Abstract
Post-translational modifications are known to be widely involved in the regulation of various biological processes, through the extensive diversification of each protein function at the cellular network level. In order to unveil the system-wide function of the protein lysine modification in cancer cell signaling, we performed global acetylation and ubiquitination proteome analyses of human cancer cells, based on high-resolution nanoflow liquid chromatography–tandem mass spectrometry, in combination with the efficient biochemical enrichment of target modified peptides. Our large-scale proteomic analysis enabled us to identify more than 5000 kinds of ubiquitinated sites and 1600 kinds of acetylated sites, from representative human cancer cell lines, leading to the identification of approximately 900 novel lysine modification sites in total. Very interestingly, 236 lysine residues derived from 141 proteins were found to be modified with both ubiquitination and acetylation. As a consequence of the subsequent motif extraction analyses, glutamic acid (E) was found to be highly enriched at the position (−1) for the lysine acetylation sites, whereas the same amino acid was relatively dispersed along the neighboring residues of the lysine ubiquitination sites. Our pathway analysis also indicated that the protein translational control pathways, such as the eukaryotic initiation factor 2 (EIF2) and the ubiquitin signaling pathways, were highly enriched in both of the acetylation and ubiquitination proteome data at the network level. This report provides the first integrative description of the protein acetylation and ubiquitination-oriented systematic regulation in human cancer cells.
Highlights
Post-translational modifications (PTMs), such as phosphorylation, acetylation, and ubiquitination, are widely known to play various important roles in cellular signaling, and more than 1000 kinds of PTMs regarding eukaryotes and prokaryotes have been registered with Unimod, a comprehensive database of protein modifications for mass spectrometry [1]
In order to grasp the network-wide status of the protein lysine modification in various types of human cancer cells, we performed a double-edged proteomic analysis of the lysine ubiquitination and acetylation by a high-resolution nanoLC-MS/MS coupled with an antibody-based enrichment of the corresponding lysine-modified peptides from each cell lysate (Figure 1)
Our results showed that the relative frequency of ubiquitinated and acetylated peptide counts dramatically varied among the investigated cancer cell lines (Figure 2B), and that the hierarchical clustering of the whole protein acetylation and ubiquitination datasets clearly indicated their cell-type dependent characteristics (Figure 2C)
Summary
Post-translational modifications (PTMs), such as phosphorylation, acetylation, and ubiquitination, are widely known to play various important roles in cellular signaling, and more than 1000 kinds of PTMs regarding eukaryotes and prokaryotes have been registered with Unimod, a comprehensive database of protein modifications for mass spectrometry [1]. Protein phosphorylation is recognized as one of the most intensively studied PTMs, and it regulates a variety of key cellular processes by transmitting diverse signals through the enzymatic reaction of kinases/phosphatases and their substrates [3]. Protein lysine acetylation is known to play essential roles in the transcriptional regulation by hub protein molecules, such as histone and p53, through the coordinated function of acetyltransferases and deacetylases [7]. The lysine ubiquitination widely contributes to the transmission of protein degradation signals, as well as the cell-cycle progression and DNA repair [8]
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