Being cut off in traffic, giving a presentation or missing a meal can all trigger a suite of physiological changes that allows the body to react swiftly to stress or starvation. Critical to this "fight-or-flight" or stress response is a molecular cycle that results in the activation of Protein Kinase A (PKA), a protein involved in everything from metabolism to memory formation. Now, a study by researchers at Penn State has revealed how this cycle resets between stressful events so the body is prepared to take on new challenges.
The details of this reset mechanism, uncovered through a combination of imaging, structural and biochemical techniques, was recently published in the Journal of the American Chemical Society.
"Some of the early changes in the fight-or-flight response include the release of hormones, like adrenaline from stress or glucagon from starvation," said Ganesh Anand, associate professor of chemistry and of biochemistry and molecular biology in the Penn State Eberly College of Science and lead author of the paper. "These hormones trigger an important molecular cycle that ultimately activates PKA, a versatile protein that can regulate more than a hundred different target proteins inside the cell. Better understanding of this cycle has implications not only for stress and starvation, but for other things we put in our bodies, like caffeine and certain drugs that initiate or extend the cycle."
In all cells of organisms from yeast to humans, PKA oscillates between active and inactive states. When hormones like glucagon or adrenaline bind to a specific location in the cell, they generate a molecule called cyclic AMP, or cAMP. This in turn binds to the inactive complex of proteins that contains PKA, switching the PKA to a more active state. But how exactly this cycle completes and the system resets itself has remained unclear.
"You don't want this cycle to be perpetually on and responding once the stressful situation has passed," Anand said. "You want to be able to reset the system. Dysregulation of PKA due to errors in this reset process can lead to cardiovascular disease, metabolic syndromes and other disorders. We wanted to know how long this system could remain active and how you can shut it off."
Using multiple imaging techniques -including electron microscopy and the higher-resolution cryo-electron microscopy as well as biochemical techniques and several different forms of mass spectrometry, which provided insights into the dynamics of the complex - the researchers uncovered at least three conformations of the complex that occur during the reset process that were previously unknown. They also clarified the physical space that these conformations use within the cell.
"Molecules are not rigid rocks; they are constantly fluctuating, almost like they are breathing," Anand said. "Cryo-electron microscopy is a powerful imaging technique that has gained a lot of traction, but it ultimately gives you a static image. If you look at a photograph of a leg, you can guess at how the knee and the ankle work, but it's just a guess. We used a variety of other techniques to really understand the mobile 'joints' of this complex. It's an iterative approach; we moved back and forth between the different techniques as we uncovered new insights so that we could really understand what the different components are doing during a variety of conformational changes."
Integral to the reset mechanism are phosphodiesterase proteins, or PDE, which remove the cAMP from the PKA complex, rendering the complex inactive once again. As the cAMP is removed, it can accumulate within the cell, allowing the number of reset cycles to be tracked.
"The system becomes more efficient as it goes through more cycles," said Varun Venkatakrishnan, graduate student in chemistry in the Penn State Eberly College of Science and first author of the paper. "Just like a runner can build up stamina with practice, there is some capacity for building endurance with this fight-or-flight response. This system has a built-in timer, which can record how many laps it has run. We would like to better understand the implications of activating the cycle many times in a row. It's also possible we could trick the system into thinking it has run more laps than it has by adding cAMP."
The researchers said that allowing the system to reset between stressors is physiologically important. For example, cAMP encourages memory formation, allowing memories to be formed around stressful events. Long-term stress can also increase the risk of developing Type 2 diabetes and impact the functioning of a variety of systems.
"In the modern world, we are continuously getting stimulated, including by substances like caffeine and drugs, and we'd like to better understand what happens when the cycle is activated for a long time," Anand said. "Caffeine is a PDE inhibitor, so it essentially pauses the reset mechanism, and we end up getting the buzz from the stress response without the actual stress. And some drugs called glucagon-like-peptides or GLPs, such as those used to alter appetite and so called 'anti-obesity' drugs, can modulate the PKA cycle. If we can find a way to keep the reset for longer, we might be able to find a way to counteract some effects of stress."
Additionally, the researchers said that the integrative, procedural framework of using cryo-electron microscopy, mass spectrometry and biochemical techniques could be adapted to understand the moving parts of a wide variety of protein complexes.
In addition to Anand and Venkatakrishnan, the research team at Penn State includes Tatiana Laremore, associate research professor and director of the Proteomics and Mass Spectrometry Core Facility; Theresa Buckley, graduate student in chemistry; and Jean-Paul Armache, assistant professor of biochemistry and molecular biology. Data for this paper was collected using the Proteomics and Mass Spectrometry Core Facility and the Cryo-Electron Microscopy Core Facility within the Penn State Huck Institutes of the Life Sciences. Funding from Penn State supported this research.
