The first and main objective of NOPHOS was the investigation of the phosphoproteomic changes in E.coli upon NO (WP1). We tuned our strategy at the beginning, based on the presumption that similarly to the eukaryotes, prokaryotes would respond to different stimuli through rapid reversible PP. Therefore, we tested NO treatments taking place for two and five minutes. At those time-points the phosphoproteomic analysis revealed 78 phospho-proteins, four of them differentially phosphorylated upon NO. In addition, we decided to further expand the sample size and explore long term effects of NO on the E.coli’s phosphoproteome by testing 15 and 45 minutes of treatment. This allowed us to uncover additional 115 phospho-proteins, 6 of them differentially regulated upon NO exposure. Functional bioinformatics analysis revealed that protein phosphorylation is involved extensively in various cellular functions in E. coli, including housekeeping processes, such as transcription and translation, central carbon metabolism, among others. In addition, alignment of the regions containing the identified phospho-sites, showed high conservation across different bacteria, suggesting that these sites could have a central role in regulating these systems. Interestingly, the most prominent of the ten phospho-proteins found to be differentially regulated upon NO – cydD, protein part of the glutathione/cysteine exporter cydDC in E. coli, has been previously found to be directly involved in NO response in this bacterium. Moreover, it seems that cydD functions as a cytoplasmic cystine reductase that sensitizes E.coli to oxidative stress and aminoglycoside antibiotics. Hence, cydD is a suitable target for the development of antibacterial drugs attacking bacterial respiration.
In WP2 we aimed to characterize the biophysical and biochemical properties of phosphoproteins involved in RNS detoxification. One such protein having central role in the detoxification of NO in E. coli, namely its flavodiiron, flavorubredoxin (norV), which reduces NO to N2O, was also found to undergo phosphorylation. The mass spectrometry analysis revealed three phospho-sites, one serine and two highly conserve histidine residues to be subjected to phosphorylation. We therefore, prepared a phospho-dead and phospho-mimetic mutants and determined that mutation of one of the histidine restudies affects significantly the NO reductase activity of norV. Biophysical characterization of the mutants revealed that histidine mutants presented general characteristics similar to the wild type enzyme, including integrity of the diiron center, while the serine mutants displayed altered EPR spectra of diiron center in its mixed valence form, when compared to the wild type. In addition, we analyzed the effect of the norV phospho-mutants in vivo (WP3). Complementation studies with a norV-deleted E. coli strain showed that norV phospho-mutants afforded in vivo protection against NO-induced stress. However, no differential effect was observed when compared to the wild type.
Findings from this project have been or will be presented (WP4) at the 12th International BioMetals Web-Symposiun, July 2020; at the 4th International conference on post-translational modifications in Bacteria, Copenhagen, Denmark, May 2022, at the TIMB3 final meeting, Tomar, Portugal, June 2022, and November 2022 at the 12th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, Japan. The results will be published in the near future in peer-reviewed journals.
