Proteome-wide identification of lysine 2-hydroxyisobutyrylation reveals conserved and novel histone modifications in Physcomitrella patens

Abstract

Protein lysine 2-hydroxyisobutyrylation (K_hib) is a newly identified post-translational modification found in animal and yeast cells. Previous research suggested that histone K_hib is involved in male cell differentiation and plays a critical role in the regulation of chromatin functions in animals. However, information regarding protein K_hib in plants is still limited. In this study, using a specific antibody and LC-MS/MS methods, we identified 11,976 K_hib sites in 3,001 proteins in Physcomitrella patens. The bioinformatics analysis indicated that these K_hib-modified proteins were involved in a wide range of molecular functions and cellular processes, and showed diverse subcellular localizations. Furthermore, an comparism of K_hib sites in histone proteins among human, mouse and P. patens found conserved sites in the H3 and H4 histone proteins and novel sites in H1, H2A and H2B histone proteins in P. patens. This is the first report on K_hib post-translational modifications in plants, and the study provides a comprehensive profile of K_hib sites in histone and non-histone proteins in Physcomitrella patens.

Figure 1

Workflow for large-scale detection of K_his modification sites in P. patens. (A) Western blotting analysis of total protein extracts to investigate the existence of K_hib-modified proteins in P. patens. (B) The experimental strategy to identify K_hib modification sites in P. patens. Total protein extracted from P. patens was digested by trypsin. Peptides containing K_hib modification sites were enriched by immunoprecipitation with a K_hib-specific antibody and subsequently analyzed by LC-MS/MS.

Figure 2

Quality control validation of MS data. (A) Distribution of the mass errors of the K_hib-containing peptides. (B) Peptide length distribution. (C) Number of modified sites in a protein.

Figure 3

Analysis of K_hib-modified proteins. (A) Functional classification of K_hib-modified proteins. (B) Subcellular localizations of K_hib-modified proteins in P. patens.

Figure 4

Sequence analyses of amino acids flanking the K_hib sites in P. patens. Heatmap shows enrichment (red) and depletion (green) of amino acids in specific positions flanking K_hib in moss.

Figure 5

K_hib sites in human, mouse and P. patens histone proteins. The protein sequences of histones in human are shown, with the modified lysines marked in red. The positions of modified lysines in mouse and moss are marked with dots and arrows, respectively. The canonical histone sequences are shown as the base sequences. K_hib modifications in variant histones are marked by asterisks.