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EMBL Team Investigates Phosphorylation-Lysine Acetylation Crosstalk in Pneumonia Bug


By Adam Bonislawski

A team led by scientists at the European Molecular Biology Laboratory has published a study in the current edition of Molecular Systems Biology exploring the relationship between protein phosphorylation and lysine acetylation in Mycoplasma pneumoniae.

Their findings suggest a broader-than-thought role for protein phosphorylation in post-transcriptional regulation as well as considerable crosstalk between phosphorylation and acetylation, showing significant linkage between the two modifications, Anne-Claude Gavin, a senior scientist at EMBL and leader of the study, told ProteoMonitor.

The researchers selected M. pneumoniae for the study due in large part to its "small toolkit," Gavin said. Given that the bacterium has only 691 protein-coding genes as well as just two protein kinases and one protein phosphatase, the EMBL team was able to develop nearly comprehensive profiles of its response to perturbations in its phosphorylation machinery.

Using a dimethyl labeling approach combined with analysis on a Thermo Scientific LTQ Orbitrap, they investigated protein expression, phosphorylation, and acetylation in wild-type M. pneumoniae as well as three isogenic mutants lacking either one of the two protein kinases or the phosphatase.

The researchers identified 564 proteins and reliably quantified 447. Of these proteins, 241 possessed either phosphorylation or lysine acetylation, with a total of 93 phosphorylation sites and 719 lysine acetylation sites identified on 72 and 221 different proteins respectively. According to Gavin, the researchers' characterization encompassed roughly 90 percent of the M. pneumoniae proteome.

Among the more surprising findings, Gavin said, was the prominence of lysine acetylation. Previous analyses of bacteria including Escherichia coli found around 200 such modifications, she noted, compared to the 719 in the MSB study.

The researchers are currently investigating this disparity to determine whether the high number of acetylation sites is a feature specific to M. pneumoniae or the result of improved detection methods, Gavin said, adding that she "suspects it is a general trend" across many types of bacteria.

"I suspect that lysine acetylation has been overlooked, and we used in this study a more sensitive technology [in the LTQ Orbitrap]," she said. "I wouldn't be surprised, if this analysis was redone in other bacteria with this type of machine, [to] see more lysine acetylation."

Also interesting, Gavin noted, was the amount of apparent crosstalk between phosphorylation and acetylation. This relationship had been previously demonstrated in certain particular examples, but not on such a global scale.

"Very, very often when a protein was phosphorylated, then we would also see a lysine acetylation … so we see them co-occur very often," she said. "What we see as well is that when we perturb phosphorylation, we affect [not only] phosphorylation, which is to be expected, but we also affect protein [expression] levels and lysine acetylation."

Overall, the researchers observed significant changes in expression levels in 39 of the 447 consistently quantified proteins in response to kinase or phosphatase deletion. Levels of mRNA were largely unaffected, suggesting the presence of post-transcriptional regulation.

"We know very well that some protein abundances are regulated by phosphorylation," Gavin said, "but to my knowledge the impact of protein phosphorylation on protein abundance globally has not been studied in a systematic way using proteomics."

The EMBL researchers also used the data from their analyses to explore network effects of phosphorylation perturbation, looking at how expression and modification patterns overlapped with protein interaction data.

Upon combining their results with interaction data from the STRING protein interaction database, they found that affected proteins interacted with each other 2.4 times more frequently than random sets of proteins. Direct interactors with the perturbed kinases and phosphatase were most affected, with a dramatic decrease in affected proteins further out in the network, demonstrating, Gavin said, "the buffering capacity of the network" that keeps such perturbations from "propagating all over."

Moving forward, the researchers aim to more fully investigate the function of protein phosphorylation and lysine acetylation in M. pneumoniae by linking proteins' specific modification states to their participation in particular protein complexes.

"One of the big problems we still have is we can't actually link the PTMs in this study to protein complexes because we don't know [from mass spec-based methods] if [we are looking at], for instance, a single protein with 10 modifications or 10 protein species each with one modification," Gavin said.

"So one interesting thing to do would be gel filtration experiments or affinity purifications where we could, for instance, look at specific proteins in the context of different oligomeric states and see what are its modifications," she said.

Such work would in principle be similar to the investigations into histone modifications currently being undertaken by many top-down proteomics researchers (PM 4/8/2011). Gavin noted that, although she has yet to contact any top-down experts, M. pneumoniae might be an ideal proving ground for their techniques.

"I think it would be a fantastic project for [top-down proteomics]," she said, "With [M. pneumoniae] they could probably cover the entire [proteome], because there are only around 600 proteins."

Indeed, in a paper published in Nature in November, researchers led by Northwestern University's Neil Kelleher used a four-dimensional separation platform attached to Thermo Scientific 12 T LTQ FT Ultra and Thermo Scientific Orbitrap Elite mass specs to identify a total of 1,043 proteins and more than 3,000 protein species in HeLa S3 cells (PM 11/4/2011).

"The level of complexity is very amenable to [a top-down] approach," Gavin said. "If you don't manage in [M. pneumoniae], it's unlikely you manage in human cells, right?

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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