Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

Abstract

Background: Lysine 2-hydroxyisobutyrylation (Khib), a newly identified post-translational modification, is known to regulate transcriptional activity in animals. However, extensive studies of the lysine 2-hydroxyisobutyrylome in plants and animals have yet to be performed. Results: In this study, using LC-MS/MS qualitative proteomics strategies, we identified 4163 Khib sites on 1596 modified proteins in rice (Oryza sativa) seedlings. Motif analysis revealed 10 conserved motifs flanking the Khib sites, and subcellular localization analysis revealed that 44% of the Khib proteins are localized in the chloroplast. Gene ontology function, KEGG pathway, and protein domain enrichment analyses revealed that Khib occurs on proteins involved in diverse biological processes and is especially enriched in carbon metabolism and photosynthesis. Among the modified proteins, 20 Khib sites were identified in histone H2A and H2B, while only one site was identified in histone H4. Protein-protein interaction (PPI) network analysis further demonstrated that Khib participates in diverse biological processes including ribosomal activity, biosynthesis of secondary metabolites, and metabolic pathways. In addition, a comparison of lysine 2-hydroxyisobutyrylation, acetylation, and crotonylation in the rice proteome showed that 45 proteins with only 26 common lysine sites are commonly modified by three PTMs. The crosstalk of modified sites and PPI among these PTMs may form a complex network with both similar and different regulatory mechanisms. Conclusions: In summary, our study comprehensively profiles the lysine 2-hydroxyisobutyrylome in rice and provides a better understanding of its biological functions in plants.

Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang)

“…The overall trend of GO classification (including biological process, molecular function and cellular components) and subcellular localization of K hib proteins identified in rhubarb were similar to previous findings obtained in other plants such as P. patens [10] and rice [12], although the percentages of each category were slightly different.…”

“…Previous studies on both eukaryotic and prokaryotic cells have found a preference for specific amino acid residues around the K hib site [10][11][12]14]. Therefore, we utilized the Motif-X tool, software for extracting Fig.…”

“…These findings suggest that K hib should be present in nonhistone proteins and should have functions independent of nucleosomes. To date, K hib has been detected in eukaryotes, including human and animal cells [ 4 , 8 ] and tissues [ 9 ], yeast ( S. cerevisiae ) [ 6 ], plants ( Physcomitrella patens [ 10 ], Oryza sativa [ 11 , 12 ]), and bacteria, including Proteus mirabilis [ 13 – 15 ] and Toxoplasma gondii [ 16 ]. These findings indicate that K hib exists on histone proteins in the nucleus but also widely exists on nonhistone proteins in various parts of the cells.…”

Systematic analysis of lysine 2-hydroxyisobutyrylation posttranslational modification in wheat leaves

“…The protein extraction and trypsin digestion were performed referring previous report with some modifications [ 20 ]. In brief, wheat leaves were grinded into powder in a mortar with the addition of liquid nitrogen.…”

“…After incubation, rinse the beads for 4 times with NETN buffer and 2 times with purified water. Elute the captured modified peptides with 0.1% Trifluoroacetic acid and vacuum dry them [ 20 ].…”