New Insights into Radio-Resistance Mechanism Revealed by (Phospho)Proteome Analysis of Deinococcus Radiodurans after Heavy Ion Irradiation

Abstract

Deinococcus radiodurans (D. radiodurans) can tolerate various extreme environments including radiation. Protein phosphorylation plays an important role in radiation resistance mechanisms; however, there is currently a lack of systematic research on this topic in D. radiodurans. Based on label-free (phospho)proteomics, we explored the dynamic changes of D. radiodurans under various doses of heavy ion irradiation and at different time points. In total, 2359 proteins and 1110 high-confidence phosphosites were identified, of which 66% and 23% showed significant changes, respectively, with the majority being upregulated. The upregulated proteins at different states (different doses or time points) were distinct, indicating that the radio-resistance mechanism is dose- and stage-dependent. The protein phosphorylation level has a much higher upregulation than protein abundance, suggesting phosphorylation is more sensitive to irradiation. There were four distinct dynamic changing patterns of phosphorylation, most of which were inconsistent with protein levels. Further analysis revealed that pathways related to RNA metabolism and antioxidation were activated after irradiation, indicating their importance in radiation response. We also screened some key hub phosphoproteins and radiation-responsive kinases for further study. Overall, this study provides a landscape of the radiation-induced dynamic change of protein expression and phosphorylation, which provides a basis for subsequent functional and applied studies.

Keywords: Deinococcus radiodurans; dynamic change; kinase; phosphoproteome; proteome; radiation.

Figure 1

Experimental design, quality control and overview of (phospho)proteome data. (A) Experimental design for (phospho)proteome analysis of heavy ion irradiated D. radiodurans. Strains were treated with 3 doses of radiation from 20 to 160 Gy. Samples were collected at two time points (0 h, 2 h) after irradiation. The color scheme for each dose remained essentially the same throughout the paper. (B) A comparison with the count of proteins identified by D. radiodurans proteomics published in the last 3 years [10,32,33]. (C) The number of proteins and phosphosites identified as a whole and the average number identified per sample. (D) PCA analysis of the global proteome samples. Different colors/shapes represent different doses/time points. (E) Fraction of proteins and phosphosites dynamically regulated after multiple dose irradiation, determined by using a two-way ANOVA test (FDR < 0.05). (F) Distribution of the scale of changes of significantly regulated proteins and phosphosites, showing that the differences in phosphorylation are generally more extensive than those at protein levels.

Figure 2

Integration analysis of irradiation sensitive proteins. (A) The number of up- or downregulated proteins at different irradiation doses and time points. (B) Heatmap of z-scored protein abundance (iBAQ value) of the DEPs. (C,D) Venn diagram of the upregulated proteins at 3 radiation doses, and 0h (C) or 2h (D) after irradiation. (E,F) Function enrichment analysis of 0h upregulated proteins with different doses (E), and of upregulated proteins at different time points (F). The red scale represents over-representation, while the blue scale represents under-representation.

Figure 3

Irradiation response-related pathways. (A) The changes in all pathways of D. radiodurans after irradiation. The ring diagram from outside to inside is as follows: pathway class (a), pathway subclass (b), proportion of proteins identified (c), proportion of upregulated proteins (d), proportion of phosphoproteins (e), and proportion of proteins with upregulated phosphorylation level (f) (Table S3A). (B) Homologous recombination (RecFOR) and mismatch repair pathways. Red color indicates upregulation; (p) means phosphorylation in the protein. The differential expressions of proteins in the pathways are labeled in the table below.

Figure 4

Integration analysis of phosphoproteins and phosphosites. (A) Fuzzy c-means clustering of 4 dynamic patterns of differential phosphosites. (B) PPI network of interactions between proteins with differential phosphosites. Displayed network includes 25 core proteins and 85 suggested proteins that have at least one interaction with other protein(s). (C) Overview of major functional classification of proteins with differential phosphosites.

Figure 5

Comparative analysis of protein expression and phosphorylation modification. (A) Classification of protein expression and phosphorylation levels after irradiation. (B) The proportion of different classes in different irradiation states. (C) Functional enrichment analysis of various classified proteins. (D) Dynamic changes in modification levels of site-specific protein RpoB.

Figure 6

The classification of proteins upregulated in at least one state after irradiation which have a kinase domain. Blue to red color gradient denotes downregulation to upregulation compared with unirradiated samples at this time point. Fold changes were log2 transformed. The text and color on the right, respectively, indicate the name and category of the kinase domain corresponding to the protein. The protein labeled with ※ is a bacteria-specific protein.