Lysine propionylation is a prevalent post-translational modification in Thermus thermophilus

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.

Fig. 1.

A, chemical structures of acetylated and propionylated lysine. B, schematic overview of identification of propionylation sites. Analysis of each biological sample involved three biological replicates (S1, S2, and S3), two anti-propionyllysine immunoprecipitations (IP1 and IP2) for each biological replicate, and two LC-MS/MS runs (Run1 and Run2) for each affinity-enriched sample. C, propionylation sites in mid-exponential and late stationary phase proteins identified in T. thermophilus; numbers adjacent to the columns indicate the number of biological replicates in which the sites were detected. ME, mid-exponential phase; LS, late stationary phase.

Fig. 2.

Verification of propionylation of the enriched peptide (ALFAEKprDGR) in vivo by comparison of elution times and MS/MS spectra of the synthetic peptide. Extracted ion current chromatograms (A) and MS/MS spectra (B) of m/z 531.782 for the affinity-enriched propionylpeptide and the synthetic propionylpeptide.

Fig. 3.

Functional classification of propionylproteins and the amino acid sequence context around propionylated lysine. A, distributions of the functional classes of 184 propionylproteins. B, distributions of functional subclasses of 108 propionylproteins in the “metabolism” class. C, the frequency of amino acid residues around propionylated lysines (p value = 0.05).

Fig. 4.

Comparison of propionylation sites between mid-exponential and late stationary phases. The numbers of identified propionylproteins (n ≥ 2) in each growth phase are shown in the upper Venn diagram, and the numbers of propionylation sites (n ≥ 2) are shown in the left Venn diagram. The numbers of propionylation sites on 40 proteins that were propionylated in both growth phases are shown in the right Venn diagram.

Fig. 5.

Comparative analyses of the expression levels of representative propionylated proteins between exponential and stationary phase. Two replicates for each growth phase were prepared. A reference gel image of mid-exponential phase of T. thermophilus, which reported spot numbers and identification results from our previous study (36), was used for comparative analysis as one of the exponential phase images in this study. Full images of 2-DE gels (pI 4 to 7, 13 cm) and each spot number are shown in supplemental Fig. S5. The left side shows the three-dimensional spot image, and the right side shows the magnified two-dimensional spot image of each propionylated protein on 2-DE gels. Red arrows indicate spots corresponding to target proteins. The number of identified propionylation sites in each propionylprotein is given in parentheses. The black number indicates the total number of identified propionylation sites (n ≥ 1) in both growth phases. The blue and red numbers indicate propionylation sites identified in mid-exponential and late stationary phase, respectively. The changes of theoretical pI values are presented in the upper 2-DE images. ME and LS indicate mid-exponential and late stationary phase, respectively. The spot numbers of specific proteins on the 2-DE reference gel are as follows: TTHA0161, Spot No. 3; TTHA0543, Spot No. 46; TTHA1028, Spot Nos. 180 and 181; TTHA1535, Spot No. 47; TTHA1838, Spot No. 150; TTHA1839, Spot Nos. 24 and 25; TTHA1840, Spot Nos. 60 and 61.

Fig. 6.

Functional classification of propionylproteins and the amino acid sequence context around propionylated lysines in mid-exponential and late stationary phases. Propionylation sites that were detected at least twice in biological triplicates were counted for these analyses. A, distributions of functional classes of propionylproteins. B, distributions of functional subclasses of propionylproteins in the “metabolism” class. C, the frequency of amino acid residues around propionylated lysines (p value = 0.05).

Fig. 7.

Comparison of propionylation and acetylation sites in T. thermophilus. The numbers of identified propionylproteins and acetylproteins are shown in the upper Venn diagram, and the numbers of modification sites are shown the left Venn diagram. The numbers of propionylation and acetylation sites in 47 acetylproprionylproteins are shown in the right Venn diagram.