Researchers at the University of New Hampshire (UNH) are taking a new look at vision, focusing on the fundamental signaling pathways in photoreceptor cells of the retina that—if disrupted by environmental or genetic factors—can lead to visual disorders, including total blindness.
Professor of Biochemistry and Molecular Biology in UNH’s College of Life Sciences and Agriculture (COLSA) Rick Cote recently received a $1,659,375 grant from the National Institutes of Health to delve into new approaches in understanding the visual signaling pathway. The results of this research will advance previous knowledge by taking a more structural look at phosphodiesterase (PDE), one of several proteins that, when mutated, can result in retinitis pigmentosa (RP).
Affecting an estimated 100,000 people in the U.S. alone, RP is a group of rare, inherited disorders that involve the degeneration of the cells in the retina. Patients with RP experience a gradual loss of vision as photoreceptor cells die when mutated genes give errant messages that influence the cells to make too little, too much, or incorrect proteins. Researchers like Cote are closing in on a better understanding of the role of PDE in RP, with hopes that knowledge about the enzyme’s structure—and how its subunits interact with one another when activated—will inform future treatments of RP.
“Many signaling pathways within cells occur with proteins that exist in a multi-protein complex, but we don’t yet know how the visual signaling complex (of which PDE is the central enzyme) is organized,” says Cote. “We know that PDE is tethered to the membrane and becomes activated upon binding to a protein called transducin, but we don’t know what other proteins are interacting with PDE and how they might modulate it. Furthermore, we don’t know the topological relationships of PDE with all its binding partners.”
Uncovering the answers to these unknowns may accelerate efforts to developing treatments for those born with RP. For many years, Cote and his team of researchers have been focusing on the step-by-step biochemical pathway that starts with light being absorbed by the visual pigment (opsin) and eventually results in an electrical response. “At this time we know all of the major components of the pathway,” says Cote. “Although PDE is central to this pathway, no one has determined the mechanism by which RP is caused by a single amino acid change in PDE.” The NIH grant allows Cote and the other researchers involved with this project to examine the structure of the PDE at the molecular level; determine how its subunits interact with one another, shifting in their conformation when the PDE is activated; and how the PDE enzyme forms a complex with other proteins.
Many protein structures can be determined by a technique called x-ray crystallography. “It’s a great technique, but no one’s ever been successful doing it with the PDE signaling complex, so we’ve had to turn to alternative approaches.”
That’s where Cote’s collaboration with Professor of Proteomics Feixia Chu comes in. Instead of trying to crystalize full-length PDE and its binding partners, Cote and Chu are using a chemical cross-linking technique to determine the structure of PDE itself. Going further, they then plan to map the interactions between PDE and the various proteins that activate and inactivate PDE during visual signaling.
“In this chemical cross-linking approach, we purify PDE and its binding partners as they exist in the photoreceptor cell, and then add chemicals that link different reactive groups together. Some of those reactive groups are close to adjacent proteins, and if they’re within a certain distance, the chemical cross-linker can create a bond between two proteins,” says Cote. “What’s really marvelous about the process is that once you’ve created those crosslinks, you’ve frozen the protein complex. Now we break down the proteins into small peptides, and you look for those peptides that are cross-linked to another peptide. From the identity of those two cross-linked peptides, you can say these two proteins approach within a certain distance in the three-dimensional protein complex.”
Identifying cross-linked peptides is made possible through the use of a mass spectrometer in Chu’s lab. Once identified, these cross-linked peptides are used to build a molecular model of PDE and the proteins to which it binds. Cote and Chu are particularly excited at the prospect of being able to use this approach to compare the dark and light-activated states of the PDE protein complex, and its potential to define the structural changes that occur during the first steps in vision.
Sometimes it only takes a single amino acid substitution in the PDE molecule to disrupt the signaling pathway and cause RP. Ultimately, that single amino acid change must be reflected in a change in the structure of the PDE. “Our hope is that if we can determine the molecular structure of PDE and the topology of the proteins with which it interacts during visual signaling, we will then be able to explain why an amino acid substitution causes PDE to not work properly” says Cote.
In addition to determining the overall molecular organization of PDE, Cote and his team are also isolating individual regions of the PDE enzyme to gain atomic-level details through a collaboration with Professor Hengming Ke, an x-ray crystallographer at the University of North Carolina. Relying on Ke’s past success in solving the structures of related proteins, Cote and Ke hope to bring atomic-level detail to understanding how PDE binds to an inhibitory protein as well as addressing fundamental differences in how rod and cone photoreceptors regulate their PDE enzymes. Cote’s research team also benefits from a longstanding, productive collaboration with UNH Professor Tom Laue, who will provide expertise in biophysical characterizations of the protein complexes central to the visual signaling pathway. Finally, as Cote notes, “We couldn’t possibly accomplish all of the work proposed in this NIH grant without the participation of many highly motivated and skilled undergraduate and graduate students here at UNH.”