title: Escherichia coli Proteome Microarrays Identified the Substrates of ClpYQ Protease.
abstract: Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases play a crucial role within this process. In Escherichia coli, ClpYQ is one of the primary ATP-dependent proteases. In addition to function with removals of abnormal peptides in the cells, ClpYQ degrades regulatory proteins if necessary and thus let cells adjust to various environmental conditions. In E. coli, SulA, RcsA, RpoH and TraJ as well as RNase R, have been identified as natural protein substrates of ClpYQ. ClpYQ contains ClpY and ClpQ. The ATPase ClpY is responsible for protein recognition, unfolding, and translocation into the catalytic core of ClpQ. In this study, we use an indirect identification strategy to screen possible ClpY targets with E. coli K12 proteome chips. The chip assay results showed that YbaB strongly bound to ClpY. We used yeast two-hybrid assay to confirm the interactions between ClpY and YbaB protein and determined the Kdbetween ClpY and YbaB by quartz crystal microbalance. Furthermore, we validated that YbaB was successfully degraded by ClpYQ protease activity using ClpYQ in vitro and in vivo degradation assay. These findings demonstrated the YbaB is a novel substrate of ClpYQ protease. This work also successfully demonstrated that with the use of recognition element of a protease can successfully screen its substrates by indirect proteome chip screening assay.
authors.affiliation: From the ‡Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan.
authors.forename: Chih-Hsuan
authors.initials: CH
authors.lastname: Tsai
authors.affiliation: §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: Yu-Hsuan
authors.initials: YH
authors.lastname: Ho
authors.affiliation: §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: Tzu-Cheng
authors.initials: TC
authors.lastname: Sung
authors.affiliation: From the ‡Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan; hfenwu@gmail.com cchen103@gmail.com.
authors.forename: Whei-Fen
authors.initials: WF
authors.lastname: Wu
authors.affiliation: §Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan; hfenwu@gmail.com 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: D004268:DNA-Binding Proteins / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D060066:Protein Interaction Maps; D020543:Proteome / Q000032:analysis; D013379:Substrate Specificity
chemical_list: C483566:ClpYQ protease, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020543:Proteome; C543927:YbaB protein, E coli; D049071:Endopeptidase Clp