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Traffic Control

Hunting aggresomes

Hunting aggresomes:  Lizabeth Allison hears about how her student, Michelle Munyikwa ’11, found some aggresomes. (Photo by Joseph McClain)

Nuclear transport isn't what you might think it is

Lizabeth Allison sometimes confuses even scientific people when she talks about her work.

“I was at a party once and I told someone that I worked on nuclear transport,” Allison said. “He worked at Brookhaven National Lab and he said ‘Hey, we’re in the same business.’ I said, ‘Well, no. We’re not.’”

Allison’s lab, on the third floor of William & Mary’s new Integrated Science Center, investigates the intracellular travels of a “messenger” protein known as the thyroid hormone receptor. “Nuclear” in “nuclear transport” refers to the cell’s nucleus, not to nuclear energy.Comic strip

“There are two major areas within cells. There’s the nucleus, where all the genetic information is, and there’s the cytoplasm, where all sorts of metabolic activities take place,” Allison explained. “We’re interested in what the mechanisms are for movement between the cytoplasm and the nucleus.”
Allison, the Margaret L. Hamilton Professor of Biology at William & Mary and chair of the biology department, refers to her work as “traffic control.”

A journey into the nucleus

The thyroid hormone receptor is the vehicle that Allison’s lab watches. Like other proteins, it’s synthesized in the cytoplasm. Then, to do its job, it needs to journey into the cell’s nucleus, steering its way through the pore complexes of the double membrane surrounding the nucleus. Once safe inside the nucleus, the thyroid hormone receptor can go about the important work of turning genes off and on.

“We’re studying how this protein gets to where it needs to be in order to function,” Allison said, “and—in certain disease situations—how it is mislocalized.”
When thyroid hormone receptors go astray, they usually exhibit a mutation or some other discernable defect. These mislocated receptors tend to get gridlocked in the cytoplasm in protein pile-ups known as aggresomes. Michelle Munyikwa, a member of the class of 2011 working in Allison’s lab, explained that some mutations of the thyroid hormone receptor are out of shape—literally. She noted that misfolding is a common characterization of mislocalized thyroid hormone receptors.

Disrupt the structure/disrupt the function

“Proteins require a certain structure to function properly, so if the structure is disrupted, then their function is disrupted, and they kind of clump together and tend not to work,” Munyikwa said. “They have a sequence of amino acids and those amino acids interact with one another to form a 3-D structure. So if for some reason something is a little strange and they don’t fold quite the way they’re supposed to—then they don’t work.”

Allison pointed out that a mislocalized, misdirected thyroid hormone receptor—stuck outside the DNA vault of the cell’s nucleus—can’t function. Nonfunctioning thyroid hormone receptors can lead to dire consequences for gene expression, the multi-step biochemical process through which the DNA contained in the genes is put to work.

“There are all sorts of things that can happen. In some cases there are particular genes that should be being expressed—but aren’t,” Allison explained. “This can lead to a lack of control of a cell dividing.”

A cell that should stop dividing, but instead continues to divide and proliferate, eventually might lead to some form of cancer, she said. Some mutations of the thyroid hormone receptor can be trouble in and of themselves, she added.

“We’ve studied one form of the receptor that’s considered cancer-causing—an oncogenic form. It has a more cytoplasmic localization. It gets recruited to these aggresomes,” Allison said.

“This oncogenic protein can also pull the normal receptor into the aggresomes with it; it interacts with the normal receptor and takes it to the wrong location,” she continued. “That’s why we talk about it as traffic control.”

Up until about eight years ago, scientists studying the thyroid hormone receptor believed that its intracellular traffic control essentially was a one-way street: The thyroid hormone receptor starts off in the cytoplasm and, if all goes well, it enters the nucleus and gets down to work. Then Allison’s lab showed that the protein moves two ways—both in and out of the nucleus.

“We found that the thyroid hormone receptor doesn’t just go in and stay there and bind to DNA and regulate genes like everyone had thought for decades: It shuttles between the nucleus and the cytoplasm,” Allison said. “We still don’t really understand the physiological significance of that, but it means that we started looking for mechanisms about how it gets into the nucleus—and then we discovered we also had to study how it gets out.”

The mysterious export process

Allison said that export—the mechanism or mechanisms by which the thyroid hormone receptor proteins shuttle out of the nucleus—is an almost completely unstudied process, but one that may be important to the understanding of how certain cancers and thyroid hormone disorders work.

Two Funding Sources“No one had thought about export,” she said. “They’ve looked at a mutation in the thyroid hormone receptor and said, oh, it affects hormone binding or maybe its affecting the way the receptor interacts with the gene. But some mutations are in regions of the cell that we’re pretty sure have to do with nuclear export. So maybe part of the defect in these cancer cells or other diseases has to do with misregulation of traffic control.”

Allison’s work has revealed a number of insights into the mechanisms used by the thyroid hormone receptor, notably an understanding that its traffic patterns are immensely more complex than she had thought. What scientists had believed to be a simple, one-way protein path has, after years of investigation, come to resemble the molecular version of a freeway interchange. Not only does the receptor move both in and out of the nucleus, but now evidence suggests that it has a number of biochemical routes to choose from.

“We thought that one nuclear localization sequence would be sufficient to bring it into the nucleus. Well, it seems to have two. Why does it have two? We’re trying to figure that out. It also appears that it might have as many as three export sequences,” Allison said. “The thyroid hormone receptor just seems to do everything in its own complicated way.”

Allison’s lab is beginning to take a closer look at the receptor’s traffic patterns, mapping out in detail each biochemical turn, twist and lane change. Manohara Mavinakere, a post-doctoral researcher in the lab, has begun what Allison calls a “very systematic dissection” of the different export sequences. He’ll be examining the various amino acids involved, working to define the minimal sequence—the shortest possible biochemical signal for the thyroid hormone receptor to follow its route.

“It’s a painstaking process and, as you can imagine, if you start altering sequences within a protein, you could potentially alter the three-dimensional structure or alter some other function,” Allison said. “You have to do very careful tests to be sure that what you’re doing is strictly related to export and not interfering with other things. It’s like solving a puzzle.” i