Shut Down, Don't Stress Out

Chemical reactions lie at the heart of many biological processes, from photosynthesis and respiration to cell signaling and drug metabolism. Thanks to an atmosphere rich in oxygen, many organisms use oxygen to carry out these life processes. But oxygen metabolism produces highly toxic by-products called reactive oxygen species. When oxidation outpaces detoxifying reactions, oxidative stress occurs, and accumulating reactive oxygen species are free to wreak havoc on cellular machinery. Cysteine, one of the 20 different amino acids that make up proteins, contains a thiol group, which can be modifi ed upon oxidation. A thiol group can stabilize protein structures by forming covalent disulfi de bonds and can mediate cysteine-regulated redox reactions. At the same time, however, the high reactivity of thiol groups makes them also particularly vulnerable to nonspecifi c reactions during conditions of oxidative stress. Over the past few years, an increasing number of proteins have been discovered that use oxidative thiol chemistry to regulate their protein activity. In PLoS Biology, Lars Leichert and Ursula Jakob describe a novel method to monitor thiol modifi cations in proteins subjected to varying redox conditions in a living organism, the bacteria Escherichia coli. This technique is capable of providing a global snapshot of the redox state of protein cysteines during normal and oxidative stress conditions in the cell. To detect proteins that have the ability to undergo stress-induced thiol modifi cations, Leichert and Jakob differentially labeled the thiol groups of thiol-modifi ed and non-thiol-modifi ed proteins. The proteins were then separated on two-dimensional gels based on their charge and molecular weight. If the technique worked, most thiol-modifi ed proteins should be detected in the oxidizing environment of the E. coli periplasm (the region between the cell's membrane layers), and they were. After proving the method's ability to detect proteins whose thiol groups were oxidized, the next logical step was to determine what proteins DsbA—the enzyme that catalyzes disulfi de bond formation in the E. coli periplasm—was targeting. In E. coli mutant strains that lack DsbA, Leichert and Jakob identifi ed a number of proteins with either substantially less or no thiol modifi cation as compared to wild-type (non-mutant) strains, suggesting that these proteins are indeed DsbA substrates. In contrast to the periplasm, the E. coli cytoplasm contains several reducing systems. When the researchers tested a mutant strain that lacked the gene for the reducing enzyme thioredoxin, they found that a large number …