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New Assay Could Aid Study of Brief Protein Interactions, Interpretation of Phosphoproteomic Work

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Medical University of Vienna researchers have developed a protein-proximity assay for investigating and validating short-lived protein-protein interactions.

Called M-Track, the technique, which they detailed in a paper published this week in Nature Methods, is a two-hybrid system based on an enzyme-substrate reaction. It offers an approach for studying protein interactions too brief to be detected via conventional methods, Egon Ogris, a Medical University of Vienna researcher and corresponding author on the study, told ProteoMonitor.

In particular, he said, the technique could prove useful for unraveling cell signaling networks, enabling research into kinase- and phosphatase-substrate interactions and validation of results generated via phosphoproteomic discovery work.

The technique relies on enzyme-catalyzed methylation of a bait protein of interest by a prey protein of interest. The bait protein is expressed as a fusion protein with a histone lysine methyltransferase and the prey protein is expressed as a fusion protein including a portion of a histone protein.

If the two proteins interact, the prey protein will methylate the bait protein. This methylation – and by extension, the proteins' interaction – can then be detected via western blotting using antibodies specific to the different histone meythlation states.

An initial version of M-Track was conceived by Medical University of Vienna researcher Gustav Ammerer – a collaborator on the Nature Methods paper – and, upon seeing a presentation by Ammerer on the method, Ogris realized its potential for his own work on serine/threonine phosphatases, and in particular for the identification of phosphatase substrates.

"When I saw [Ammerer's] stuff, I thought it was a cool thing because here was an enzyme that left a [methylation] mark [on a bait protein] that, at least in yeast, was very stable," he said. "So if [a phosphatase] goes to [its cognate substrate] and then leaves very quickly – which it usually does – maybe that [interaction] would give in a two-hybrid assay the SuVar enzyme a chance to leave its [methylation] mark."

"Basically we were trying to detect the targets of [the phosphatase] by following the methylation traces, and that turned out to work," he said.

Key to the technique, Ogris noted, is use of a specific methyltransferase – the H320R mutant of HKMT SUV39H1 – that has a more than 20-fold higher catalytic activity than the wild-type enzyme. According to calculations based on previously published data, wild-type SUV39H1 was slower-acting than the phosphatases Ogris wanted to study. The mutant version, on the other hand, was able to keep up with these enzymes, enabling the researchers to use its activity as a proxy for that of the phosphatases.

Methylation likely indicates a direct interaction between the phosphatase and its substrate, although, Ogris acknowledge, the researchers have not directly shown that dephosphorylation took place during methylation detection in vivo. However the researchers used several methods in the study to validate the technique and provide evidence for their claim of detecting phosphatase-substrate interactions.

Most notably, Ogris said, the researchers performed a test of the assay using the protein Net1 – a potential substrate of the phosphatase – and a fusion protein phosphatase that targets it, checking to see if the fusion protein phosphatase would methylate a Net1 mutant that lacked three of its usual phosphorylation sites. They found that the phosphatase would not methylate the Net1 mutant, indicating, he said, that the methylation was dependent on the phosphatase activity.

One potential issue with the technique, as with any two-hybrid technique, he noted, is that of false positives, with methylation potentially resulting from the intrinsic affinity of the detection enzyme SuVar to its cognate target sequence, This, however, can be controlled for by including the necessary control experiments, Ogris said.

The Vienna team used the technique to investigate protein interactions in the high osmolarity glycerol response pathway as well as interactions between protein phosphatase 2A and several of its substrates. While M-Track is inherently a targeted approach, Ogris suggested that it could fit into proteomics discovery work as a tool for validating or interpreting results from large-scale phosphoproteomic screens.

For instance, he said, by allowing researchers to distinguish between direct and indirect kinase and phosphatase targets, it could enable them to better determine the structure of cell signaling cascades.

The authors also noted in the paper that they "envision several future developments of the system such as substrate identification screens using mass spectrometry or the quantitative analysis of dynamic PPI changes by micro-western arrays."

Orgis said the technique might be useful in mutational analysis, as well, enabling researchers to track how particular mutations affect enzyme-substrate interactions.

Ogris said the authors have no intention of patenting or commercializing the M-Track technique itself. He said, however, that they might license the methylation-specific monoclonal antibodies used for the read-out step. He already has deals with several biotech companies for anti-protein phosphatase 2A and other monoclonal antibodies developed by his lab.

M-Track is designed for use in yeast, which lacks a cognate methyltransferase and demethyltransferase to the one used in the assay, Ogris said, noting that this "means that these [methylation] marks in [yeast] are rock solid stable. Basically they just die with the degradation of the protein."

The researchers' efforts to use the system in mammalian cells, on the other hand, were unsuccessful, he said. They attempted to use it in cells where the two SUVH homologs had been knocked out, but the prey protein "got methylated by other methyltransferases still present in the cell."

Currently, Ogris said, he and his colleagues are pursuing another enzyme pair that they hope will allow the method to work in mammalian cells, although he declined to say what enzymes they were exploring.

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