Johns Hopkins researchers have uncovered a key step in
the body's regulation of melatonin, a major sleep-related
chemical in the brain. In the advance online section of
Nature Structural Biology, the research team reports
finding the switch that causes destruction of the enzyme
that makes melatonin — no enzyme, no melatonin.
Melatonin levels are high at night and low during the
day. Even at night, melatonin disappears after exposure to
bright light, a response that likely contributes to its
normal daily cycle but plagues shift workers and
jet-setters by leading to sleeplessness. To help understand
melatonin's light-induced disappearance, the Hopkins
researchers turned to the enzyme that makes it, a protein
called AANAT.
One way cells turn proteins like AANAT on and off is
by modifying them, attaching or removing small bits, such
as phosphate groups, to particular spots along the
protein's backbone. For AANAT, the key spot turns out to be
building block number 31, the researchers have found.
"We have discovered that addition and removal of the
phosphate group at this position is the key step in
regulating the enzyme's stability," said Philip Cole, the
E.K. Marshall and Thomas H. Maren Professor and director of
Pharmacology and Molecular Sciences in Johns Hopkins'
Institute for Basic
Biomedical Sciences. "When this phosphate group is
present, the enzyme is stable."
To test the importance of the phosphate group to the
enzyme's stability, research associate Weiping Zheng
developed a mimic of the key building block with the
equivalent of a permanently affixed phosphate group.
Zheng inserted the mimic into the appropriate place in
the enzyme, and research associate Zhongsen Zhang injected
the altered enzyme into cells. The altered enzyme stayed
intact in the cells much longer than the normal enzyme,
whose phosphate group can easily be removed, the scientists
report.
The researchers' next step is to determine how
exposure to light accelerates removal of the phosphate and
destruction of the enzyme, leading to a rapid drop-off in
melatonin. "Now we can fish for unknown players in the
degradation of the enzyme and hopefully find the trigger
that leads to its light-activated destruction," Zheng
said.
They've already shown that the phosphate group on
building block number 31 also improves the enzyme's ability
to bind to a protein known as 14-3-3, further increasing
the enzyme's stability and delaying its degradation.
Cole added that the mimic Zheng developed will do far
more than just ease study of melatonin's daily cycles.
Literally thousands of important proteins are controlled by
the addition or removal of phosphate groups, he said,
offering thousands of opportunities to use the mimic to
help understand cellular processes and their controls.
Funding for the study was provided by the National
Institutes of Health and the Ellison Medical Foundation.
Aspects of the work were carried out at the AB Mass
Spectrometry/Proteomics Facility at the Johns Hopkins
School of Medicine, which is funded by the U.S. National
Center for Research Resources, the Johns Hopkins Fund for
Medical Discovery and the Johns Hopkins Institute for Cell
Engineering.
Authors on the study are Zheng, Zhang and Cole of the
Johns Hopkins School of Medicine, and Surajit Ganguly,
David Klein and Joan Weller of the National Institutes of
Health.
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