Saturday, February 18, 2012
Exhibit Hall A-B1 (VCC West Building)
It is believed that a decrease in mitochondrial NAD(P)H:quinone oxidoreductase (NQR) activity enhances free radical damage which accumulate, resulting in diseases of old age. In normal mitochondrial function, NQR reduces ubquinone to ubiquinol, a lipid-soluble antioxidant. A bacterium Vibrio harveyi designated MYR-4A was isolated from the surface mucus of a shallow-water coral during a previous study from the Great Barrier Reef in Australia, and it was found that NAD(P)H:quinone oxoreductase (NQR) activity surprisingly increases in this bacterium when exposed to UV, suggesting that NQR protects against UV-induced oxidative stress. In animals, NQR expression is regulated by the transcriptional activator Nrf2 which has never been reported previously in bacteria. A bioinformatics study has identified a hypothetical protein of unknown function in the predicted proteome of MYR-4A. The aim of our present study was to confirm the existence of this protein from the expressed proteome of MYR-4A. MYR-4A was spread inoculated on marine broth agar plates. Inoculated plates were irradiated for 5 hours under twin UVB lamps. MYR-4A survived up to 5 hours compared to E. coli which died within 15mins of irradiation. MYR-4A UVB resistant survivors (called MYR-4A Irvine) were grown aerobically overnight in 100 ml of marine broth. After reducing the optical density to 0.8, MYR-4A Irvine was re-suspended in artificial sea water containing glucose. The bacteria then underwent irradiation and samples were taken at 0, 0.5, 1 and 5 hrs. Cells were collected by centrifugation and lysed in TSB /Triton by vortex mixing with glass beads. The soluble and insoluble protein fractions were analyzed by LC-MS using a MIDAS proteomics approach, which is based on the selective detection of modified peptides from a known target protein. As expected, the hypothetical protein A7N436 was confidently identified in the insoluble fraction at time point zero (before irradiation) and time point 30mins in the soluble fraction. A7N436 is bound to the cell membrane through the binding motif and hence we detect the protein only in the insoluble fraction of the proteome. On exposure to UVB, this stress induces reactive oxygen species and/or nitric oxide which binds to the YYY motif causing dissociation of the protein from the membrane, hence we detect the protein only in the soluble fraction at t=30 min’s. A7N436 then binds to DNA via the helix-turn-helix motifs, this is very strong binding and may account for why we do not see the protein in the soluble or insoluble fractions at t=1 and 5 hr. A7N436 acted as the transcriptional activator for NQR which presented that it can be Nrf2 for bacterial cells. These results pave the way for a more in-depth analysis of the regulation of NQR activity in UV-tolerant bacteria for use as a model of human mitochondrial function.