Regulatory Protein Shown to Indicate Stress in Skeletal Muscle
Calmodulin points to factor contributing to inflammation
Results: Researchers at Pacific Northwest National Laboratory have demonstrated that calmodulin (CaM) a central regulator of metabolism, is a reliable indicator of oxidative stress in mammalian tissues. Their work demonstrated oxidative modification of CaM indicating that skeletal muscle operates under a high oxidative load as compared with other tissues. Furthermore, this team identified the molecular mechanism underlying the altered regulation by oxidized CaM of a calcium channel, the ryanodine receptor (RyR). This study appeared in the January 8 issue of Biochemistry.
Why it matters: Its high oxidative load suggests that skeletal muscle is likely to play a major role in amplifying inflammation induced by stressors such as pathogens, environmental pollutants and ionizing radiation. This result agrees with current proteomic and immunodetection data showing high levels of oxidatively modified proteins in muscle. However, as a result of the multiple oxidizable groups (methionines) in CaM, this single protein provides a progressive measure of the oxidative stress in vivo. Thus, CaM may be used as a potent sensor of stress in a variety of cells and tissues.
Methods: As reported in a series of four papers published in Biochemistry in 2007 and 2008, the PNNL team developed a novel fluorescent probe of CaM conformation that revealed both abnormal and normal modes of CaM binding. This provided insight into the effects of CaM oxidation on the major target of CaM regulation in muscle, the RyR. This calcium release channel (see figure) is essential in muscle as it initiates muscle contraction and is important in maintaining calcium homeostasis.
As shown in the figure, a crystal structure-derived model of calmodulin (CaM) with fluorescent pyrenes (blue ring structures) attached to their sites on N- and C-terminal domains, respectively. When cellular calcium levels rise, calcium (the grey balls) binds CaM, and CaM (in most, but not all cases) wraps itself around a specific sequence on the protein that it will regulate. In the second structure, the CaM binding sequence is shown as a blue helical structure. In the wrapped conformation, the C- and N-domains of CaM are close together, which brings the pyrenes into proximity. When they overlap, they emit a distinct fluorescence signal. Thus, there are specific signals associated with the extended and "wrapped" conformations of CaM that can be monitored simultaneously.
From this, the PNNL team could see the abnormal binding of oxidized CaM to various target proteins. This will help answer a major question in the field: How does the same protein (CaM) bind to a variety of different proteins to up- or down-regulate their activities, and because the total CaM in the cell is limited, how does CaM modification (like oxidation) alter the distribution of CaM among its various target proteins?
The researchers found that with this extent of oxidation, CaM retains high-affinity binding to the RyR1 calcium channel, but weakens preferentially in its ability to facilitate channel closure. This contrasts with unoxidized CaM, which acts as a local sensor of calcium level that regulates RyR1 by promoting channel opening at low calcium concentrations and channel closure at high calcium concentrations. Thus, oxidized CaM leads to prolonged channel opening (slower closure) and increased cellular calcium concentrations.
What's next: This work demonstrates the usefulness of CaM both as an internal and external sensor of stress. Further, the novel fluorescent probe developed in these studies might be used for multiplex detection of the effects of physiological stressors on binding conformation and affinity of CaM to its many regulated cellular targets.
Acknowledgments: The research team includes Curt Boschek, Heather Smallwood, Thomas Squier, and Diana Bigelow. The work was supported by the National Institutes of Health.
Boschek CB, TE Jones, HS Smallwood, TC Squier and DJ Bigelow. 2007. "Loss of calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin" Biochemistry 47(1):131-142.
Boschek CB, H Sun, DJ Bigelow, TC Squier. 2008. "Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels. Biochemistry. Published online January 18, 2008, 10.1021/bi701987n S0006-2960(70)01987-6
Boschek CB, TC Squier, and DJ Bigelow. 2007. "Disruption of interdomain interactions upon partial calcium activation of calmodulin." Biochemistry 46:4580-4588.
Boschek CB, TE Jones, TC Squier, and DJ Bigelow. 2007. Calcium occupancy on N-terminal sites within calmodulin induces inhibition of the RyR1 calcium release channel. Biochemistry 46:10621-10628.