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 Kd between 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.
Highlights
Protease is responsible for protein degradation that can digest long protein chains into shorter fragments by splitting the peptide bonds [1]
Because the substrate of a protease can reflect its role in a bacterial cell, identifying potential enzyme substrates is essential in protease research
The transformed bacterial cells were cultured in Luria Broth (LB) broth until the OD600 reached to 0.3– 0.4, and 1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) was added to induce protein expression for 30 min
Summary
Few substrates have been identified for the ClpYQ protease in comparison with other ATP-dependent proteases [25,26,27]. Using substrateenzyme interactions as indicators for screening enzyme substrates in large batches has become a prevalent approach in recent studies on ATP-dependent protease substrates [28, 29]. Because ClpY is responsible for substrate recognition, in this study, we used E. coli proteome microarrays to identify ClpY recognition proteins and the results showed that the ClpY exhibited strong interactions with the YbaB. These results proposed the YabB is a novel target of ATP-dependent protease ClpYQ
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