Nitric Oxide May Change Basic Understanding of Cellular Machinery

Durham, N.C. -- Standard methods used to discover the role of proteins in the cell do not take into account a key player in that function: nitric oxide, according to new research by Howard Hughes Medical Institute and Duke Medical Center researchers. The researchers have found that the molecule, naturally present throughout the human body, plays a major role in assembling protein networks that direct fundamental cellular activities, such as whether cells live or die.

Cell death, or apoptosis, is involved in a wide range of human ailments, including heart failure, Alzheimer's disease and asthma, said Jonathan Stamler, M.D., HHMI investigator at Duke and senior author of the study. Therefore, the finding may lead to new insights into the underlying causes of disease, he added.

The Duke-based team, including HHMI associate and lead author Akio Matsumoto, M.D., report their findings in the August 1, 2003, issue of Science.

New tools developed by the team -- which will allow researchers to observe proteins in action under conditions that include natural levels of nitric oxide (NO) -- could change scientists' view of how proteins work together to drive essential cellular reactions. The scientists' results are particularly relevant today as researchers begin to decipher the function of the "proteome" -- the thousands of proteins encoded by the human genome, which is the body's complete set of genetic instructions, Stamler said.

"Understanding dynamic interactions among proteins is critical to understanding biological function," Stamler said. "When proteins come into contact with one another too much or too little, the result can be disease. The picture we have now of how life reactions occur is just the tip of the iceberg." The new method, he added, will enable scientists to fill in more of the missing biological details, yielding new insights into how healthy cells function and new opportunities for the treatment of disease.

Chemical 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 appeared with the addition of such tiny amounts of NO," Stamler said.

Two of the novel interactions discovered in yeast cells occurred in mouse cells only in the presence of NO, the researchers found. "The interactions seen in yeast turned out to be dramatically important in deciding the fate of mouse cells," he said.

Stamler's group suggests that NO regulates a broad spectrum of cellular reactions. NO might be as fundamentally important as phosphate in controlling cellular behavior, Stamler said. The addition of phosphate, known as phosphorylation, is the primary on-off switch for proteins that control basic life processes.

The Duke-based team's earlier work had revealed an unexpected role for nitric oxide. In blood cells, the NO molecule attaches to hemoglobin molecules, influencing oxygen delivery to tissues. However, said Stamler, the new studies suggest a broader role for NO in the body by revealing that the molecule influences interactions of a multitude of proteins.

The finding might have additional ramifications for understanding disease processes, said Stamler. Levels of NO commonly change in patients who have disease. For example, NO concentrations increase in patients with asthma. The finding that NO directly affects fundamental life reactions suggests that changes in cellular concentrations of the molecule might underlie some disease symptoms, Stamler said. Elucidating those effects could lead to new therapies.

Other participants in the study include Karrie Comatas and Limin Liu, Ph.D., both at Duke University Medical Center.