Abstract
Recent studies of protein post-translational modifications revealed that various types of lysine acylation occur in eukaryotic and bacterial proteins. Lysine propionylation, a newly discovered type of acylation, occurs in several proteins, including some histones. In this study, we identified 361 propionylation sites in 183 mid-exponential phase and late stationary phase proteins from Thermus thermophilus HB8, an extremely thermophilic eubacterium. Functional classification of the propionylproteins revealed that the number of propionylation sites in metabolic enzymes increased in late stationary phase, irrespective of protein abundance. The propionylation sites on proteins expressed in mid-exponential and late stationary phases partially overlapped. Furthermore, amino acid frequencies in the vicinity of propionylation sites differed, not only between the two growth phases but also relative to acetylation sites. In addition, 33.8% of mid-exponential phase-specific and 80.0% of late stationary phase-specific propionylations (n ≥ 2) implied that specific mechanisms regulate propionylation in the cell. Moreover, the limited degree of overlap between lysine propionylation (36.8%) and acetylation (49.2%) sites in 67 proteins that were both acetylated and propionylated strongly suggested that the two acylation reactions are regulated separately by specific enzymes and may serve different functions. Finally, we also found that eight propionylation sites overlapped with acetylation sites critical for protein functions such as Schiff-base formation and ligand binding.
Highlights
From the *Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
We demonstrated for the first time that lysine propionylation is a prevalent post-translational modifications (PTMs); we identified 361 propionylation sites in 183 proteins expressed in the midexponential and late stationary phases of T. thermophilus
We identified a total of 361 unique propionylation sites in 183 proteins in the mid-exponential and late stationary phases (Table I and supplemental Table S1)
Summary
Media and Culture Conditions—Tryptone and yeast extract were purchased from Nihon Seiyaku (Tokyo, Japan). Preparation of Crude Extracts and Tryptic Digests—A cell pellet harvested at either mid-exponential or late stationary phase was suspended in 5 ml of lysis buffer (50 mM Tris-HCl containing 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride, pH 8.0) and disrupted using an ultrasonic disruptor (UD-201, TOMY, Tokyo, Japan) at 4¦°C. The enriched propionylpeptides were eluted three times with 200 l of 0.1% trifluoroacetic acid, and the eluates were pooled These immunoprecipitation steps were repeated twice per biological replicate. Validation of Identifications Using Synthetic Propionylpeptides— Two propionylpeptides, ALFAEKprDGR from TTHA1742 and LLFKprDEVR from TTHA1552, were selected for verification of propionylation sites that we identified in this study These peptides were synthesized by Hokkaido System Science Co. Ten amino acids on the N-terminal and C-terminal sides of propionylated lysine residues were subjected to amino acid sequence context analysis, and the results were plotted as the percent difference (p value Ͻ 0.05)
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