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St. Jude Researchers Develop Method To ID Mysterious Unstructured Proteins

Researchers at St. Jude’s Children’s Research Hospital in Memphis have developed a technique to better identify a little-understood class of proteins called intrinsically unstructured proteins, whose existence was widely accepted by the scientific community only about a decade ago.
Unlike other proteins, IUPs lack three-dimensional shapes. While they are known to play important biological roles in the cell by transmitting signals and coordinating biochemical and genetic activities, little else is known about them.
In their report, the authors said that their method, especially coupled with the most sensitive mass spectrometry-based techniques “can be used for the detection and functional characterization of disordered signaling and regulatory proteins involved in pathogenesis.”
According to authors, their study is a “gateway” for clinically relevant studies such as those exploring the possibility of developing prognostic tools using IUPs.
Up to 40 percent of the 30,000 proteins in humans are believed to be IUPs, but only 460 have been identified and catalogued in the Database of Protein Disorder, according to DisProt’s Web site.
Using their technique, St. Jude researchers were able to identify about 200 such proteins, including 150 new ones, said Richard Kriwacki, an associate member at St. Jude’s and the lead author of a report describing the method appearing in the online issue of the Journal of Proteome Research.
The researchers are still developing “newer, more sensitive, and more powerful proteomics methods to identify instead of 200, on the order of 1,000 IUPs at a time,” he said. When that has been accomplished, the next step would be further diagnostic and clinical research on the IUPs.
“One of the obvious applications of this technology … is to look for IUPs that are biomarkers of disease. That’s a direction in which we’re going,” Kriwacki said, adding that research by other groups and individuals suggests a correlation between IUPs and cancer.
Data from other researchers, he said, have found that about 70 percent of proteins known to be associated with cancer are likely to be intrinsically unstructured. One IUP, p27, has already been found to be cancer-associated.
P27 functions to prevent cells from dividing, and cancer cells have low levels of the protein resulting in their ability to proliferate uncontrolled.
“This is an example of an IUP whose levels are reduced very deliberately, biochemically, inside cancer cells,” Kriwacki said. “Because of the involvement, in general, of IUPs in signaling and regulation and since a lot of what goes wrong in cancer cells is disruption of naturally occurring signaling and regulatory mechanisms, it’s not surprising that there’s this association between IUPs and cancer.” 
Structure is Biology …Or is it?
So little is known about IUPs because the general scientific community has only recently acknowledged their existence, said Kriwacki. While researchers first noticed the prevalence of proteins that seemed to be structure-less about 25 years ago, the notion that proteins could function biologically without structure was viewed as “heretical” in the biological community, he said.
“There’s a very well accepted concept that a protein’s structure confers its biological function. It’s central dogma in biochemistry,” Kriwacki.
The concept of functional IUPs began to emerge in the 1990s, but the broad scientific community began to accept the idea only within the past few years, Kriwacki said. It has been only in the past year that researchers have used proteomic methods and technologies to analyze IUPs “rather than just sort of inferring the existence of proteins on the basis of gene sequences.”

“One of the obvious applications of this technology … is to look for IUPs that are biomarkers of disease. That’s a direction in which we’re going.”

The method developed by the St. Jude’s researchers is based on using heat treatment and other denaturing conditions such as organic solvent and low pH to enrich and separate the IUPs from other protein classes. Heated protein extracts were analyzed by SDS-PAGE to determine the extent of protein precipitation.
Heat treatment “resulted in an enrichment of IUPs and depletion of [structured proteins],” the authors wrote in their report. “IUPs comprised only 11.8 percent of the proteins identified in the untreated cell extract [4 degrees Celsius] but 41.9 percent following heat treatment at 98 degrees Celsius.
“Conversely, mixed ordered, or disordered proteins, and intrinsically folded proteins which comprised 42.8 and 45.4 percent of proteins in the untreated cell extract, were substantially depleted to 27.4 and 30.6 percent, respectively, after heat treatment at 98 degrees Celsius,” they wrote.
Once the IUPs were separated from the other class of proteins, Kriwacki and his colleagues used mass spectrometry to identify them, then checked them against the DisProt database and the RCSB Protein Data Bank to sort known IUPs from novel ones. Most of the IUPs identified were cytoplasmic or nuclear proteins involved in cell signaling and regulation, the authors wrote in their report.
As a result, “it can be expected that many of these proteins will be present at low levels within cells.” The inherent low sensitivity of 2D gels also will hinder the detection of IUPs using their technique, the authors wrote.
However, heat treatment for the enrichment of IUPs combined with the use of alternative more advanced proteomic techniques such as MudPIT “should lead to the identification of significantly larger number of IUPs from mammalian cell extracts,” the authors suggest.

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