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U of Chicago Staph Study Suggests Cysteine Phosphorylation More Important, Widespread Than Thought

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A team led by researchers at the University of Chicago has identified cysteine phosphorylation as playing a key role in regulating virulence and antibiotic resistance in the human pathogen Staphylococcus aureus.

The findings, which they detailed in a paper published last week in the Proceedings of the National Academy of Sciences, offer new insight into bacterial signaling and suggest that cys phosphorylation may be a more widespread and prominent protein post-translational modification than previously thought, Chuan He, a University of Chicago researcher and author on the paper, told ProteoMonitor.

While protein phosphorylation has for some time been a key area for proteomics work and drug-development efforts, researchers have typically focused on phosphorylation of serine, threonine, tyrosine, histidine, and aspartate residues while paying less attention to cys phosphorylation.

Indeed, He and his colleagues didn't set out to study cys phosphorylation, but came across it while looking into the role of cysteine oxidation in regulating S. aureus' response to oxidative stress.

"Because [S. aureus] is a human pathogen, one of its intrinsic properties is the ability to adapt to oxidative stress – because it has to adapt to [the human] immune response," he said. "In the last several years my lab has discovered a range of factors and metabolic proteins that seem to respond to oxidation, and they use cysteines to sense oxidative stress and fine-tune" the bacteria's response.

Cys oxidation alone, however, didn't seem enough of a factor to fully account for S. aureus' response to oxidative stress, He noted, which led the researchers to speculate that other modifications might be involved.

"This bothered us for several months," he said, "until we suddenly realized that the cysteines might be phosphorylated. And, indeed, when we looked closely, the cysteines were phosphorylated."

The scientists identified, in particular, cys phosphorylation on the transcriptional regulators SarA, MgrA, and SarZ, part of the SarA/MgrA family of regulators, which play a role in controlling S. aureus virulence and response to changes in host environment.

To detect the cys phosphorylation, He and his colleagues performed in vitro phosphorylation assays on SarA, MgrA, and SarZ using S. aureus cell extract, finding that the three proteins were indeed phosphorylated. They also found that no protein phosphorylation occurred when the proteins' cysteine residues were mutated to serines – indicating that cysteine was in fact the site of the detected phosphorylation.

They followed up on this in vitro assay with LC-MS/MS analysis of the proteins on a Thermo Fisher Scientific LTQ Orbitrap Velos instrument, again detecting phosphorylated cysteines.

The researchers also established that phosphorylation took place only on reduced forms of the proteins, not the oxidated varieties, suggesting, they wrote, that "phosphorylation of these proteins is highly redox-dependent."

This, He suggested, is evidence of cross-talk between redox signaling and phosphorylation networks. "What's going on is there are surface receptors — two component systems that receive signals from antibiotics or other [stimuli] — and then they basically control gene expression through cysteine regulation."

The researchers also identified cys phosphorylation on other global transcriptional regulators including CymR and a variety of proteins beyond transcription factors, suggesting a potentially broad role for the modification.

"In [S. aureus] there's a lot more cysteine phosphorylation beyond the transcription regulators, and there are also other bacteria in which we found cysteine phosphorylation, so it appears to be more widespread than we previously thought," He said.

He noted that the broad conservation in Gram-positive bacteria of the Stk/Stp kinase/phosphatase pair responsible for the observed cys phosphorylation offers another indication of the modification's likely presence across a wide range of organisms.

"I think our paper points in the direction that people studying [Mycobacterium tuberculosis] or other Gram-positive bacteria should really think about [cys-phosphorylation]," he said. "Some of these [Stk/Stp pairs] might be [modifying] serine and threonine, but maybe many of these homologs are actually cysteine kinases and phosphatases."

Moving forward, He said, he and his colleagues hope to further flesh out cys phosphorylation's role in S. aureus as well as attempt to detect it in other bacteria and eukaryotes. For this work they will likely use mass spec to enable a proteome-scale investigation of cys phosphorylation.

"When you look at the mass spec pattern, you can see them if you are paying attention," he said. "That's the key – most people never thought cysteine would be phosphorylated so they weren't really paying attention."

Given this, it could be interesting to reinterrogate previously generated phosphoproteomic datasets to look for cys phosphorylations that the original researchers might have passed over, He said. Given that cys phosphorylation is more labile than serine or threonine phosphorylation, though, the usefulness of such an approach will likely depend on the conditions under which the original data was generated, he said.

The modification is stable under physiological conditions, He said, but noted that it "can be labile if you use very extreme [pH] conditions or strong reducing agents." He added that titanium dioxide – which is commonly used to enrich phosphopeptides – might not be compatible with analysis of cys phosphorylation.

He noted that existing antibodies to phospho-cys are also in need of optimization. "We haven't developed an antibody [specifically] for cysteine phosphorylation," he said. "We have some antibodies that appear to work but aren't working great, so I think there is still a lot of room to develop more selective tools to study [the modification.]"

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