Screening And Identification Of Active Antisense Homology Box Peptides In Death Receptors And Their Ligands

Various evidences have shown that the peptide coded by the sense (positive) strand of DNA can make specific interaction to the peptide coded by the antisense (negative) strand of DNA. This kind of interaction is mediated by specific, pair-wise interaction between amino acids. Based on these research works, the Proteomic Code Theory was generated by Miller in 2002. The fundamentals of the theory are: The information of recognitions and interactions between proteins are stored in their DNA sequences, the sense coding regions for receptors/antigens on one strand of DNA are complemented by those encoding regions for ligands/antibodies on another strand of DNA. On the basis of this theory, some research groups have developed bioinfomatic software and found antisense homology box peptides in C5a anayphytoxin,IL-1β,Fas ligand that can interact with their cognate receptors through these software.Death receptors (DR) belong to the second group of the TNF receptor superfamily. This group concludes TNFRI, Fas, DR3, DR4, DR5, and DR6. The common sense of these proteins is that they all have two to six cystein-rich domains (CRD) in their extracellular part and a death domain (DR) in their intracellular part. Their corresponding ligands TNFα, TRAIL, and FasL etc., belong to the TNF superfamily. While binding to the death receptors, they can induce the apoptosis of the cells.Endothelial monocyte-activating peptide-II (EMAP-II) is a proinflammatory -cytokine generated and secreted by cells under apoptotic condition or in stress. In recent years, studies of this cytokine have shown that it has multiple biological activities such as procoagulant and anti-angiogenetic properties. It can also induce the apoptosis of some cells (lymphocytes and tumor cells). In vivo and in vitro experiments have shown that EMAP-II can diminish the solid tumor masses. It can inhibit the proliferation and induce apoptosis of endothelial cells of blood vessels in the tissues of solid tumors. These results show that EMAP-II is a prospective cytokine in tumor immunotherapy. The research work about the cloning and purifing of recombinat EMAP-II and its function on apoptotic effects on endothelial cells are not as many as the work of the natural EMAP-II.As the first part of this study, we design a bioinfomatic software based on the Proteomic Code Theory which can find antisense homology box sequences between two proteins. Through this software we searched for antisense homology sequences between TNFRI-DR5 and their correponding ligands TNFαand TRAIL. After that we analysed the searching results using the structucal information of these moleculars and select 9 sequences to synthesize peptides. Then we indentified these synthesized peptides using ELISA and cellular experiments. As the second part of this study, we cloned and expressed the recombinat EMAP-II using pMAL expression system. Then we purified the recombinant protein and detect its apoptotic effect on MCF-7.Part I: Screening, analyzing and Synthesizing of Antisens homology box peptidesThis software is named Internet applications of Screening Functional Peptides Base on Proteomic Code and Analysis 2.0. Its copyright is protected by the China Copyright Office (Registration No. 2003SR5611). It can help users to find antisense homology box sequenses between two proteins. The protein sequences can be imported through standard text on internet or an RTF file. The sequences can also be imported through protein databases.Using this software we searched for antisense homology box peptides. TNFαand TRAIL were imported as Sequence I, TNFRI and DR5 were imported as Sequences II The ratio of matching is set to 70%. On the basis of the searching result of the software, we analyzed these sequences mainly using informations of the 3D-structures of these proteins as well as those of ligand-receptor complex . Then we selected 9 of them to synthensize.Part II: Identification and Characteration of these peptidesThree of the sythesized peptides are from TNFα, three are from TNFRI, one is from TRAIL and another two are from DR5. Firstly, we investigated these peptides by ELISA binding assay. In the binding assay, we find a TNFRI derived peptide LV-7 that can make specific binding with TNFαand a DR5 derived peptide FR-11 that can bind to TRAIL.Then we investigated these peptides on cellular experiments. These included: peptides inducing apoptosis effect on cells; peptides inhibiting apoptotic effect induced by ligand; peptides inhibiting proliferation of tumor cells. The DR5-derived peptide FR-11 peptide was found to partly inhibit the apoptotic effect of TRAIL on SW1990 cell. This inhibitory effect also depends on the concentration of TRAIL. When the concentration of TRAIL is high (1~20μg/ml), we can hardly see any difference. Only when the concentration of TRAIL is low (20~500 ng/ml), the inhibitory effect is significant. In the presence of 500 ng/ml TRAIL, 1.5μg/ml actinomycin D, the inhibitory effect of FR-11 peptide (100μg/ml) can reach up to 30%. Then we held the concentration of TRAIL and changed the concentration of FR-11 peptide. We found that the inhibitory effect of FR-11 became more significant as the concentration of peptide increased. The same result can be seen in FACS. At last, we detected the level of caspase-9. When adding FR-11 peptide, the level of caspase-9 is much lower than the sample without FR-11 peptide.Part III: Cloning, expression, purification of human EMAP-II and study of its activityAs the second part of our work, we cloned and expressed recombinant human EMAP-II . At first, we cloned the gene of the protein from human liver cDNA library. Then we digested the PCR fragment and the pMAL plasmid with EcoRI and BamHI. After that, we ligated the two fragment into one recombinant plasmid and transformed it into BL-21 E.coli. We identified the colonies on the plate and sequence it comercially. Then we expressed the recombinant EMAP-II in BL-21 and purified it. After that we determined the biological activity of recombiant EMAP-II by its ability to induce TF expression on cell line ECV304 and apoptosis in MCF-7 cells using a concentration range of 20~40 ng/ml.