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Scripps Researchers Identify New Protein PTM Linked to Glycolysis

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Scientists at the Scripps Research Institute have identified a new protein post-translational modification that appears to play a role in glycolysis.

Described in a paper published last week in Science, the modification occurs when the primary glycolytic metabolite 1,3 bisphospoglycerate reacts with protein lysine residues to form 3-phosphoglyceryl-lysine, or pgK.

According to Raymond Moellering, first author on the paper and a post-doc in the lab of Scripps researcher Benjamin Cravatt, co-author on the paper, the modification could prove of diagnostic and therapeutic interest in understanding the remodeling of cellular metabolism in diseases like diabetes and cancer.

Enzyme-dependent PTMs like phosphorylation are a primary area of proteomics research. Enzyme-independent PTMs like the pgK modification identified by Moellering and Cravatt, however, have been less thoroughly investigated.

A number of recent publications, Moellering said, have identified enzyme-independent PTMs generated by secondary metabolites – molecules not directly involved in the normal function of an organism, but, he told ProteoMonitor, "we were wondering whether there were primary metabolites that were intrinsically reactive and might be able to modify proteins."

The researchers did a survey of potential metabolites from which 1,3-BPG emerged as a likely candidate due to its highly electrophilic acylphosphate group – a component known to react with lysine residues.

Testing the metabolite in vitro, Moellering was able to demonstrate that 1,3-BPG did, in fact, modify lysine, and, based on this finding, the researchers began to look for the pgK modification in vivo.

"We went forward and started devising ways to pull [the modified proteins] out of cells to see if it was there," Moellering said. "And with every step we took, it became more and more intriguing because not only were we finding it, but we were finding it on functional proteins and on functional lysines."

In particular, he said, they observed the modification on lysines in the active sites of glycolytic enzymes, suggesting it may play a role in glycolysis. The 1,3-BPG metabolite responsible for the pgK modification is a product of catalysis by glyceraldehyde-3-phosphate dehydrogenase, GAPDH – a key glycolytic enzyme, and, Moellering suggested, pgK modifications could be governed by the presence of GAPDH.

"We think that what may be happening is that where GAPDH goes, it introduces this [1,3-BPG] molecule, so if GAPDH is localized to a complex, those proteins are more susceptible to pgK modification," he said.

Moellering added that identification of the pgK modification might also shed some light on other functions of GAPDH, which, beyond its function in glycolysis, has also been linked to processes including gene transcription and apoptosis.

"We think that maybe part of what it is doing in those other spots is delivering this [1,3-BPG] molecule and therefore this [pgK] modification," he said.

Moellering said it was very unlikely that pgK would be as widespread as common modifications like phosphorylation, but, he noted, the findings of the Science study likely didn't represent its full scope given the lack of an optimized method for enriching the modification upfront of mass spec analysis.

Essentially, Moellering said, the researchers used standard phosphopeptide enrichment methods, operating under the assumption that the pgK peptides were similar enough to phosphopeptides that such an approach would prove effective.

This similarity between the two modifications also enabled the researchers to reinterrogate previously generated phosphoproteomic datasets to look for the presence of pgK-modified proteins.

"That was something we found really powerful and really illuminating," Moellering said, noting that he and Cravatt looked at phosphoproteomic data in nine different mouse lines generated by the lab of Harvard University researcher Steven Gygi.

"We figured that would be a good place to look because it was a very expensive, well-done proteomic experiment," he said. And, Moellering noted, "because they used different upfront fractionation methods, different instruments, even a different organism," the Gygi dataset offered a chance to see if, given these different experimental parameters, they would still find the modification.

Indeed, he said, "we saw it all over the place" in the Gygi data. "So it was really clear that this [pgK] data is embedded in the phospho data of other proteomics groups."

Nonetheless, a more pgK-specific enrichment scheme would likely enable more comprehensive study of the modification. Enrichment is necessary due to the relative scarcity of the PTM, as well as the fact that, because of the affect of the modification on the lysine residue, pgK-modified peptides will typically be poor candidates for mass spec analysis.

"So, if you don't have an enrichment step upfront, your [modified] peptide will get swamped out [by other molecules] in the mass spec," Moellering said.

The researchers have made pan-pgK antibodies to allow such enrichment that they are currently in the process of optimizing, he said. "We think that will give us not only more specific enrichment but also a better picture of where these [modifications] are. I think we'll see more sites once we remove the background of phosphopeptides."

Moving forward, Moellering said, they are interested in looking at what role the modification might play in remodeling metabolism in various diseases. For instance, he said, in diabetes "extracellular glucose is chronically deregulated, so it would be interesting to know if this PTM is involved in that and if there is a way to use it diagnostically or therapeutically."

Likewise, in cancer, tumor cells are known to significantly upregulate their glycolytic processes. "So it would be interesting to see whether or not these modifications are increased under those conditions, and what kind of functional role they might play," he said.

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