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Toronto Team Hoping New ABC Transporter Interaction Map Will Support Drug Research, Other Clinical Efforts

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Researchers from the University of Toronto have created a map of the protein interactions of 19 members of the ATP-binding cassette transporter class in a species of yeast, combining this data with previously-reported ABC transporter interactions into a comprehensive "interactome."

Though the group did the mapping with ABC transporters from a yeast species — Saccharomyces cerevisiae — many of the proteins are homologous to disease-linked human ABC proteins. The group hopes the map, published online this week in Nature Chemical Biology, will be a resource for future applied research, providing insight into these molecules' roles in a variety of cellular processes with implications for drug development and other clinical areas.

Igor Stagljar, the study's senior author, told ProteoMonitor this week that his team has been interested broadly in how integral membrane proteins interact at the genome-wide level.

"ABC transporters are one of these pharmaceutically very important proteins because they play a role in health and disease, specifically drug resistance," Stagljar explained.

"Overall, membrane protein interactions are difficult to study because of their biochemical features," he said. "It won't work if you want to co-immunoprecipitate them or apply any proteomic biochemical method because you will have to use detergents, and then you destroy all the protein complexes."

Stagljar and his colleagues previously developed a method, called a membrane yeast two-hybrid, or MYTH assay to allow them to study interactions of full-length membrane proteins in their natural cellular environment. They harnessed that method in this recent study to build the yeast ABC transporter map.

The group described the procedure in Nature Protocols in 2010. Briefly, MYTH adapts a previously developed split ubiquitin method as an in vivo sensor of protein–protein interactions. One half of a split ubiquitin molecule is attached to an ABC transporter (or another cellular protein of interest) and the other half to its potential interaction targets. If the two bind, the ubiquitin molecule reforms, triggering the yeast cell to signal the interaction by turning blue, Stagljar explained.

In the group's ABC transporter study, Stagljar and his team used MYTH to map the interactions of all of S. cerevisiae's 19 non-mitochondrial ABC transporters.

They then combined the MYTH screening results with previously reported ABC transporter physical interactions available through the BioGRID database.

Overall, the study yielded a map covering 537 unique binary interactions across 366 proteins, the study authors wrote, which they annotated with functional classifications to create a standard map, as well as a map showing protein conservation in humans and known disease associations.

According to the study authors, the annotated interactome contains 14 functional groupings with varying sizes. The largest group — about 26 percent of the interactions — corresponds to proteins involved in transport and related processes. The second largest, with 16 percent, marks interactions with proteins of unknown function. And the third group — 15 percent— is involved primarily in metabolic processes, the study authors wrote.

Remaining groups range in size from one percent to seven percent of the whole, covering a diverse range of processes, the researchers reported.

This broad association "suggests that [ABC transporters] involvement in cellular function is more complex than previously demonstrated," the authors wrote.

Important for the map's potential usefulness in future clinically-oriented research, 50 percent of the interactions mapped involve a target with an identifiable human ortholog, about 40 percent of which are known to be associated with human disease.

Stagljar said that his team is interested in following up on some insights from the map that are promising for human pharmaceutical research.

The group went on in its recent study to investigate two potentially interesting associations revealed in the yeast ABC transporter interactome map: physical interactions of ABC transporters with one another and the interaction of these molecules with members of the zinc transport system.

The latter, Stagljar said, is an important area for drug development, because it opens the possibility that you could block ABC transporter activity by blocking associated zinc transport proteins.

"We showed [in the paper] for the first time this interaction between some ABC transporters of the PDR subfamily and zinc transporters," Stagljar said. "Because ABC transporters play a role in drug resistance, for instance by pumping out chemotherapeutic drugs, it would be of pharmacological importance if you could potentially block ABC transporters by blocking zinc transporters."

However, Stagljar said, his team's main goal is to generate interactome data so that other groups can also harness it.

He said his lab expects to publish several more papers in the near future with results of MYTH screening in yeast for other relevant proteins being investigated as drug targets, such as G-protein coupled receptors.

The team is also seeking funding for a study of the interactions of all human ABC transporters using an adapted version of MYTH, called maMYTH, designed to work with human cell lines.

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