Global identification of CobB interactors by an <italic>Escherichia coli</italic> proteome microarray

Abstract

Protein acetylation is one of the most abundant posttranslational modifications and plays critical roles in many important biological processes. Based on the recent advances in mass spectrometry technology, in bacteria, such as Escherichia coli, tremendous acetylated proteins and acetylation sites have been identified. However, only one protein deacetylase, i.e. CobB, has been identified in E. coli so far. How CobB is regulated is still elusive. One right strategy to study the regulation of CobB is to globally identify its interacting proteins. In this study, we used a proteome microarray containing ∼4000 affinity-purified E. coli proteins to globally identify CobB interactors, and finally identified 183 binding proteins of high stringency. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in carboxylic acid metabolic process and hexose catabolic process, and also enriched in transferase and hydrolase. We further used bio-layer interferometry to analyze the interaction and quantify the kinetic parameters of putative CobB interactors, and clearly showed that CobB could strongly interact with TopA and AccC. The novel CobB interactors that we identified could serve as a start point for further functional analysis.

Protein Array-Based Approaches for Biomarker Discovery in Cancer

“…When >60% of full length proteins encoded by a given organism are presented on a functional protein array, they can be referred as proteome arrays. To this date, proteome arrays have been constructed for several important model organisms, such as Escherichia coli [16], Saccharomyces cerevisiae [17], and humans [3], [18]. In recent years, proteins over-expressed in tissue culture or in vitro translated without further purification have also been used to fabricate functional protein arrays [19], [20].…”

Discovering cancer biomarkers from clinical samples by protein microarrays

“…In contrast, functional protein microarray usually covers a huge amount of proteins on a single slide [29], sometimes near the total proteome of an organism, for example Escherichia coli [30,31], Saccharomyces cerevisiae [18,32], and humans [33]. In general, the immobilized proteins on functional protein microarray are expressed in engineered bacteria or yeast and most of them are not well characterized.…”

Studies of Nε-Lysine Acetylation Modification on Escherichia coli Topoisomerase I

“…TopA has been demonstrated in a previous study to be a CobB binding protein using a proteome microarray as well as bio-layer interferometry analysis (Liu et at.,2014). Purified recombinant TopA and His-tagged CobB were allowed to interact before pull-down by HisPur Cobalt Agarose Resin.…”

“…Proteomics studies have revealed multiple lysine sites on E. coli topoisomerase I as targets of lysine acetylation (Yu et al, 2008;Zhang et al, 2009a). Moreover, the proteome microarray assay identified a strong interaction between a deacetylase and E. coli topoisomerase I suggesting the importance of regulation of lysine acetylation in E. coli topoisomerase I (Liu et al, 2014). Similarly, eukaryotic topoisomerases have been found to be post-translationally modified by acetylation (Choudhary et al, 2009), phosphorylation (Wells et al, 1994), sumoylation (Mao et al, 2000) and ubiquitylation (Mao et al, 2001) that can alter the protein activity and localization.…”

“…Therefore, it appears that TopA lysine acetylation can occur through the nonenzymatic acetyl phosphate dependent mechanism. The NAD +dependent sirtuin CobB deacetylase, the predominant deacetylase in E. coli, is reported to strongly interact with TopA (Liu et al, 2014). Given the frequency of observation of TopA (Cheng et al, 2005).…”