Screening Of Proteins Interacting With BRAP And Its Influence On Cell Oxidative Metabolism

Bombesin receptor subtype3(BRS-3), one of the family members of bombesin-like peptides, BLPs, may play an important role in the stress response regulation of the lung and airway epithelium facing the oxidative stresses. Our laboratory found a new protein interacting with BRS-3which was named bombesin receptor activated protein (BRAP). Our previous study found that BRAP mainly expressed in the lung tissue membrane and cytoplasm. BRAP was upregulated in the lung of ozone stressed animals, suggesting that BRAP, as a cytoplasm protein, may participate in the transduction of stress signal and the regulation of some special physiological functions under stress. In order to explore the mechanism and biological functions of BRAP, this study screened the interacting proteins of BRAP by yeast two-hybrid technology. We classified the screened proteins according to their functions and speculated that BRAP was closely linked to the function of cell oxidative metabolism. Subsequently, we detected a series of related indexes of cellular oxidative metabolism.Objective:Screen the proteins interacted with BRAP and research the influences on cell oxidative metabolism to further explore the function and the mechanism of BRAP.Methods:(1) The control experiments were performed to check the yeast two-hybrid system.(2) Construct the bait plasmid pGBKT7/BRAP, expressing BRAP and GAL4DNA-BD fusion protein in the yeast cells. Transform the bait plasmid into Y2HGold yeast cells; mate it with transformed general cDNA library yeast cells (Y187) to get the diploid cells. Screen the clones by observing the expression of report genes in the auxotroph plates.(3) Prepare the concentrated overnight yeast culture, extract and simply purify the total DNA. Transform the E.coli DH5a with the total DNA. Extract and sequence the plasmid from e. coli and blast the sequenced results with the national center for biotechnology information database in order to find the proteins which may interact with BRAP. More than20kinds of gene products have been concerned of interacting with BRAP.Among the sequenced results, six genes were screened which might have the functions associated with cell oxidative metabolic pathways.(4) Isolate the total RNA in human bronchial epithelial cells (HBEC) and reverse transcribed it into cDNA. Clone the cDNA of the target gene and transform it into Y2HGold yeast cells together with pGBKT7/BRAP.to confirm the existence of the interactions between the oxidative metabolic related genes and BRAP.(5) Meanwhile, detect the intracellular reactive oxygen species (ROS) level in293T cells under both resting state and ozone stressed state to investigate the influence of BRAP.(6) Detect the level of intracellular oxidative metabolism in293T cells using the reported gene pARE-Luc, to investigate the influence of over-expressed BRAP.(7) The influence on HEK293T cell apoptosis of BRAP is observed by the flow cytometry and fluorescence spectrophotometer.(8) Detect the influence on caspase-9activation (the mitochondrial pathway of apoptosis) of BRAP under the ozone stress via chemiluminescence.(9) Detect the influence on mitochondrial membrane potential of BRAP under the ozone stress via fluorescence spectrophotometer.Results:(1) The existing yeast two-hybrid system is available.(2) Successfully constructed the yeast two-hybrid bait plasmid pGBKT7/BRAP, The experiments confirmed that the pGBKT7/BRAP could be expressed in yeast, without neither self-activated nor obvious toxicity. (3)2×106cells have been screened and358clones can grow on auxotroph tablet positive clones, which can directly or indirectly interact with BRAP.(4) Part of the positive clone plasmids containing the exogenous gene fragment have been extracted and purified. Then transform the plasmids into yeast strains. The screened cells have been sequenced, including enzymes that may play an important role in the oxidative metabolism in the cells and other proteins acting in other functions in cellular activities.(5) Successfully cloned part of the full length of target genes, constructed the carrier, and transformed it into yeast cells together with the BRAP. Up to now, the experiments have preliminarily verified that three target genes could interact with BRAP.(6) Over-expressed BRAP could down regulate not only the intracellular reactive oxygen species (ROS) level under ozone stress, but also the level in cell oxidative metabolism.(7) Over-expressed BRAP could lower the cell apoptosis level, and maintain the mitochondrial membrane potential under ozone stress.(8) Over-expressed BRAP could reduce the caspase-9activation level in cells, partially inhibited the apoptosis of cells.Conclusions: (1) BRAP can interact with a variety of proteins in the cell and has the effects on the functions of oxidative metabolism.(2) BRAP can significantly reduce the level of intracellular oxidative metabolism in the state of oxidative stress, as well as oxidative damage and cell apoptosis.