Abstract
BackgroundLysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb.ResultsIn this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date.ConclusionsA total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.
Highlights
Protein posttranslational modifications (PTMs) are reversible and dynamic modifications of proteins that are crucial to protein maturation, protein structure and function
To detect the Keywords: Lysine 2-hydroxyisobutyrylation (Khib) modification status in rhubarb, total protein samples were isolated from rhubarb leaves and submitted to proteome-wide identification conducted by combining affinity enrichment by the Khib antibody with nano-HPLC-MS/MS
Most proteins contain only one Khib site, 4 proteins were found to have more than 20 Khib sites, including elongation factor 2-like (22 sites), hypothetical protein CCACVL1_04477 (22 sites), ribosomal protein (23 sites), and heat shock cognate protein 80 (Supplemental Table S1), indicating that Khib is a type of multiple PTM
Summary
Protein posttranslational modifications (PTMs) are reversible and dynamic modifications of proteins that are crucial to protein maturation, protein structure and function. Khib 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 [13,14,15] and Toxoplasma gondii [16] These findings indicate that Khib exists on histone proteins in the nucleus and widely exists on nonhistone proteins in various parts of the cells. Khib modification has not been reported far in rhubarb
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