Ionizing Radiation-induced Proteomic Oxidation in Escherichia coli

Steven T Bruckbauer,Benjamin B Minkoff,Deyang Yu,Vincent L Cryns,Michael M Cox,Michael R Sussman

doi:10.1074/mcp.ra120.002092

Abstract

Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli, the most significant target of IR by a wide margin was glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life.

Highlights

Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteomewide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration
Quantification of IR-induced modification to the E. coli and D. radiodurans proteomes— much previous work has examined biological responses to IR exposure, we have chosen to focus on the immediate, abiotic chemical effects of IR on the in vivo proteome before any biological response
To ensure our observations of IR-induced changes to the E. coli proteome are abiotic, cultures were cooled to 4°C before irradiation and extensively washed in 13 phosphate-buffered saline (PBS) (PBS) to halt metabolism and remove nutrients in growth media that may act as a radioprotectant

Summary

Introduction

Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteomewide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Hydroxylation (115.99 Da) was the most prevalent covalent adduct detected In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. A significant number of proteins have evolved to repair DNA damage, there do not appear to be ROS degrading enzymes and protein repair machinery yet evolved which address oxidative damage to the proteome caused by the large scale generation of highly reactive and short lived (nanoseconds) hydroxyl radicals generated by IR [16]. This is likely because of the presence of both widely conserved and unique DNA repair enzymes and to the extraordinary ROS scavenging capacity of the D. radiodurans metabolome [3, 5, 6, 18,19,20,21,22]

Methods

Results

Conclusion