Nitric oxide may change basic understanding of cellular...( l modification of proteins by the addit...)

l modification of proteins by the addition of NO, a process known as nitrosylation, changes their shape and function, thereby altering how they interact with one another in precise signaling networks that control the machinery of the cell. The composition of the signaling networks governs cellular behavior such as proliferation and response to outside chemical signals.

The researchers uncovered NO's ability to alter protein interaction by screening protein behavior both with and without levels of NO naturally present in cells.

"When we incorporated NO into protein screens in the laboratory, we observed scores of new interactions that had never been seen before," Stamler said. "By screening protein-protein interactions in the absence of NO, as is standard practice, an enormous amount of information has been missed."

The researchers first adapted the standard method of screening protein-protein interactions in yeast. By this method, known as "yeast 2 hybrid screening," genes of interest are inserted into yeast cells. The cells are manipulated such that those in which the inserted proteins physically interact have a growth advantage, allowing researchers to identify protein pairs that work together.

In the current study, the team first genetically altered yeast to enable them to screen proteins for interactions in an environment that included NO at levels naturally present in human cells. Yeast cells normally die in the presence of the molecule. Therefore, the screens typically exclude NO.

As a test case, Stamler's team screened the interaction of a mouse version of procaspase-3 -- a protein enzyme that controls cell death in response to inflammation -- with other cellular proteins. In the presence of NO, the protein exhibited dozens of novel protein interactions. A more exhaustive screen of protein-protein interactions turned up many more that occur only in the presence of NO

"It's mind-boggling how many new interactions
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Contact: Kendall Morgan
kendall.morgan@duke.edu
919-684-4148
Duke University Medical Center
31-Jul-2003

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