The Response of Rhodotorula mucilaginosa to Patulin Based on Lysine Crotonylation

Abstract

Patulin (PAT) is a mycotoxin produced by some Penicillium, Aspergillus, and Byssochlamys species. Rhodotorula mucilaginosa is able to degrade PAT in vivo as well as in vitro, up till date, the process and molecular mechanism(s) involved patulin degradation still remains unknown. Protein lysine crotonylation (Kcr) plays an important role in regulating chromatin dynamics, gene expression, and metabolic pathways in mammals and eukaryotes. Investigation of the Kcr changes accompanying degradation of patulin in R. mucilaginosa were observed to investigate the mechanisms of patulin inhibition. Tandem mass tag (TMT) labeling and Kcro affinity enrichment, followed by high-resolution LC-MS/MS analysis, were used to perform quantitative lysine crotonylome analysis on R. mucilaginosa. Consequently, 1691 lysine crotonylation sites in 629 protein groups were identified, among which we quantified 1457 sites in 562 proteins. Among the quantified proteins, 79 and 46 crotonylated proteins were up-regulated and down-regulated, respectively. The differentially up expressed modified proteins were mainly involved in tricarboxylic acid cycle and gluconeogenic pathway. The differentially down expressed Kcr proteins were mainly classified to ribosome and carbohydrate transport and metabolism. Bioinformatic analyses were performed to annotate the quantifiable lysine crotonylated targets. Moreover, interaction networks and high confidence domain architectures of crotonylated proteins were investigated with the aid of bioinformatic tools, and these results showed that there was an increase in the number of yeasts with crotonylated proteins. The results also provided information on the various roles of crotonylation, which are involved in PAT degradation.

Keywords: Rhodotorula mucilaginosa; degradation; histone lysine crotonylation; patulin; post-translational modification.

FIGURE 1

The subcellular up-regulated and down-regulated proteins (Y+PAT/Y). (A) Subcellular localization of the up regulated proteins by patulin. (B) Subcellular localization of the down regulated proteins by patulin.

FIGURE 2

Analysis the characters of the identified peptides. (A) Crotonylation motifs were constructed with Motif-X software. The central K (at position 0) indicates the crotonylation lysine. All the surrounding amino acid residues are indicated with the letters in different heights which is consistent with their frequencies in respective positions. (B) Heat map showing the frequency of the amino acid residues around the crotonylated site based on analysis of all the identified peptides. (C) Number of the crotonylated peptides in each motif. (D) Heat map showing the enriched motifs in the representative KEGG pathways.

FIGURE 3

GO-based enrichment analysis of up-regulated and down-regulated proteins (Y+PAT/Y). (A) Classification of the up-regulated proteins with molecular function and biological process. (B) Classification of the down-regulated proteins with molecular function and biological process.

FIGURE 4

Bioinformatics analysis of the lysine crotonylated proteins of R. mucilaginosa (Y+PAT/Y). (A) Classification of the down regulated lysine crotonylated proteins with molecular function and biological process. (B) Classification of the up regulated lysine crotonylated proteins with molecular function and biological process.

FIGURE 5

The metabolic pathway influenced by patulin and Protein interaction network of Kcr proteins. (A) Lysine crotonylation of metabolic enzymes identified by MS based proteomics in TCA cycle. (B) Protein interaction network of TCA cycle. (C) Protein interaction network of Kcr proteins. Size of the node represent different number of modification sites. Different colors represent differential regulated type: up-regulated protein (red); down-regulated protein (green); up-regulated and down-regulated protein (yellow); no differential expressed protein (light blue).