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

Abstract

Background: Lysine 2-hydroxyisobutyrylation (K_hib) 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, K_hib modification has not been reported thus far in rhubarb.

Results: In this study, we performed the first global analysis of K_hib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping K_hib modification peptides matched on 1525 K_hib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these K_hib-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 K_hib site, and a total of 13 K_hib 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.

Conclusions: A total of 4333 K_hib-modified peptides on 1525 proteins were identified. The K_hib-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 EK_hib/K_hibE, GK_hib and k.kkk….K_hib….kkkkk, were extracted from a total of 13 K_hib-modified peptides. This study provides comprehensive K_hib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of K_hib modification, and the K_hib proteome data will facilitate further investigations of the roles and mechanisms of K_hib modification in rhubarb.

Keywords: Chinese herb rhubarb (Dahuang); Lysine 2-hydroxyisobutyrylation (Khib); Motif; Posttranslational modification (PTM).

Fig. 1

Distribution of identified lysine 2-hydroxyisobutyrylation (K_hib) peptides and proteins. (A) Distribution of K_hib-modified peptides based on their length; (B) Distribution of K_hib-modified proteins based on the number of K_hib peptides identified

Fig. 2

GO functional classification of the K_hib-modified proteins based on biological process, molecular function, and cellular component. The number of proteins and the percentages of each category are indicated

Fig. 3

Subcellular localization distribution of K_hib-modified proteins

Fig. 4

GO enrichment analysis of the K_hib-modified proteins. The y-axis denotes the categories of GO terms. The x-axis denotes the fold enrichment

Fig. 5

KEGG pathway and protein domain enrichment analysis of the K_hib-modified proteins. The y-axis denotes the categories of KEGG pathways (A) and protein domains (B). The x-axis denotes the fold enrichment.

Fig. 6

K_hib-modified enzymes enriched in the TCA cycle in rhubarb. The identified K_hib enzymes are indicated with a red background

Fig. 7

Properties of the identified K_hib peptides. (A) The K_hib sequence motifs and conservation of K_hib sites. The size of the letter indicates the frequency of the amino acid residing at that position. (B) Frequency of each motif occurring in peptides with K_hib modification. Motif score was also indicated in the histogram. (C) Heat map showing the frequency of the amino acid residues around the K_hib sites. The enrichment [11] and dispersion (green) of amino acids from −10 to +10 flanking the K_hib sites.