Cysteine Oxidation

As the most reactive member of nature’s standard stable of amino acids, cysteine is commonly tapped as a nucleophile in enzyme active sites. But cysteine’s reactivity also makes it uniquely susceptible to oxidation by reactive oxygen species in the cell.

Some cysteine oxidations are simply the result of collateral damage incurred during times of oxidative stress. But others appear to be highly regulated smoke signals sent in the name of cellular communication.

Armed with a range of new chemical tools for detecting and isolating proteins bearing oxidized cysteines, scientists are trying to figure out which ones are which.

“The importance of addressing such questions becomes clear when one considers that oxidative stress and cysteine modifications are prominent features of many acute and chronic diseases, as well as the normal aging process,” says Kate S. Carroll, an assistant professor of chemistry at the University of Michigan. Her lab is one of a handful of groups trying to design better ways of detecting oxidized cysteines. “We think that oxidized cysteines may play important biological and pathological roles,” she says.

When compared with phosphorylation, acetylation, and other posttranslational modifications that regulate protein function, cysteine oxidation is the new kid on the block. But its potential as a regulatory switch is gaining wider appreciation.

Carroll points to tyrosine phosphatases as evidence of cysteine oxidation’s regulatory potential. These enzymes play key roles in cell growth by controlling the phosphorylation of tyrosine, a chemical modification that cells use as a signal. There’s good evidence to suggest that oxidization of an active-site cysteine thiol inactivates certain tyrosine phosphatases, she notes. As a result, a key phosphorylated-tyrosine signal persists until the oxidized enzyme is degraded or repaired.

“Oxidation of cysteine at sites other than the active site can also have complex regulatory functions,” says Cristina M. Furdui, an assistant professor of molecular medicine at Wake Forest University School of Medicine. In some kinase enzymes, for example, oxidation of a cysteine outside of the active site causes that oxidized cysteine to form a disulfide bond with a neighboring cysteine residue, she notes. Such disulfide bond formation can dramatically change the conformation of an enzyme, thereby altering its activity or targeting it for degradation.

The list of proteins suspected to undergo cysteine oxidation has dramatically increased in recent months, thanks to the development of chemical tools for detecting and isolating the “gateway” cysteine oxidation: sulfenic acid.

Cysteine sulfenic acid (–SOH) is the initial product of oxidation of cysteine by cellular reactive oxygen species such as hydrogen peroxide. Most sulfenic acids enjoy only a fleeting existence, quickly undergoing disulfide bond formation or further oxidation to sulfinic (–SO₂H) or sulfonic (–SO₃H) acids. So detecting sulfenic acids is a challenge.

All of the recently developed tools for sulfenic acid detection build on an observation first made by biochemist William Allison back in the 1970s. Figuring that sulfenic acid’s sulfur should be more electrophilic than that of cysteine or further oxidized species, he began scouting for nucleophiles that would react specifically with it. His hunt turned up the cyclic diketone 5,5-dimethyl-1,3-cyclohexadione, better known as dimedone.

Dimedone conjugates to protein-borne sulfenic acids by a mechanism that still has not been nailed down, Carroll notes. That hasn’t stopped her group and others from adapting the scaffold to tag and isolate proteins bearing sulfenic acids.

One of the first groups to do so was that of Leslie B. Poole, a professor of biochemistry at Wake Forest University. Her group has since reported dimedone-based reagents bearing a fluorophore label or a biotin handle for easy detection and isolation of proteins bearing sulfenic acids from cell extracts. Philip Eaton’s group at King’s College London has developed similar bio­tinylated dimedone-based reagents.

More recently, Carroll has made azide analogs of dimedone that can be used to label sulfenic-acid-containing proteins directly in live cells, minimizing the potential for oxidative artifacts during cell lysis. The azidodimedone-labeled proteins can then be conjugated to biotin or another tag via copper-catalyzed chemistry.

Last month, Carroll’s lab reported antibodies that recognize dimedone-labeled sulfenic acids (Proc. Natl. Acad. Sci. USA 2009, 106, 16163).

With sulfenic-acid-specific reagents in hand, scientists have begun profiling the proteins that undergo this modification in cells. For example, Carroll recently used her azidodimedone probes in live human cell lines to show that as many as 200 different cellular proteins undergo cysteine oxidation (ACS Chem. Biol. 2009, 4, 783).

Despite this and other hints that cysteine oxidation might be a more widespread phenomenon than previously appreciated, the functional relevance of cysteine oxidation remains untested in all but a few cases, notes Christopher J. Chang, an associate professor of chemistry at the University of California, Berkeley. His lab is building small-molecule cellular probes for peroxide and other reactive oxygen species.

“As with other posttranslational modifications, just because a cysteine is oxidized doesn’t necessarily mean it plays a functional role,” Carroll says. Nailing down whether a particular oxidized cysteine plays a functional role or is just damaged will require detailed genetic and biochemical follow-up, Eaton adds.

Commercialization of sulfenic acid detection tools will help people figure out what role cysteine oxidation plays in particular biological processes, Carroll predicts. To that end, she is working with Ann Arbor-based Cayman Chemical to commercialize her azidodimedone reagents and with Ann Arbor-based Assay Designs to bring her sulfenic acid antibodies to market. Eaton expects that a similar antibody developed in his lab will get to market this fall. And Poole tells C&EN that her group is working to commercialize their sulfenic acid detection reagents.

A key challenge for the future, scientists in the field note, will be to find chemical reactions that are specific for sulfinic acid and other oxidized species that trace their roots back to sulfenic acid. For example, sulfenic acids can in some cases form cyclic sulfenamides on the peptide backbone. The role of such sulfenamides remains poorly characterized, but they may serve to protect sulfenic acid from further oxidation, says John D. Helmann, a professor of microbiology at Cornell University.

Because sulfonic acids (–SO₃H) are the irreversible oxidative end-of-the-road for cysteine, mass spectrometry will likely suffice for their detection, Carroll notes. But she’s already thinking of ways to detect sulfinic acids (–SO₂H). “This kind of tool would help us figure out what fraction of sulfenic acids are susceptible to further oxidation to sulfinic acid,” she says. It might also help scientists determine whether such further oxidation might also play a regulatory role in cells.

“There’s a significant amount of incidental protein oxidation that occurs,” Helmann says. “We need to find ways to pinpoint the modifications that really matter as signals, because a better understanding of how cysteine oxidation can affect protein function might improve diagnosis of disease and potentially intervention.”