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Published on: June 21, 2021
Quantifying changes in the thiol redox proteome upon oxidative stress in vivo
Lars I Leichert1, Florian Gehrke, Harini V Gudiseva
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109, USA.
Summary
Reactive oxygen species (ROS) are key to host defense, targeting bacterial proteins. A new technique, OxICAT, precisely quantifies these oxidative thiol modifications in vivo, revealing distinct oxidant targets and essential genes in E. coli.
Area of Science:
- Biochemistry
- Microbiology
- Cellular Biology
Background:
- Host defense mechanisms utilize reactive oxygen species (ROS) to combat bacterial infections.
- Protein thiol groups are primary targets of ROS-mediated oxidative damage in vivo.
- Understanding these interactions is crucial for developing new antimicrobial strategies.
Purpose of the Study:
- To develop and validate a quantitative method for identifying and measuring in vivo oxidative thiol modifications.
- To identify specific protein targets of hydrogen peroxide (H2O2) and hypochlorite (NaOCl) stress in Escherichia coli.
- To elucidate the role of redox-sensitive proteins in bacterial response to oxidative stress.
Main Methods:
- Development of OxICAT (Oxidative In-Cell Cysteine Assay with Thiol-trapping), a quantitative thiol trapping technique.
- Application of OxICAT to identify and quantify oxidative modifications in hundreds of proteins in a single experiment.
- Determination of redox-sensitive cysteine residues within target proteins.
Main Results:
- Identified a subset of E. coli proteins with 30-90% oxidatively modified thiol groups under H2O2 or NaOCl stress.
- Demonstrated that distinct oxidants target specific proteins in vivo.
- Found that one-third of redox-sensitive proteins are encoded by conditionally essential genes, potentially explaining bacteriostasis.
Conclusions:
- OxICAT is a powerful tool for in vivo monitoring of oxidative thiol modifications across various organisms.
- Oxidative stress selectively targets distinct proteins, influencing bacterial physiology and essential gene function.
- Redox-regulated proteins play a critical role in protecting E. coli against oxidative challenges.
