|
US researchers have used NMR to identify a previously undetected trigger point on a naturally occurring "death protein" that helps the body get rid of damaged or diseased cells. The researchers suggest that their findings may offer a novel target for new drugs that could be used to treat cancer by forcing malignant cells to undergo apoptosis, or cellular suicide. Loren Walensky, Evripidis Gavathiotis, Marguerite Davis, Kenneth Pitter, Gregory Bird, and Samuel Katz of the Dana-Farber Cancer Institute, the Children's Hospital Boston, and Harvard Medical School, Motoshi Suzuki and Nico Tjandra of the National Institutes of Health, Bethesda, Maryland, and Ho-Chou Tu, Hyungjin Kim, and Emily Cheng of Washington University School of Medicine, Saint Louis, Missouri, report in the journal Nature how they have directly activated the trigger on the "executioner" protein BAX. This kills cells in the laboratory by initiating the cell's own self-destruct mechanism. "We identified a switch that turns BAX on, and we believe this discovery can be used to develop drugs that turn on or turn off cell death in human disease by targeting BAX," explains Walensky. "Because BAX lies at the crossroads of the cell's decision to live or die, drugs that directly activate BAX could kill diseased cells like in cancer and BAX-blocking drugs could potentially prevent unwanted cell death, such as in heart attack, stroke, and neurodegeneration," adds Walensky. BAX is just one of the BCL-2 family of proteins, all of which seem to be involved in different ways with the life and death of the cell. An imbalance in BCL-2 proteins in cancer cells enables them to avoid apoptosis triggers , ensuring unchecked cellular survival. The BAX protein lies dormant until the cell is exposed to stress. So, to test the trigger theory, the team synthesized a peptide that directly bound the trigger site, revealing its location and topography. Binding of this peptide to BAX spurs it into action and leads to the activated protein poking holes in the cell's energy apparatus, the mitochondria, which leads to apoptosis. The late Stanley Korsmeyer, an apoptosis pioneer for whom Walensky worked at the Dana-Farber Cancer Institute, suggested that killer proteins like BAX were somehow activated by a subset of "death domains" called BH3. Korsmeyer's hypothesis was that the interaction leading to activation was so fleeting that it would prove very difficult to study in detail. Indeed, the natural binding process did not succumb to conventional methods of detection and analysis. "When you tried to measure binding of the BH3 subunits to BAX, you couldn't detect the interaction," notes Walensky. However, a clue as to how the interaction might be observed emerged from the discovery by Walensky and his colleagues that the BH3 peptides being used in the laboratory do not maintain the coiled shape of the natural BH3 domains that participate in BCL-2 family protein interactions. With this in mind, the team designed so-called "stapled" BH3 peptides, which bear a crosslink that locks them into the helical form, thus sustaining their biological activity. Obtaining a structure, however, requires that the binding complex be stable to investigation, but by definition the fleeting BH3 binding event that leads to the killer BAX state is unstable. So, the question then becomes how to retain the potency of the binding process but slow the subsequent conversion of BAX to active BAX sufficiently to render it visible to NMR spectroscopy. By combining pure BAX protein in solution with increasing concentration of the stapled BH3 peptide, it was possible to use NMR to detect a specific group of BAX amino acids that were affected by the initial binding event. "The discrete subset of amino acids that shifted upon exposure to the stapled BH3 peptide mapped to a completely unanticipated location on BAX," explains Walensky. They had found the elusive BAX binding site that triggers its killer instincts. Writing in an accompanying editorial, Douglas Green and Jerry Chipuk of the Department of Immunology, at St Jude Children's Research Hospital, in Memphis, Tennessee, suggest that with this initiating event now defined, the subsequent steps of BAX activation remain the focus of ongoing investigation. "There might actually be two interaction sites between BAX (or BAK) and their activators, one at the back and one at the front. We do not know the conformations of the activated forms...nor can we reasonably model the nose-to-nose dimers at this point," they explain, "But the pieces are starting to come together as we puzzle out how these killers do the dastardly deed of triggering apoptosis." Reference:
Related links:
|
|
