Function Of A Human Autophagy Associated Gene PRR7

Objectives:Autophagy has a role in biological processes, including embryogenesis, aging, and many diseases. In order to find novel human genes involved in autophagy, we use high-throughput cell-based screening technique to indentify 1000 human function unknown genes. A gene named PRR7 which can reduce luciferase report gene Renilla luciferase( PRL) activity was seleted to further study for the function and mechanism,the results will lay a solid foundation for development of functional genes with values of clinical application.Methods:1. screening genes of interested:Express 1000 novel human genes in HEK293T cell, and screens on HTS platforms based on dual-luciferase report assay. Selected human novel genes PPR7 which down regulate the PRL activaty for further study.2. Bioinformatics and software were used to forecast the gene's nucleic, alternative splicing and protein sequence, signal peptide, sub-cellular localization, interacted genes, sequence compares among the species, structural domain.3. The ORF of interested gene PPR7 was sub-cloned into the mammalian expression vectors, including: pcDNA3.1b(-)-myc-his, GFPN1 and GFPC3.4. The expression pattern of PPR7 in various human cell lines were analysed by reverse transcription-PCR (RT-PCR).5. We used CCK8 assay to monitor the cell death.6. To determine the effects of PPR7 in cell death, we used morphology observation and Calcein AM/EthD-1 2-color fluorescence probes which could distinguish living and dead cells.7. PS externalization and Dioc6 analysis were used to evaluate cell death involves hallmarks of apoptosis.8. PARP was analyzed by western blot, and the cleaved PARP was observed in PPR7 transfected cells. The pan-caspase inhibitor, Z-VAD-FMK was used to analysis if it could prevent cell death induced by PRR7.9. RT-PCR was used to assay the PPR7 on the mRNA expression level of apoptosis related molecular P53 and DRAM.10. Transfection of PPR7 with a GFP tag into HEK293T cell to examine the localization by using confocal microscopy.Results:1. Based on the dual-luciferase report assay, we identified a novel gene named PRR7, which could down-regulate PRL activity.2. Human PRR7 full cDNA with 1543bp length containing 2 exons and 2 introns is located in the human chromosome 5q35. The predicted protein encoded by this gene contains 274 high conserved amino acids with a theoretical molecular weight of 31 kDa; There is a signal peptide and a putative transmembrane region near the N-terminus of this protein analyzed by bioinformatics., and it is conserved in Human, Pan, Mus, Rabbit, Bos, Canis and Danio.3. Over expression of PRR7 or BAX in HEK293T cell induced apoptosis characteristics, including marked rounding, cell shrinkage and nuclear condensation.4. Calcein AM/EthD-1 2-color fluorescence-based assay revealed that PRR7 could induce cell death. The result of the flow cytometry showed that HEK 293T cells transfected with PRR7, the propotion of Annexin V and PI positive cells was increased compared with control. The flow cytometry analysis suggested that overexpression of PRR7 in HEK293T cells could disrupt mitochondrial transmenbrane potential.5. Cleaved PRAP was not observed in PRR7 tarnsfected cell, but which was found in BAX transfected cells. The pan-caspase inhibitor Z-VAD-FMK could not prevent cell death induced by PRR7.6. The result of the flow cytometry showed that H1299 cells transfected with PRR7, the propotion of Annexin V and PI positive cells was not increased compared with control, but BAX was increased, this result show that PRR7 could not induced H1299 cell death, which lack expression of P53 protein.7. To further determine the role of P53 in PRR7 induced cell death, we used the RT-PCR, the result suggested that the PRR7 could increase mRNA expression of P53.8. Further study showed that PRR7 could induce autophagic characteristics in HeLa cells, including dotted GFP-LC3 localization and increase LC3-Ⅱ/I ratio shown by western blot.9. RT-PCR results suggested that the PRR7 could increase mRNA expression of DRAM.10. The confocal microscopy showed that PRR7 encode a membrane protein.Conclusions:1. Screening on HTS platforms based on dual-luciferase report assay, we indentified a human novel gene PRR7.2. Human PRR7 is a membrane protein, and the nucleic acid sequence of PRR7 in the mult-species was high conserved.3. PRR7 could induce cell death and caspase cascade was not involved in PRR7 induced apoptosis.4. Human PRR7 play an important role in the regulation of autoptagy.5. PRR7 may induce autophagy through"P53-DRAM-autophagy"pathway, typeⅡcell death.