. The present study is a exploratory task. Our goal is to exploit a novel small molecules peptide based on natural structure of selenoprotien P (SelP) protein, which can induce HCC cell apoptosis.The study consisted of four parts.Part I. The developmental expression pediment of SelP and its significance in clinical pathology. The expression of SelP was detected in normal foetual tissues, normal adult tissues and HCC tissues with polyclone antibody by using tissue microarrys and immunohistochemistry. The expression of SelP was analysis with clinical pathology. The results show that SelP possessescomprehensive biological function and play an important role in the carcinogenesis and development of HCC. Thus, SelP was considered as a valuable leading compound for exploiting novel anti-HCC drugs.Part II. Studies on Bioinformatics of SelP. By the aid of Bioinformatic analysis, two main domains were found in SelP. One is N-terminal domain (SelP_N), and another is C-terminal domain (SelP_C). SelP_N contains a conserved Sec40thxxCys motif, which is similar to the CysxxCys found in thioredoxins. SelP_N also contains many motifs (small domains), which are related with apoptosis. Furthermore, the function reagon of SelP_N (SelP137) was confirmed. It is considered that Sec40th may impart unique biological properties to SelP137.Part III. According to the analysis results of Part II, it is considered that deleted Sec residue may impart apoptosis to SelP 137. Then, the mutuant of SelP 137 (SelP 137m) that altered Sec40th-Cys was designed. To elucidate the possible involvement of SelP 13 7m in apoptosis, BEL-7402 cells was transfected with pEGFP-C3 fused with SelP 137m and examined the effect of over-expression of SelP137 in these cells after screening a stable cell line. It was demonstrated that SelP 13 7m can induce BEL-7402 cells apoptisis by the technic of molecular and cellular biology.Part IV. Studies on the mechanism of the apoptosis indeced by SelP137m. SelP137m gene was subcloned into pET30a plasmid to form the pET30a-SelP137m prokaryotic expressing vector. The recombinant plasimd was transfected into the host strain E.Coli BL21. Induced with IPTG, the strain controlled by T7 promoter expressed the fused target protein with a hexahistidine tail in its N-terminal. Wepurified SelP137m by affinity chromatograthy and studied its function on liver mitochondrial MTP. We found SelP137m can provoke liver mitochondrial MTP open and decrease in inner-membrane potential ( m). Current evidence show sthat SelP137m induce apoptosis by provoking mitochondrial PTP opening.Now, we find a novel small molecules apoptosis inducer based on natural structure of selenoprotien P (SelP) protein, which act against mitochondrial MTP.