Designing Peptide Array Based On Sequence Of APE1 And P53 Proteins And The Peptide Screening For Mitochondrial Targeting Sequence

Mitochondria are the only organelle with genomic DNA besides nucleus in human cells, and mitochondrial DNA(mtDNA) is extremely susceptible to oxidative damage. Oxidative stress is closely related with a variety of pathological conditions, including cancer, aging and cardiovascular disease. Cellular DNA is a biologically important target for ROS and DNA oxidative damage was mainly repaired by the base excision repair (BER). APE1 and the tumor suppressor gene p53 are the classic dual-targeted mitochondrial proteins for nuclear-mitochondrial. Previous studies showed that both of the two proteins were mainly localized in the nucleus and when subjected to different stimuli ofoxidative stress APE1 and p53 would translocate to mitochondrial, which demonstraed they involved in DNA damage repair process. Human apurinic/apyrimidinic endonuclease(APE1) plays a central role in the cellular response to oxidative stress. Previous studies showed APE1/Ref-1 stimulates the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as AP-1 (Fos/Jun), HIF-1a, CREB, p53 and so on. Recently report revealed WTp53 is a negative regulator of APE1 expression, that is to say p53-induced APE1 repression is mediated by interference with Sp1 binding to the APE1 promoter. Thus the repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.As classical dual-targeted mitochondrial proteins, APE1 and tumor suppressor gene p53, being located in nuclear-mitochondrial. A growing body of evidence has shown that the subcellular distribution of APE1 can be cytoplasmic in some cell types with high metabolic or proliferative rates, with predominant localization in the mitochondria and the endoplasmic reticulum (ER) (5) (6-7). It is a dual-targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. Dual-targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS) which makes them difficult to predict by current bioinformatics approaches. Thus, its mitochondrial localization sequence (MTS) is not clear. Translocase of the outer mitochondrial membrane(TOM) is closely related to proteins translocation into mitochondria, which is to be required for two translocation contact sites. That is to say, the nuclear-encoded mitochondrial-targeted preproteins are recognized by the receptors on the mitochondrial surface and are subsequently translocated across the outer mitochondrial membranes through a general import pore (GIP) that assembles into a high molecular weight complex, termed the preprotein translocase of the outer mitochondrial membrane. The three Tom subunits, Tom20, Tom22, and Tom70, expose major portions on the cytosolic side of the outer membrane and function as import receptors for distinct classes of the preproteins in vivo and in organello. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Protein import studies with isolated mitochondria indicate that Tom20 and Tom22 function as heterodimer receptors for the typical mitochondrial preproteins that carry the cleavable N-terminal mitochondrial targeting sequence (MTS). Tom70 is reported to be required for the import of non-cleavable preproteins that have been shown to have specificity for carrier proteins destined for the inner mitochondrial membrane. The purified cytosolic domains of these import receptors were validated to specifically bind the mitochondrial preproteins in vitro.Our study will mainly investigate to design on peptide array based on the sequence of proteins APE1 and p53 and its peptide screening for mitochondrial targeting sequence. To further reveal the mitochondrial translocation mechanism of DNA damage repair pathway. Our experimental approaches were combined in this study to identify the MTS of APE1 and p53 by molecular biology, cell biology, biochemistry and bioinformatics to establish peptide library and make a good use of it. First, the interactions between the peptides from APE1 and the three purified translocases receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. All the peptide sequences originated from APE1 and in 15 amino acids'lengths overlapped by 12 residues that were covalently attached to the cellulose membrane. All the peptide sequences originated from p53 by the same method of 15 amino acids'lengths overlapped by 8 residues, and use Spotfinder to predict the most possible MTS of the both proteins above for the future design of the blocking peptide inhibited mitochondrial localization with providing a theoretical basis, which sheds light on future prediction of MTS of p53 and APE1. Thus the p53-mediated suppression of APE1 expression could offer a potential approach for sensitizing tumor cells to drugs.The main results and conclusion of the study are as follows:1. To construct Tom receptor preteins of expression vector for mitochondrial outer membrane. Expression of the Cytosolic Domains of Tom20,Tom22, and Tom70—(His)10- tagged cytosolic domains of Tom20,Tom22 and Tom70 were induced using IPTG and Saccharomyces cerevisiae genomic DNA as template. The three kinds of Tom proteins were cloned into pET19b vector to obtain recombinant plasmid pET19b-yTom20cd-His10,pET19b-yTom22cd-His10 and pET19b- yTom70cd - His10.2. To validate the purity of the Tom proteins with SDS-PAGE and Coomassie BrilliantBlue R250 with His tag by Ni-NTA Resin. To evaluate the concentration of the purified protein by Bradford and detecte the three fusion proteins with His labeled antibody with HRP-tag by western blot. The results provided the fusion protein with high purity and concentration to make affinity test.3. Subcellular localization of APE1 under different stimuli. To confirm the subcellular fractions of the nucleus, cytoplasm, and mitochondria from each group were subjected to western blot. The results indicate that the major localization of APE1 in the HeLa cell is the nucleus, and that oxidative stress or the menadione induced the translocation of APE1 to the mitochondria.4. To construct the peptide screen assay. Together with results of hybridization and the pull-down assay demonstrate that APE1 possesses higher affinity to Tom20 by peptide array for the three kinds of Tom protein. And Tom20cd was distributed in the regions of the first one-third of both the N-terminus and C-terminus.Tom22cd and Tom70cd were strikingly similar, the overall intensity of the interactions with Tom22cd and Tom70cd were significantly lower.TOM respectively APE1 protein peptide library microarray chip hybridization. The peak regions of APE1 including peptides of residues were observed in the peptide scans by Spotfinder, and analyzed by SAM3.02 and Cluster3.0 to filter the candidate MTS 211-240,238-258,265-279 and 289-312.5. The results provided by the peptide screen assay together with the pull-down assay demonstrate that APE1 possesses higher affinity to Tom20 than to Tom22 or Tom70, which led to the hypothesis that mitochondrial translocation of APE1 may be through a Tom20–dependent pathway.6. To construct the subcellular location system with gene GFP as the reporter gene. The results by laser confocal microscope showed wild-type p53 located in the nucleus, and the subcellular distribution of p53 and transcription factor A mitochondria (TFAM) were tested as nuclear- and mitochondrial targeting controls to validate the correction of our observation system.7. To construct the peptide screen assay by the same method. The result demonstrated that p53 possesses higher affinity to Tom20 by peptide array for the three kinds of Tom protein, which hitten the hypothesis that mitochondrial translocation of p53 may be through a Tom20–dependent pathway. The peak regions of p53 including peptides of residues were observed in the peptide scans by Spotfinder, and analyzed by SAM3.02 and Cluster3.0 to filter the candidate MTS 351-365,358-372,365-379 and 372-386.