Research at the Cooke Lab
Motor proteins perform roles in numerous biological processes, including: muscle contraction, cell division and the movement of intracellular organelles. We study the mechanism of action of two classes of motor proteins, the myosin family and the kinesin family, which share a structural homology. One long-range goal of our laboratory is to understand the molecular mechanisms by which these two classes of motor proteins generate force.
The interaction of two proteins, actin and myosin, produces the force of muscle contraction and is also involved in the motility of all eukaryotic cells. We have used spectroscopic probes, both paramagnetic and fluorescent, to measure the orientation and conformation of both actin and myosin in active muscle fibers.
Kinesin motors are smaller than myosin but generate forces and displacements that are similar. To monitor conformational changes in this motor we have placed paramagnetic probes on active elements. This work, carried out in collaboration with the laboratories of Dr. Ron Vale, and Dr. Robert Fletterick led to the first molecular model of kinesin motility.
Another goal of the lab is to determine how muscle fibers adapt to intense activity and fatigue. Using muscle fibers that are permeable, we can simulate the intracellular conditions that occur in fatigued muscle. These studies have shown that the accumulation of a number of metabolic products in the interior of the muscle cell can account for some of the fatigue felt by the long distance runner, but it does not explain all of it. Our laboratory has recently pursued a new area, the energetics of resting muscle. We have found a new mechanism for generating thermogenesis in skeletal muscle fibers. The metabolic activity of resting skeletal muscle is of interest, because it plays a significant role in the whole body resting energy expenditure. Muscle metabolism is involved in cold induced thermogenesis, in consumption of calories from excess food intake and is a major regulator of blood sugar levels. The mechanism of muscle thermogenesis and its regulation remain an active area of investigation.
We started from the premise that thermogenesis was linked to an old observation that myosin in a test tube had a much greater activity than it has in living resting fibers. No one had previously found an in vitro system that replicates the in vivo activity. We employed quantitative fluorescence spectroscopy to measure single nucleotide turnovers in relaxed skinned fibers, and finally we observed the elusive inhibited myosin. We called the new state of myosin with the highly inhibited ATPase activity the "Super-Relaxed State" (SRX). In the SRX it takes about 250 seconds for myosin to turnover one ATP. The SRX is in dynamic equilibrium with a second relaxed state in which the myosin heads are disordered, with an ATP turnover rate that is 10 times faster than for the SRX. We call this the DisorderedRelaxed State (DRX). We propose that the inhibition of myosin ATPase activity in the SRX is achieved by binding of the myosin heads to the core of the thick filament in a structure previously identified using electron microscopy by Craig, Padron and co-workers. By analogy with another motor, active myosin is akin to a car racing down the road. Myosin in the DRX is similar to a car stopped at a traffic light with the motor idling, and the counterpart of the SRX is a car parked beside the road with the motor off.
We have also found that cardiac muscle has an SRX but the properties of this state differ from the skeletal state. In cardiac cells the SRX is involved in modulating the contractility of the myocardium. Modulation could occur via phosphorylation of myosin and myosin binding protein C (MBP-C). We have recently shown, in collaboration with James McNamara, working in the laboratory of Dr. Cris dos Remedios, that knock out of MBP-C in mice or mutations in human MBP-C reduce the population of the SRX. The exciting aspect of this new state is that manipulating it can provide therapies to treat human cardiac diseases. Agents which alter the stability of the SRX in cardiac cells could be used to modulate the contractility of the myocardium providing effective treatments for many cardiac disorders including heart failure and cardiomyopathies. We are currently collaborating with MyoKardia and the laboratory of Dr. James Spudich to identify compounds that would modulate the SRX in cardiac cells to treat cardiomyopaties.
Our discovery of the SRX has important implications for the development of an entirely new target for the treatment of obesity and Type 2 diabetes, via the up-regulation of whole body energy usage by perturbation of the population of the myosin in the SRX. The SRX is in equilibrium with a disordered state of myosin, which has a higher ATPase activity called the DRX. This difference in energy consumption provides an entirely new method for modulating the rate at which calories are metabolized, and thus impact obesity and Type 2 diabetes. Observations of the metabolic rate of relaxed, in vivo skeletal muscle indicate that the SRX is the physiologically dominant state, leading to the energy economy of relaxed muscle. A pharmaceutical that destabilized the SRX, populating the DRX, would be capable of increasing whole body metabolic rate by up to 1000 Cal/day. These pharmaceuticals could provide more effective therapies for obesity and Type 2 diabetes. Muscle makes an excellent target for increased thermogenesis due to its large reserve metabolic capacity.
To identify new molecules that destabilize the SRX, we developed a fluorescent probe that could measure the population of the SRX in a matter amenable to high throughput screens. We screened 2128 compounds discovering one hit. The hit was piperine an alkaloid found in black pepper. In a series of in vitro experiments we characterized the interactions of piperine with muscle fibers. We showed that the compound increased the ATPase activity of skinned muscle fibers. It has a Kd in the ATPase assay of ~2μM. We showed that it was effective in fast skeletal muscle, but had no effect in cardiac tissue. It also had no effect on the mechanics of active fibers. After identifying piperine with the screen, a search of the literature showed that piperine mitigates both obesity and Type 2 diabetes at very high doses in rodents. However, the mechanism for this effect had not been identified. We propose that the mechanism is its effect on the SRX. Piperine is also reported to affect a number of other targets, including liver enzymes and trans-membrane channels. It is small, too hydrophobic and binds to too many targets to make a good therapeutic compound in humans. Thus piperine itself will not function effectively as a therapeutic in humans, but it would make a good lead compound for the development of similar molecules that will function effectively.
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