| Summary: | 碩士 === 國立中央大學 === 系統生物與生物資訊研究所 === 104 === Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Due to its metal-chelating property, histidine residues are easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesize that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >5000 proteins. The 10 interacting proteins were successfully validated using fluorescence polarization assay, a third peptide probe of different sequence, as well as binding constant measurements. A consensus binding motif was identified among these 10 bacterial interacting proteins based on bioinformatic prediction, which also appeared to be present on human S100A1 protein. The preferential binding of S100A1 with 2-oxohistidine over histidine was successfully validated using all three peptide probes, suggesting that the capacity to recognize 2-oxohistidine modification may be evolutionarily conserved from bacteria to humans. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, sensitive enough to detect even the insertion of a single oxygen atom in this case.
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