A new method for mapping ‘social networks’ of proteins

Left to right: Joseph Ecker (image courtesy of Salk Institute) and Shelly Trigg (image courtesy of Austin Trigg)

Scientists at the Salk Institute for Biological Studies reported that they have developed a new technique to determine which proteins in a cell interact with each other.

Current mapping methods of cell interactions, also known as “interactome,” have been slow because the number of interactions that could be tested at once was limited. The new approach, called CrY2H-seq, was published on June 26 in Nature Methods.

CrY2H-seq allows researchers to test millions of relationships between thousands of proteins in a single experiment.

The interactome of a cell is similar to a map of social networks. It enables scientists to see “who’s working with who” in the world of proteins. By understanding the network, scientists are able to determine the roles of different proteins and piece together the different players in molecular pathways and processes.

For example, if a newly discovered protein interacts with lots of other proteins involved in cellular metabolism, researchers can deduce that’s a likely role for the new protein and potentially target it for treatments related to metabolic dysfunction, according to the La Jolla-based not-for-profit, scientific institute.

“The power of this new approach is the ability we now have to scale it up,” says senior author Joseph Ecker, professor, and director of Salk’s Genomic Analysis Laboratory and investigator of the Howard Hughes Medical Institute. “This assay has the potential to begin to address questions about fundamental biological interactions that we haven’t been able to address before.”

A new mapping method let researchers discover new links (gray lines) between two groups of plant proteins (yellow and blue) that have a common structure (the BBX domain), suggesting many different combinations of interactions, rather than a few, are involved in coordinating cellular programs like flowering time and circadian rhythm.
(Credit: Salk Institute)

Researchers have typically relied on standard high-throughput yeast two-hybrid (Y2H) assays to determine the interactions between proteins.

The system requires using a single known protein—known as the “bait”—to screen against a pool of “prey” proteins. However, finding all the interactions between, for instance, 1,000 proteins, would require 1000 separate experiments to screen once for each bait’s interaction partners.

“Current technologies essentially require that interactions detected in primary screening get retested individually,” says Shelly Trigg, an NSF Graduate Research Fellow at the University of California, San Diego, in the Ecker lab, and first author of the new paper. “That may no longer be necessary with the screening depth this approach achieves.”

Other researchers on the study were Renee Garza, Andrew MacWilliams, Joseph Nery, Anna Bartlett, Rosa Castanon, Adeline Goubil, Joseph Feeney, Ronan O’Malley, Shao-shan Carol Huang, Zhuzhu Zhang, and Mary Galli of the Salk Institute.

The method developed by the team adds a twist to the standard Y2H assay for a much more effective way of measuring the interactome.

The genes for two proteins, each on their own circle of DNA, are added to the same cell.

If the proteins of interest interact inside the cell, a gene called Cre is activated.

When turned on, Cre physically splices the two individual circles of DNA together. This pairs the genes of interacting proteins together so the team can easily find them through sequencing.

The team can generate a massive library of yeast cells—each containing different pairs of proteins by introducing random combinations of genes on circular DNA called plasmids.

When cells are positive for a protein interaction, the researchers can use genetic sequencing to figure out what the two proteins interacting are, using new high-throughput DNA sequencing technologies similar to those used for human genome sequencing.

With this method, the scientists are not limited to testing one “bait” protein at a time. Instead, they can now test the interactions between all the proteins in a library at once.

The group tested the CrY2H-seq method on all the transcription factors—a large class of proteins—in the plant Arabidopsis.

“When you take 1,800 proteins and test the interactions among them, that’s nearly 4 million combinations,” said Ecker. “We did that ten times in a matter of a month.”

They discovered more than 8,000 interactions among those proteins tested. The data helps answer longstanding questions about whether certain groups of transcription factors have set functions, according to the scientists.

The team members believe the CrY2H-seq method can be scaled to test larger sets of proteins. Human cells, for instances, contain about 20,000 different proteins.


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