title: Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips.
abstract: Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Because of its metal-chelating property, the histidine residue is easily oxidized in the presence of Cu/Fe ions and H2O2via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesized 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 >4200 proteins. Ten interacting proteins were identified, and successfully validated using a third peptide probe, fluorescence polarization assays, as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed that our interacting proteins were enriched in oxido-reduction processes, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. Besides, we found that the consensus binding motif among our identified proteins, including bacteria and human, were located within α-helices and faced the outside of proteins. 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, and sensitive enough to detect even the insertion of a single oxygen atom in this case.
authors.affiliation: From the ‡Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan.|§Department of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan.
authors.forename: Jun-Mu
authors.initials: JM
authors.lastname: Lin
authors.affiliation: ¶Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Tsai
authors.affiliation: ¶Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Liu
authors.affiliation: ¶Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan.
authors.forename: Yun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: ¶Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan hctai@ntu.edu.tw cchen103@gmail.com.
authors.forename: Hwan-Ching
authors.initials: HC
authors.lastname: Tai
authors.affiliation: From the ‡Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan; hctai@ntu.edu.tw cchen103@gmail.com.|§Department of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan.
authors.forename: Chien-Sheng
authors.initials: CS
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D060066:Protein Interaction Maps; D017433:Protein Structure, Secondary; D020543:Proteome / Q000378:metabolism; D009418:S100 Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome; D009418:S100 Proteins; C110294:S100A1 protein; C083725:2-oxohistidine; D006639:Histidine