Proteomic analysis of thioproline misincorporation in Escherichia coli

Recently it was discovered that thioproline, an unnatural analog of proline, can arise in vivo from the reaction of cysteine and formaldehyde in cells under oxidative stress. Sequence-specific bioincorporation of thioproline into proteins was studied via shotgun proteomics of Escherichia coli (E. coli) cells. In a strain auxotrophic for proline, thioproline was found widely incorporated in lieu of proline when the cells were incubated with thioproline. In total 1428 proteins and 235 distinct thioproline-containing peptides were identified. Label-free relative quantitation revealed 102 differentially expressed proteins (82 up-regulated and 20 down-regulated) in the thioproline-treated group (with thioproline in the medium) relative to the control group (with proline in the culture medium). Pathway enrichment analysis of the differentially expressed proteins showed that amino acid biosynthesis and protein synthesis has been most affected by thioproline exposure, as expected. Phenotypically, the thioproline-treated group was found to exhibit slower cell growth and stronger antioxidant capacity relative to the control. SIGNIFICANCE: Thioproline is a secondary metabolite of formaldehyde and a structural analog of proline. It is also known to exhibit a wide variety of pharmaceutical properties, but its exact biochemical role in the cell has not been elucidated. In this paper, we studied thioproline misincorporation (in lieu of proline) events during protein synthesis in E. coli. Global proteome profiling revealed that thioproline is extensively misincorporated throughout the proteome in E. coli cells exposed to thioproline, and pathways related to amino acid and protein biosynthesis are up-regulated. In addition, we demonstrated that pretreatment with thioproline appeared to increase E. coli cells' capacity to tolerate oxidative stress. Our findings suggest a novel explanation of thioproline's known antioxidative properties. This is, to our knowledge, the first ever study of thioproline misincorporation at the proteome level in any organism.