Now, in a study that took more than five years to complete, Rockefeller University researchers, in collaboration with a team of bacteriologists at the University of Wisconsin, Madison, have become the first to solve the structure of a protein complex that protects these cells from singlet oxygen. The findings, which appear in the September issue of Molecular Cell , not only advance knowledge of how cells sense the presence of singlet oxygen, but also how they turn on critical genes to defend themselves from its effectsing photosynthetic bacteria that generate this toxic form of oxygen, Elizabeth Campbell, a research associate in Seth Darst's Laboratory of Molecular Biophysics, and her colleagues determined the unique arrangement of atoms in this two-protein complex, called s E /ChrR - and in the process, clarified the key to the structure's function: a single zinc ion. While the Wisconsin team, led by Timothy Donahue, showed that ChrR binds zinc, Campbell and her team used X-ray crystallography to reveal how the two proteins, s and anti-s, work together to ward off singlet oxygen. The crystal structure revealed that the anti-s protein, ChrR, has two domains, each of which could bind the zinc ion. Under thriving conditions, when cells lack singlet oxygen, Campbell and her collaborators found that anti-s needs this ion to latch onto its s counterpart. In its clutch, anti-s prevents s from activating the genes that instruct the cell to neutralize singlet oxygen's harmful properties. The researchers further showed that anti-s's first domain, which has been preserved evolutionarily, does this by blocking the groove where s binds RNA polymerase, the protein that initiates gene transcription. When the bacteria sense the presence of singlet oxygen, though, the anti-s's second domain responds by causing its first domain to release sigma. Now free, s can initiate the events that protect the cell from damage. What had come as a surprise to Campbell and her collaborators was that the second domain, the so-called sensing domain, had a zinc ion of its own, a particular feature not found previously in any other anti-s. When Campbell's collaborators at the University of Wisconsin, Madison removed the zinc-containing portion of the second domain and exposed the bacteria to light, the bacteria died, possibly suggesting that this second zinc is needed to initiate a process that protects the cell from this singlet oxygen.
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To find out if the droopy-tailed fish were asleep, Yokogawa checked to see if they experienced sleep rebound, the drive to try to catch up on lost sleep, after being sleep-deprived. So first he had to make sure the fish stayed awake. Tapping on the aquarium walls and using an underwater speaker didn't work, but he found a gentle electrical pulse kept fish active. He then created a computerized system to stimulate a fish each time it started to doze off. Once a sleep-deprived fish returned to a peaceful, dark aquarium, it compensated for lost rest with longer napping.
Unfortunately, some of the researchers had to stay awake along with the fish. "Originally, we didn't have the automated sleep-deprivation system, so I manually sleep-deprived them, becoming sleep-deprived myself," Yokogawa added.
The new model has already provided insights into the function of sleep-regulating molecules and brain circuits in zebrafish. Compared with normal zebrafish, the sleep mutants with neurons lacking hypocretin receptors experienced something akin to insomnia rather than narcolepsy. Although in dogs the loss of the receptor results in full narcolepsy, in zebrafish only nighttime activity was affected. Overall sleep decreased 30 percent in mutant fish, and when they finally did drift off, they remained asleep only half as long as normal fish.
Future research will search for fish mutants that might oversleep or lack sleep completely, in hopes of discovering new regulatory molecules and brain networks passed on through evolution to humans. "Many people ask the questions, 'Why are we sleeping?' and, 'What is the function of sleep?'" Mignot said. "I think it is more important to figure out first how the brain produces and regulates sleep. This will likely give us important clues on how and maybe why sleep has been selected by natural evolution and is so universal."
The Howard Hughes Medical Institute and the McKnight Foundation funded the study. The other authors are graduate students Wilfredo Marin, Jian Zhang, Guillaume P?İzeron; research associate Juiette Faraco, PhD; postdoctoral scholar Lior Applebaum, PhD; and professor Fr?İd?İric Rosa, PhD, at the Ecole Normale Sup?İrieure in Paris.
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